The Role of Molecular Chaperones in Mitochondrial Protein Import and Folding

The Role of Molecular Chaperones in Mitochondria1 Protein Import and Folding Michael T. Ryan,*Nt Dean J. Naylor,t Peter B. Herj,t Margaret S. Clark,* and Nicholas J. Hoogenraad*"

*School of Biochemistry, La Trobe University, Bundoora, Victoria 3083, Australia; and ?Department of Horticulture, Viticulture and Oenology, The University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia

Molecular chaperones play a critical role in many cellular processes. This review concentrates on their role in targeting of proteins to the mitochondria and the subsequent folding of the imported protein. It also reviews the role of molecular chaperones in protein degradation, a process that not only regulates the turnover of proteins but also eliminates proteins that have folded incorrectly or have aggregated as a result of cell stress. Finally, the role of molecular chaperones, in particular the mitochondrial chaperonins, in disease is reviewed. In support of the endosymbiont theory on the origin of mitochondria, the chaperones of the mitochondrial compartment show a high degree of similarity to bacterial molecular chaperones. Thus, studies of protein folding in bacteria such as fscherichia coli have proved to be instructive in understandingthe process in the eukaryotic cell. As in bacteria, the molecular chaperone genes of eukaryotes are activated by a variety of stresses. The regulation of stress genes involved in mitochondrialchaperone function is reviewed and major unsolved questions regarding the regulation, function, and involvement in disease of the molecular chaperones are identified. KEYWORDS: Molecular chaperone, Mitochondria, Protein import, Protein folding, Proteolysis, Heat shock.

1. Introduction According to the endosymbiont hypothesis, a eubacterium-like ancestor was engulfed by an anaerobic urkaryotic cell (Gray, 1989). These two To whom correspondence should by addressed: Fax: 61-3-9479-2467. E-mail: [email protected]. lnrernarional Review of Cyrology, Vol. 174 0074-7696/97 $25.00

127

Copyright 0 1997 by Academic Press. All rights of reproduction in any form reserved.

128

MICHAEL T. RYAN ET AL.

cells formed a partnership whereby the progenitor of the eukaryote, the protoeukaryote, was created. This cell became aerobic while most of the original eubacterial genes were transferred into the urkaryote’s genome and the eubacteria evolved into specialized double-membraned compartments, the mitochondria. The mitochondrion of the modern day eukaryote acts as a compartment to provide the cell with ATP and assists in the compartmentalization of potentially overlapping metabolic pathways. As a consequence of this evolutionary process, most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol on free ribosomes as preproteins, and subsequently imported into the mitochondria. A number of obstacles are confronted by the preprotein during its translocation into mitochondria. It needs to be targeted to mitochondria, bind to membrane receptors, and subsequently be imported through the proteinaceous channels of the outer membrane and, in most cases, also the inner membrane. Furthermore, these channels are slim and the imported preprotein must therefore be in an extended, unfolded conformation in order to pass through. Whether the newly synthesized preprotein is maintained in an unfolded conformation prior to its import into the mitochondria or whether it is unfolded at the mitochondrial surface is not clear, but in any case, following translocation the preprotein must fold in an environment containing high protein concentrations. This review addresses some of the ways the preprotein accomplishes the arduous journey from cytosolic preprotein to a folded mitochondrial protein. Central to the process is a group of proteins that have been highly conserved during evolution-the molecular chaperones. Proteins destined for mitochondria are maintained in an import-competent state in the cytosol, at least partly, by molecular chaperones. Furthermore, molecular chaperones located in mitochondria are responsible for the translocation and subsequent folding of these proteins into their active states. Much of our knowledge about the mechanism by which molecular chaperones act in the eukaryotic cell has arisen through studies of molecular chaperones from Eschericia coli. Molecular chaperones exhibit both structural and functional identity between species. This is particularly evident when comparing molecular chaperones of bacteria and mitochondria, perhaps not surprising given their common eubacterial ancestry. This review also addresses the role of molecular chaperones in protein folding and provides an assessment of the role these molecules may play in disease.

II. Historical Perspective A. How Are Proteins Folded? Anfinsen (1973) proposed that the native, folded conformation of a protein represents its minimum global free-energy state that is dictated solely by

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

129

the amino acid sequence. This conclusion stemmed from studies on the spontaneous refolding of chemically denatured ribonuclease in vitro and was further supported by the finding that in soluble proteins, approximately 80% of hydrophobic side chains are buried within the protein to minimize contact with polar water molecules (Anfinsen, 1973;Lesser and Rose, 1990). Although many proteins can fold into their active state in vitro, they are synthesized in a crowded environment in vivo that is vastly different to the situation in a test tube and less favorable for spontaneous folding. The cellular milieu has a very high protein concentration (up to 500 mg/ ml in some mitochondria; Hackenbrock, 1968) and contains a multiple array of membranous components. This provides a high potential for interaction with the exposed hydrophobic regions of a newly synthesized protein that may initiate “off-pathway” interactions and compromise protein folding. At the site of protein synthesis, nascent polypeptides are also present in high local concentrations, adding to macromolecular crowding, whereby much of the cellular volume is physically occupied and thus unavailable for other macromolecules (Ellis and Hartl, 1996). A consequence of such crowding would be a tendency for hydrophobic regions of the nascent polypeptide to improperly associate and aggregate. Protein refolding in vitro is initiated from the complete, unfolded polypeptide, whereas proteins are synthesized vectorially in vivo. Therefore, protein folding in vivo has the potential to be initiated during polypeptide synthesis, a situation that has been observed for multidomain proteins such as 0-galactosidase (Seckler and Jaenicke, 1992). In other cases, the interaction of the N-terminal region of a polypeptide with its C-terminal region has been suggested to be important in folding pathways (Ptitsyn, 1981). Thus, it may be undesirable for the N-terminal region of the polypeptide to undergo unfavorable and premature folding events or improper interactions with itself, or other cellular components, prior to its completion of synthesis. A similar situation applies to a polypeptide that is translocated through membrane channels into organelles such as mitochondria. The preprotein resembles an incompletely synthesized polypeptide during its vectorial import while in an unfolded conformation. What prevents these translocating polypeptides from associating prematurely with other organellar components in a fashion that would otherwise lead to their aggregation? Detailed biochemical and genetic studies strongly suggest that molecular chaperones provide the answer to this question. 6 . What Are Molecular Chaperones?

Molecular chaperones have been defined as a family of proteins that bind to and assist in the folding of proteins into their functional states. They do not form part of the final protein structure nor do they possess steric

130

MICHAEL T. RYAN E r AL.

information specifying a particular folding or assembly pathway (Ellis, 1987). The term molecular chaperone was first used by Laskey et al. (1978) to describe the action of the protein nucleoplasmin in the assisted folding of nucleosome assemblies. Although nucleosomes could be formed in vitro from DNA and histones at low ionic strength, a precipitate was formed when assembly was attempted at physiological ionic strength. The presence of nucleoplasmin at physiological ionic strength promoted nucleosome assembly by preventing incorrect ionic interactions between the histones and DNA. Other proteins performing molecular chaperone-like functions, such as members of the Hsp70 family and the chaperonins [Chaperonin 60 (Cpn60) and Chaperonin 10 (CpnlO)], were subsequently identified (Ellis, 1987) and the list is continually increasing. Consistent with their role in the folding of newly synthesized and newly translocated proteins, many molecular chaperones are expressed in the cell constituitively. Under conditions that compromise protein folding and cell physiology, for example, heat shock, the synthesis of most molecular chaperones is induced to even higher levels. Although many molecular chaperones were first discovered as heat shock proteins, many of the genes encoding molecular chaperones are also induced by other cell-stressing agents such as glucose deprivation, calcium ionophores, amino acid analogues, ethanol, and heavy metals. The genes encoding molecular chaperones are therefore classified as stress genes and the associated induction of expression during periods of stress is referred to as the stress response. The common trigger for the stress response is the presence of abnormal proteins in the cell. For example, the stress response is observed after denatured (but not native) proteins are injected into Xenopus oocytes (Ananthan et al., 1986).

C. Escherichia coli: A Model System for the Study of Molecular Chaperones Unlike the highly compartmentalized eukaryotes, bacteria contain few molecular chaperones with overlapping functions. The relative simplicity of the bacterial chaperone complement has facilitated both the genetic and biochemical characterization of these essential gene products. Furthermore, given that molecular chaperones constitute a remarkably conserved family of proteins with homologues in all organisms studied, the characterization of bacterial molecular chaperones has been particularly instructive. In keeping with the endosymbiont model for the origin of the mitochondrion, this is perhaps most evident when comparing the repertoire of molecular chaperones in bacteria and mitochondria. The molecular chaperones of E. coli and their eukaryotic counterparts are listed in Table I and described below.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

131

1. DnaK, DnaJ, and GrpE Chaperones of E. coli The E. coli DnaK, DnaJ, and GrpE heat shock proteins were first identified and subsequently purified as host factors involved in A-phage DNA replication (Georgopoulos et al., 1990). Amino acid sequence comparisons indicated that DnaK is a member of the Hsp70 family. All members of this family exhibit molecular weights of approximately 70 kDa and a strong binding to ATP but only a weak ATPase activity (Zylicz and Georgopoulos, 1984; Welch and Feramisco, 1985; Liberek et al., 1991a). The Hsp70 family members are involved in the folding, assembly, and disassembly of nascent and denatured proteins chiefly by preventing unwanted interactions and aggregations (Pelham, 1986). Like other Hsp70 members, DnaK can undergo limited proteolysis in vitro to produce two distinct domains (Chappell et af., 1987). A highly conserved 44-kDa N-terminal domain is involved in ATP binding and hydrolysis, whereas a less conserved C-terminal domain binds polypeptides. The structures of these domains have been determined individually by X-ray crystallography (Flaherty et aL, 1990;Zhu et al., 1996). The ATPase domain resembles the ATPase domains of hexokinase and actin (Bork et aL, 1992). In intact DnaK, binding of Mg-ATP to this domain results in a conformational change in the C-terminal substrate binding domain (Liberek et d.,1991b; Buchberger et aL, 1995). The substrate binding domain of DnaK consists of a compact P-sandwich subdomain followed by an extended structure of @-helices(Zhu et al., 1996). A seven-residue polypeptide with high affinity for DnaK (Gragerov et al., 1994) was cocrystallized with this domain and shown to bind in an extended conformation in the @sandwich cleft. The peptide substrate is encapsulated within this cleft by helical segments in DnaK that serve as a lid but do not contact the peptide. The lid may be opened upon ATP binding and hence facilitate polypeptide binding and release (Zhu et af., 1996). Interestingly, this ahelical region may also bind to the cofactor DnaJ (Wawrzyndw and Zylicz, TABLE I Nomenclature of Molecular Chaperones from E. coli and Their Mitochondria1Homologues from Fungi and Mammalian Mitochondria

E. coli

Fungi

Mamma1s

DnaK DnaJ GrpE GroEL GroES

mt-Hsp70/SSCl MDJlp YGElplMGElp Hsp60 HsplOlCpnlO

mt-Hsp70/GRP75 ?

mt-GrpE Cpn60/Hsp60 CpnlOlHsplO

132

MICHAEL T. RYAN ET AL.

1995), thereby providing assistance in the opening and closing of the lid and access of the polypeptide substrate to DnaK. DnaJ is a heat shock protein with a monomeric molecular weight of approximately 40 kDa. DnaJ possesses a molecular chaperone function of its own because it can protect denatured polypeptides from aggregation in vitro and binds to nascent polypeptides (Langer et al., 1992a; Schroder et al., 1993; Hendrick et al., 1993; Szabo et al., 1994). Binding to substrate proteins is achieved through a central cysteine-rich region containing two zinc atoms (Szabo et al., 1996). DnaJ also binds to the substrate-binding domain of DnaK through an approximately 70-amino acid long J domain. This J domain is a common motif in a number of proteins with suspected molecular chaperone activity (Cyr et al., 1994).DnaJ stimulates the hydrolysis of DnaK-bound ATP. The interdependence of DnaJ and DnaK is further underscored by the organization of their genes into an operon. However, the ATPase activity of DnaK is only stimulated 2- or 3-fold by DnaJ alone but is stimulated up to 50-fold in the presence of both DnaJ and GrpE (Liberek et al., 1991a). GrpE has a monomeric molecular weight of approximately 23 kDa and binds to the ATPase domain of DnaK (Buchberger et al., 1994), where it stimulates the release of bound ADP (Liberek et al., 1991a). Unlike the dnak or dnaj genes, the bacterial grpe gene is essential for cell viability (Ang et al., 1986). DnaK, DnaJ, and GrpE combine to assist in the folding of a number of proteins via a reaction cycle whereby their association affects the affinity for both ATP and substrate polypeptides (Szabo et al., 1994; Schmid et al., 1994; Langer et al., 1992a; Buchberger et al., 1995; Gamer et al., 1996). Some proteins probably assume their final folded state following interaction with the DnaK/DnaJ/GrpE machinery, but in many cases the folding pathway requires further participation of two other chaperones, GroEL and GroES. 2. GroEL and GroES

GroEL and GroES are members of the chaperonin family of molecular chaperones (Hemmingsen et al., 1988; Ellis, 1996). The genes encoding GroEL and GroES constitute an operon in E. coli and are essential for cell viability (Tilly et al., 1981; Fayet et al., 1989). Electron microscopy indicates that the quaternary structure of GroEL consists of two stacked seven-membered rings arranged into a tail-to-tail conformation containing a central cavity 50 wide (Langer et al., 1992b; Braig et al., 1993; Chen et al., 1994). The crystal structure of a mutant form of GroEL, which exhibits similar characteristics to wild-type GroEL (Braig et al., 1994), and the structure of GroEL (14 ATPyS) (Boisvert et al., 1996) show that each

A

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

133

60-kDa subunit contains an equatorial, an intermediate, and an apical domain. The equatorial domain comprises the ATP binding site (Boisvert et al., 1996), whereas the apical domain is involved in the binding of both GroES and polypeptide substrates (Fenton et al., 1994). The intermediate domain may confer flexibility between the two other domains (Braig et al., 1995). GroES is a heptamer of 10-kDa subunits and the structures of the E. coli (Hunt et al., 1996) and Mycobacterium leprae (Mande et al., 1996) homologues indicate that the heptamer is dome shaped. Each subunit contains a flexible loop region that projects from the bottom of the outer rim of the dome (Hunt et al., 1996) and is involved in binding to GroEL (Landry et al., 1993). Upon binding to GroEL, this loop is thought to become immobilized because it is no longer susceptible to proteolysis (Landry et al., 1993). The mechanism by which GroEL and GroES act in the folding of proteins has been studied intensely (Clarke and Lund, 1996; Hartl, 1996) and their mode of action has been a subject of some controversy. The further refinement and elucidation of chaperonin structures in the presence of both nucleotides and bound substrates may lead to a consensus on their action. Although a detailed description of the chaperonin-mediated folding cycle is beyond the scope of this review, there are four essential features: (i) GroEL and GroES associate in the presence of Mg-ATP and K+ facilitating the binding of substrate to GroEL (Bochkareva and Girshovich, 1992; Todd et al., 1993, 1994; Burston et al., 1995; Corrales and Fersht, 1996); (ii) the unfolded polypeptide binds within the central cavity of GroEL and binding is stabilized by hydrophobic residues at the apical region of the central cavity (Braig et al., 1993; Fenton et al., 1994); (iii) in the presence of ATP, GroES binds to GroEL and may displace the polypeptide into an enlarged central cavity (Hartl, 1994; Chen et al., 1994) capable of accommodating a protein of up to 50-60 kDa (Braig et al., 1994); and (iv) ATP hydrolysis facilitates the release of GroES, polypeptide, and ADP (Todd et al., 1994). Although some proteins may fold while enclosed in the central cavity of GroEL (Weissman et al., 1996;Mayhew et al., 1996), other proteins may require multiple rounds of binding and release (Todd et al., 1994; Weissman et al., 1994, 1995). Although the in vitro folding rates of molecular chaperones may differ substantially from those observed in vivo, it is likely that not all proteins require chaperonins for folding. Careful calculations based on in vitro folding rates suggest that GroEL and GroES are likely to facilitate the folding of no more than 5% of all E. coli proteins in actively dividing cells (Lorimer, 1996). However, GroEL and GroES are essential for cell viability, and approximately 50% of chemically denatured proteins from an E. coli lysate will form complexes with GroEL (Viitanen et al., 1992a) and about 30%

134

MICHAEL T. RYAN ET AL.

of proteins in GroEL-deficient cells fail to attain their native conformation (Horwich et al., 1993).

3. Molecular Chaperones Recognize Different Structural Features in Their Substrates Different homologues of molecular chaperones may be found in different subcellular compartments within a eukaryotic cell (Table 11). Although molecular chaperones bind a range of substrate proteins that have no apparent sequence similarities, several studies indicate that members of each family recognize similar structural motifs. Although most Hsp70 members have affinity for hydrophobic peptides (Flynn et al., 1991; Gragerov et al., 1994),their binding specificities are not always interchangeable between homologues (Gething and Sambrook, 1992). The peptides are bound to Hsp70 in an extended conformation (Palleros et al., 1991; Langer et al., 1992a; Landry et al., 1992; Blond-Elguindi et al., 1993), consistent with the affinity of Hsp70 members for nascent polypeptides (Beckmann et al., 1990; Nelson et al., 1992; Frydman et al., 1994; Hansen et al., 1994). Structural characterization of the DnaK substrate-binding domain complexed with a polypeptide indicates that binding occurs primarily through five hydrogen bonds that are contributed by peptide backbone groups. A central pocket at the polypeptide binding site of DnaK accommodates large aliphatic residues (Zhu et al., 1996). GroEL can bind to proteins containing different structural motifs, including a helical peptide (Landry and Gierasch, 1991) and immunoglobulins that contain P-sheet structures only (Schmidt and Buchner, 1992). Studies of GroEL mutants indicate that substrate binding by GroEL is mediated TABLE II Molecular Chaperone Homologues of Bacterial and Eukaryotic Cells

Fungi E. coli

Cytosol

DnaK

SSA1-4 Hsp70 (SSC1)

DnaJ

Ydjlp SISlp

Mitochondria

SSB1-2

GrpE GroEL GroES

MDJlp YGElpMGElp Hsp60 HsplO/CpnlO

Mammals ER

Cytosol

Mitochondria

BiP

Hsc70 (Hsp73) mt-Hsp70

Lhslp

Hsp70 (Hsp72)

SEC63p Hsp4O SCJlp

?

mt-GrpE Cpn60/Hsp60 CpnlO/Hsp10

ER BiP/ Grp78 MTJl?

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

135

through hydrophobic residues on its apical domain (Fenton et al., 1994), consistent with the high affinity of GroEL for hydrophobic amino acids. This suggests that GroEL recognizes the hydrophobic regions of a substrate that are normally buried when it is in its native state (Richarme and Kohiyama, 1994). This is further exemplified by GroEL’s ability to bind and facilitate the refolding of a trapped folding intermediate of malate dehydrogenase (Peralta et al., 1994). These kinetically trapped intermediates have been suggested to be in a form in which significant energy barriers prevent proper folding (Todd et al., 1996). Such intermediates are often bound to GroEL in a molten globule state (Martin et al., 1991; Hayer-Hart1 et al., 1995) but not always (Okazaki et al., 1994). Nuclear magnetic resonance (NMR) spectroscopy indicated that, although a peptide derived from the vesicular stomatitis virus glycoprotein was bound to DnaK in an extended conformation, it was a-helical when bound to GroEL (Landry et al., 1992). Binding of the polypeptide to DnaK restricted the mobility of the peptide backbone, whereas binding to GroEL restricted the mobility of the side chains. Hsp70 members interact with early folding intermediates, whereas the chaperonins target intermediates that exhibit both secondary and tertiary structure. Taken together, these findings suggest that protein folding can be mediated by a sequential action of molecular chaperones, and in vitro experiments support such a model in the folding pathway of some proteins (Langer et al., 1992a). The extent to which such a sequential folding pathway is adopted in vivo would depend on both the structural features of the substrate protein (Rospert et al., 1996) and the physiological conditions of the cell.

111. Targeting of Proteins to the Mitochondrion A. Preprotein Import into the Mitochondria Can Occur Posttranslationally Early studies on mitochondrial protein import suggested that, like most proteins targeted to the endoplasmic reticulum, import can occur cotranslationally (Verner, 1993). For example, yeast cells treated with cyclohexamide in order to arrest translation were found to contain large numbers of polysomes bound to mitochondria (Kellems and Butow, 1972). Analysis of these polysomes revealed that they were enriched in mRNA for a number of mitochondrial proteins (Ades and Butow, 1980). Although a number of preproteins may be imported into mitochondria cotranslationally in vivo, it is not obligatory because both in vitro and in vivo studies have shown that mitochondrial protein import can also take place posttranslationally

136

MICHAEL T. RYAN ET AL.

(Reid and Schatz, 1982; Verner, 1993). Subsequent studies into mitochondrial protein import have concentrated on the characterization of the factors involved in the posttranslational import of proteins.

6.Components Involved in the Import of Proteins into Mitochondria

A great deal of evidence supports the proposal that protein import into mitochondria is very sensitive to the conformation of the preprotein. Because molecular chaperones are involved in cellular protein folding, it is therefore not surprising that they are involved in the import pathway. The identification and characterization of molecular chaperones and other components involved in mitochondrial protein import has largely been achieved using the fungi Saccharomyces cerevisiae and Neurospora crassa as model systems. This is because mutants defective in components of the import apparatus can be created and subsequently characterized by genetic and biochemical means (Kiibrich et al., 1995; Lithgow et al., 1995). Identification of components involved in mitochondrial preprotein import and folding in mammalian cells has been facilitated by sequence similarities with fungal and bacterial counterparts, whereas their functions in the import and folding process have been ascertained through in vitro approaches. However, many of these components were initially identified in the mammalian cell through the study of the stress response. Factors involved in the targeting of preproteins to the mitochondrial surface are illustrated in Fig. 1.

1. Chaperones at the Ribosomes The association of molecular chaperones with translating ribosomes suggests that they are intimately involved in the synthesis and/or protection of nascent polypeptides. Two members of the yeast Hsp70 family, Ssbl and Ssb2, are found associated with translating ribosomes (Nelson et al., 1992). These proteins are 99.3% identical and are highly expressed at times of optimal growth. However, in contrast to other Hsp70 proteins, their synthesis is reduced under heat shock conditions (Craig and Jacobsen, 1985).Both Ssbl and Ssb2 are released from ribosomes upon the addition of puromycin, an inhibitor of protein synthesis (Nelson et al., 1992).Ribosomebound Hsp70s may be involved in preventing improper interactions of the nascent polypeptide with ribosomal proteins or preventing premature folding. Alternatively, they may act by binding to and pulling the nascent polypeptide out of the ribosomal pore, analogous to the roles played by organellar Hsp70 members during protein translocation (see below). Christopher and Baldwin (1996) have further proposed that ribosome-bound

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

137

FIG. 1 Preprotein targeting to the mitochondrion. Following translation in the cytosol, the mitochondrially targeted preprotein (in black) is maintained in an import-competent conformation by cytosolic factors such as Hsc70, Ydjlp, MSF, PBF, and possibly Hip. Depending on its conformation and the factors bound to it, the preprotein binds to the appropriate outer membrane receptors. The association with these receptors facilitates the transfer of the preprotein to the translocation machinery and release of cytosolic factors from the preprotein.

Hsp7O may anchor the N-terminal region of the emerging polypeptide to the ribosome and thereby prevent the formation of structures (“knots”) in the polypeptide that might otherwise prevent successful folding and hence result in aggregation. A number of distinct DnaJ homologues exist in the cytosol of eukaryotes (Cyr et al., 1994). One of these homologues is the heat-inducible yeast protein Sislp (Luke et al., 1991). Sislp is essential for cell viability and associates with ribosomes. In cells with impaired Sislp function, the level of polysomes decreases and evidence points to an involvement of Sislp in the initiation of translation (Zhong and Arndt, 1993). Sislp may be involved in the targeting of Ssbl/2 to nascent polypeptides at the ribosomes; however, an interaction of Sislp with these Hsp70 homologues has not been established (Luke et al., 1991). By analogy to other DnaJ homologues, Sislp may associate with nascent polypeptides and prevent their aggregation with other components.

138

MICHAEL T. RYAN ET AL.

Proteinaceous factors may ensure targeting of transported proteins to their correct cellular compartment (Lithgow et al., 1993a). Indeed, a protein that seems to perform such a function has been identified and termed nascent polypeptide-associated complex (Wiedmann et al., 1994). This protein is loosely bound to ribosomes and shields nonsignal peptide regions of nascent polypeptides from interacting promiscuously with the signal recognition particle, thereby preventing inappropriate translocation into the endoplasmic reticulum.

2. Mitochondria1 Preproteins and Their Interaction with Cytosolic Chaperones Most mitochondrial preproteins are synthesized on cytosolic ribosomes with N-terminal mitochondrial targeting sequences. Artificial preproteins consisting of genuine mitochondrial targeting signals attached to cytosolic passenger proteins are capable of being imported into the mitochondria, suggesting that the information contained within these signals is sufficient to support protein targeting (Hartl et al., 1989). Early surveys revealed that mitochondrial targeting signals exhibited little or no sequence identities. They are typically 15-40 residues long and are rich in hydrophobic and basic residues (von Heijne, 1986; Hartl et al., 1989). Von Heijne (1986) predicted that mitochondrial targeting signals adopt a common structural feature, namely, a positively charged amphiphilic a-helix. The structural characterization of mitochondrial targeting signal peptides has firmly supported this proposal (Endo et al., 1989; Karslake et al., 1990; Bruch and Hoyt, 1992; Thornton et al., 1993; Hammen et al., 1994; MacLachlan et al., 1994; Jarvis et al., 1995). Translocation of the preprotein across the inner mitochondrial membrane into the matrix is initiated by the mitochondrial targeting signal in a manner that requires a membrane potential (AT:)(Schleyer et al., 1982), perhaps to provide an electrophoretic force directing the positively charged targeting signal toward the negatively charged matrix. Although the targeting signal targets the preprotein to the mitochondria, it alone is not sufficient to ensure import of the entire protein. Elegant experiments by Schleyer and Neupert (1985) and Eilers and Schatz (1986) demonstrated that a preprotein must be in a relatively unfolded state for successful import through the translocation channels. This can be achieved by factors that either unfold the preprotein prior to its entry into the mitochondria or alternatively maintain the newly synthesized preprotein in a relatively unfolded state in the cytosol. Consistent with the latter proposal, a number of early observations indicated that factors in reticulocyte lysate preparations could stimulate the in vitro import of preproteins into isolated mitochondria (Argan et al., 1983; Miura et al., 1983; Ohta and Schatz, 1984; Murakami et al.,

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

139

1988a). Because loosely folded preproteins required little or no ATP for import, whereas more compact proteins did, such cytosolic factors were believed to require ATP to maintain their unfoldase activity (Chen and Douglas, 1987; Pfanner et af., 1988; Ostermann et af., 1989; Neupert et af., 1990). These early findings laid the foundation for the identification of cytosolic factors involved in protein import. C. Cytosolic Chaperones

1. Hsp70 Homologues Two isoforms of Hsp70 exist in the cytosol of mammalian cells: a constitutively expressed form of Hsp7O (Hsc70 or Hsp73) and a highly inducible form that is synthesized during or after stress (Hsp70 or Hsp72; Welch, 1990). In yeast, there are two forms of each of these members encoded by a total of four genes, ssal-4 (Werner-Washburne et al., 1987). Simultaneous inactivation of all four genes is lethal to the cell. Hsc70 was first purified through its ATP-dependent activity to uncoat clathrin from coated vesicles and clathrin cages (Schlossman et al., 1984), but Hsc70 has since been shown to carry out a variety of other roles including protein folding, prevention of protein aggregation, progesterone receptor assembly, protein translocation, and protein degradation (Gething and Sambrook, 1992). The finding that Hsc70 could bind to mitochondria1 precursor proteins and stimulate preprotein import in v i m suggested that this was the ATPdependent factor involved in maintaining precursor proteins in an importcompetent state (Murakami et aL, 1988a). Beckmann et af. (1990) provided further support for these findings by coimmunoprecipitating nascent polypeptides with Hsc70. Moreover, depletion of Hsc70 in yeast led to the accumulation of at least the precursor form of the /3 subunit of the mitochondrial F1-ATPase (Deshaies et al., 1988). In addition to Hsc70, a soluble and NEM-sensitive factor in reticulocyte lysate was also shown to be required for the in vitro import of a number of preproteins into mitochondria (Murakami et af., 1988a; Randall and Shore, 1989; Sheffield et al., 1990). 2. DnaJ Homologues DnaJ homologues seem to reside in all compartments containing a Hsp70 member. The cytosolic homologue is called Ydjlp (or MASS) in yeast (Caplan and Douglas, 1991; Atencio and Yaffe, 1992) and Hsp40 in mammals (Hattori et al., 1992). Yeast cells that lack a functional YDJl gene are viable; however, they lack normal growth characteristics, exhibit altered

140

MICHAEL T. RYAN ET AL.

cell morphology, and are temperature sensitive (Caplan and Douglas, 1991; Atencio and Yaffe, 1992).Ydjlp contains a farnesyl moiety at its C terminus, which results in an increased membrane localization at high temperatures (Caplan et al., 1992a).Cells containing a mutant form of Ydjlp that lacks this moiety are temperature sensitive, indicating that this membrane association may be important for its function (Caplan et al., 1992a). A direct role for Ydjlp in mitochondrial preprotein import is likely because cell lines containing mutant forms of Ydjlp have a reduced ability to import preproteins into mitochondria (Atencio and Yaffe, 1992; Caplan et al., 1992b), although its exact role has yet to be defined. Like bacterial DnaJ, both yeast and human cytosolic DnaJ homologues stimulate the ATPase activity of Hsc70 and hence facilitate the dissociation of polypeptides from Hsc70 (Cyr et al., 1992;Cheetham et ab, 1994).Because Ydjlp is farnesylated and shows membrane-binding abilities, it may be involved in targeting Hsc70-bound preproteins to mitochondrial membrane receptors. Indeed, a population of Hsc70 is located on the outer membrane of rat mitochondria (Lithgow et al., 1993b) and this may be due to association with DnaJ. Alternatively, these molecular chaperones may be involved in stabilization of the preprotein receptor domains located on the outer mitochondrial membranes, especially during cell stress.

3. Is There a Cytosolic GrpE Homologue? In E. coli, GrpE acts as a nucleotide exchange factor, releasing ADP from DnaK and thus allowing for ATP rebinding and subsequent polypeptide release. Based on the activities of cytosolic forms of Hsp70 and DnaJ, Hohfeld et al. (1995) sought to identify a cytosolic GrpE homologue using the yeast two-hybrid system. This led to the identification of one protein that was termed Hip (Hsc70-interacting protein). However, in contrast to GrpE, which facilitates the removal of ADP from Hsp70, Hip seemed to stabilize the ADP-bound state of Hsc70. Nevertheless, Hip increased the efficiency of luciferase refolding with Hsc70/Hsp40 almost three-fold. The ATPase of Hsc70 differs from that of DnaK because both polypeptides and DnaJ stimulate ATP hydrolysis and nucleotide exchange (Sadis and Hightower, 1992; Ziegelhoffer et al., 1995), suggesting that a cytosolic GrpE homologue may not be necessary.

4. Presequence Binding Factor A factor in rabbit reticulocyte lysate was found to stimulate the in vitro import of the purified precursor to ornithine transcarbamylase (p-OTC) (Murakami et al., 1988b). This factor was shown to bind to p-OTC but not

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

141

to the unfolded mature form (OTC), indicating that the mitochondrial targeting signal represents the binding motif (Murakami et af.,1988b, 1990). This protein, termed presequence binding factor (PBF), was subsequently purified and found to consist of 50-kDa subunits (Murakami and Mori, 1990). Import of p-OTC in the presence of PBF was further enhanced by the addition of purified Hsc70 (Murakami and Mori, 1990). Rabbit reticulocyte lysate depleted of PBF supported neither the import of p-OTC nor the precursors of aspartate aminotransferase and malate dehydrogenase, but did support import of 3-oxoacyl CoA thiolase (Murakami et af., 1992). The thiolase preprotein does not contain a cleavable mitochondrial targeting signal and these results therefore suggest the existence of PBFdependent and -independent pathways for mitochondrial protein import.

5. Mitochondria1 Import Stirnulatory Factor Initial studies on the role of cytosolic factors in preprotein import implied that, in addition to Hsc70, an NEM-sensitive proteinaceous factor was required (Murakami et af., 1988a; Randall and Shore 1989). This protein was eventually identified by studying the in vitro import of the precursor form of adrenodoxin (pAd) into mitochondria. pAd synthesized in vitro in wheat germ lysates could not be imported into rat liver mitochondria, but the addition of rat cytosolic extracts to this translation mix promoted its import (Hachiya et af., 1993). Based on this assay, the factor that could restore the import capability of pAd was subsequently purified and named mitochondrial import stimulatory factor (MSF; Hachiya et af., 1993). MSF is a heterodimer and can unfold aggregated pAd utilizing the hydrolysis of ATP (Hachiya et af., 1993; Komiya et af., 1996). ATP hydrolysis results in the dissociation of MSF from the preprotein, which suggests that multiple rounds of binding and release occur in order for the polypeptide to remain import competent (Hachiya et af., 1993). The MSF ATPase was inhibited specifically by mitochondrial outer membrane proteins, suggesting that it interacts with receptor components (Hachiya et af., 1994). Indeed, recent studies suggest that MSF transfers the preprotein to components of the translocase of the outer membrane (Tom; Pfanner et af., 1996), the Tom37/ Tom70 receptor subunits, and, as a consequence of ATP hydrolysis, is displaced from the membrane itself (Hachiya et af., 1995). In the absence of ATP, a stable complex can be formed between MSF/pAd and the Tom receptor components. In contrast, urea-denatured pAd can bind directly to a different set of outer membrane receptor subunits (Torn20flom22). The relative contribution of PBF and MSF to the mitochondrial protein import pathway remains to be established (Komiya et al., 1996; Mihara and Omura, 1996).

142

MICHAEL T. RYAN ET AL.

IV. Protein Translocation across Mitochondria1Membranes

In a typical eukaryotic cell, about 10% of all proteins are targeted to mitochondria, At the mitochondrial surface, further sorting to the outer membrane, intermembrane space, inner membrane, and matrix compartments occurs. A discussion of protein sorting into the various mitochondrial compartments has been provided by Glick et al. (1992) and will not be further dealt with here. Early studies suggested that the translocation of preproteins into mitochondria occurred at a single import channel fixed at contact sites between the outer and inner mitochondrial membranes (Schleyer and Neupert, 1985; Pfanner et al., 1990). However, recent evidence indicates that there are two separate and independent protein import channels-one located in the outer membrane and one located in the inner membrane (Horst et al., 1995). Thus, mitochondria stripped of outer membranes (i.e., mitoplasts) are able to import preproteins (Ohba and Schatz, 1987) as are mitochondrial outer membrane vesicles free from inner membrane remnants (Mayer et al., 1993). The two translocation machineries can be assayed in succession: A fusion protein containing targeting information to both the intermembrane space and the matrix can be imported into the intermembrane space in the absence of a membrane potential and then be chased into the matrix upon its restoration (Segui-Real et al., 1993). Furthermore, some preproteins destined for the intermembrane space require the outer membrane translocation machinery but not that of the inner membrane (Lill et al., 1992). Although the translocation machineries of the outer and inner membrane can come in close contact at times of preprotein import, it seems that their interaction is dynamic rather than static (Glick et al., 1991; Pfanner et al., 1992). Figure 2 illustrates the organization of translocation components in yeast.

A. Translocation Components of the Outer Membrane Preproteins that are targeted to mitochondria associate with a translocase in the outer membrane containing at least nine different components in yeast (Lithgow et al., 1995; Kiibrich et al., 1995). The subunits Tom20, Tom22, Tom37, and Tom70 [the number refers to the approximate molecular size (in kDa) of the species wherein the translocase component was first discovered] contain large cytosolic domains in order to perform receptor functions. Tom72, which exhibits more than 90% identity to Tom70, is also likely to function as a receptor (Bomer et al., 1996). Although the

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

143

FIG. 2 Translocation of preproteins through the outer and inner mitochondrial membranes. The preprotein binds to translocase components of the outer membrane (Tom) and is transferred to the general import pore (GIP). Following insertion into the intermembrane space, the preprotein associates with translocase components of the inner membrane (Tim). In most cases the preprotein contains a positively charged N-terminal targeting signal that can adopt an a-helix. This targeting signal is believed to initiate the import of the preprotein into the negatively charged mitochondrial matrix via an electrophoretic effect. The remainder of the preprotein enters the matrix via the action of mt-Hsp70. Preproteins that contain folded domains on the outer face of the mitochondria are pulled into the matrix by mt-Hsc70 anchored to Tim44, whereas other preproteins may enter the matrix via the action of Brownian motion accompanied by mt-Hsp70 binding and release. mt-GrpE facilitates this cycling via nucleotide exchange from mt-Hsp70. In many cases, the mitochondrial targeting signal is cleaved from the preprotein by the mitochondrial processing peptidase (MPP). A legend defining the components of the translocases is shown in the insert.

identification of all these receptor components has come from fungal studies, homologues have been identified in mammals and plants (Goping et al., 1995; Pchelintseva et al., 1995; Perryman et al., 1995; Seki et al., 1995; Hanson et al., 1996; Komiya et al., 1996). The entire Tom complex can be isolated by coimmunoprecipitation (Moczko et al., 1992). Within this large assembly, a number of receptor subunits form subcomplexes whose functions have been investigated. Tom37 and Tom70 associate and bind preproteins that may require the assistance of

144

MICHAEL T. RYAN E r AL.

cytosolic chaperones including MSF for targeting (Gratzer et af., 1995; Hachiya et af., 1995). It has been proposed that this subcomplex interacts with the mature part of some preproteins. perhaps by binding to unfolded or hydrophobic regions (Gratzer et af., 1995). Tom20 and Tom22 also associate (Bolliger et af., 1995; Mayer et af., 1995). This subcomplex binds the targeting signals of preproteins through electrostatic interactions (Moczko et af., 1994; Haucke et af., 1995). An association between the cytosolic domains of Tom70 and Tom20 has also been inferred using coimmunoprecipitation and the yeast two-hybrid system (Haucke et af., 1996). Following recognition by receptors, the insertion of preproteins into the outer membrane occurs at the general insertion pore (GIP). Tom40, the major and essential component of this pore, was originally identified by chemical cross-linking to a chimeric preprotein (Vestweber et af., 1989). Antibodies against Tom40 inhibited preprotein import in vitro (Kiebler et af., 1990) and the gene encoding Tom40 was found to be essential for cell viability (Baker et af., 1990). Tom40 is an integral membrane protein and immunoprecipitation analysis indicated that it associates with Tom20 and Tom70 (Kiebler et af., 1990). Tom22 is also essential for cell viability (Lithgow et af., 1994). It is thought to function at the GIP and be involved in both the recognition and translocation of preproteins (Kiebler et af., 1993; Bolliger et af., 1995; Honlinger et af., 1995; Mayer et af., 1995). The translocase also contains three small subunits: Tom5, Tom6, and Tom7. Although a role for Tom5 in the translocase is yet to be defined, genetic evidence and coimmunoprecipitation techniques suggest that Tom6 stabilizes the interaction of the receptors within the GIP (Alconada et af., 1995). In contrast, Tom7 destabilizes this interaction (Honlinger et af., 1996). The small Tom proteins thus seem to mediate the dynamic interactions between the translocation components during preprotein transfer and sorting.

6 . Translocation Components of the Inner Membrane A number of translocation components of the mitochondrial inner membrane have been identified (Pfanner et al., 1994). This has been achieved through the genetic analysis of yeast mutants that fail to import an artificial preprotein into mitochondria. Maarse et af. (1992) constructed a strain in which the cytosolicenzyme, orotidine-5’-monophosphate(OMP) decarboxylase, was synthesized with a mitochondrial presequence. The import of OMP decarboxylase into mitochondria made yeast cells auxotrophic for uracil. Mitochondria1protein import (mpi)mutants were created by random mutagenesis and selected for their ability to grow on uracil-free media. Three of the four complementation groups corresponded to essential genes

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

145

encoding mitochondrial inner membrane proteins. These proteins are components of Tim (Pfanner et al., 1996). Tim17 and Tim23 are integral membrane proteins synthesized without cleavable presequences (Dekker et al., 1993; Maarse et al., 1994). Antibodies directed against these components inhibit import of preproteins into mitoplasts (Emtage and Jensen, 1993; Berthold et al., 1995) and both Tim components can be cross-linked to a preprotein in transit to the matrix (Kubrich et al., 1994;Berthold et al., 1995). The third component of the inner membrane translocase defined from mpi mutants is Tim44, a hydrophilic peripheral membrane protein that contains a cleavable presequence (Maarse et al., 1992). Biochemical studies, in which a preprotein in transit between the outer and inner membrane was cross-linked to a 44-kDa protein, first suggested a role for Tim44 in preprotein import (Scherer et al., 1992). Tim44 predominantly interacts with preproteins carrying a complete presequence (Blom et al., 1993). Curiously, antibodies against Tim44 inhibited the import of preproteins into mitoplasts, whereas Tim44 could be released from the inner membrane into the matrix by salt or high pH treatments (Scherer et al., 1992; Kronidou et al., 1994; Blom et al., 1993). This suggests that Tim44 lines the inner membrane translocation pore and performs receptor and translocase functions. Indeed, Tim44 binds to both incoming preproteins and the mitochondrial-matrix Hsp70 homologue (mt-Hsp70) (Schneider et al., 1994; Kronidou et al., 1994; Rassow et al., 1994; Horst et al., 1995). Mt-Hsp70 binds to the imported preprotein as it enters the matrix, thereby preventing the preprotein from sliding back into the cytosol (Pfanner and Meijer, 1995). Mt-Hsp70 was the fourth component identified from the mpi mutants to be involved in preprotein import (Dekker et al., 1993).

C. Proteolytic Maturation of Mitochondria1 Preproteins Upon import into the mitochondrial matrix, the cleavable targeting signals of preproteins are removed by the matrix processing peptidase (MPP), which consists of two subunits-aMPP and pMPP (Kalousek et al., 1993). Both subunits have been purified from a number of organisms (Hart1 et al., 1989) and their essential nuclear genes have been defined by screening and complementation of fungal mutants that are defective in preprotein import (Witte et al., 1988; Yang et al., 1988; Jensen and Yaffe, 1988). Analysis of the cleavage site motifs recognized by MPP reveals that an arginine is often found at positions -11, -10, -3, or -2 relative to the cleavage site (located between positions -1 and + l ) , but otherwise little or no sequence identities exist between presequences (Gavel and von Heijne, 1990). This lack of sequence specificity has led to the notion that MPP recognizes a structural motif (Hammen et al., 1994). The elucidation of the

146

MICHAEL T. RYAN E r AL.

structures of a number of processed and non-processed targeting signals by NMR spectroscopy reveals that MPP may recognize an amphiphilic helix-linker-helix motif (Hammen et al., 1994); however, this is not always sufficient for cleavage (Jarvis et al., 1995; Waltner and Weiner, 1995).

V. Role of Matrix Chaperones in Protein Import A. Requirement for Matrix-Located Hsp70 Mt-Hsp70 has been purified and identified from a number of sources including mammals (Leustek et al., 1989; Mizzen et al., 1989; Webster et al., 1994) and yeast (Craig et al., 1989). The yeast mt-Hsp70 gene is expressed moderately under normal growth conditions and is induced approximately 10-fold upon heat shock, whereas mammalian mt-Hsp70 is induced very little upon heat shock but may be induced 2- to 5-fold under other stress conditions (Mizzen et al., 1989). Mt-Hsp70 performs a compulsory cellular function because its gene is essential for the viability of yeast (Craig et al., 1989). Temperature-sensitive yeast mutants deficient in mt-Hsp70 accumulated preproteins at translocation sites in such a manner that they were accessible to the matrix-located protease and to externally added protease (Kang et al., 1990). Furthermore, in vitro studies showed that preproteins arrested at translocation sites could be cross-linked to mt-Hsp70 (Ostermann et al., 1990; Scherer et al., 1990). Although the translocation defect of mitochondria containing mutant mt-Hsp70 could be circumvented in vitro by chemically denaturing an artificial preprotein, its subsequent folding was impaired (Kang et al., 1990). These findings indicate that mt-Hsp70 is involved in both preprotein translocation and protein folding. Two separate models for mt-Hsp70 action during preprotein import have been proposed (Glick, 1995;Pfanner and Meijer, 1995) and are summarized in the following sections.

1. Brownian Ratchet Model The mitochondria1presequence initiates the translocation of the preprotein into the matrix in a manner that requires a membrane potential (A*) (Schleyer et al., 1982).However, the entry of the remainder of the preprotein into the matrix is not dependent on A*. The preprotein in transit between the translocation channels has been proposed to oscillate forwards and backwards by Brownian motion (Simon et al., 1992; Ungermann et al., 1994). By itself, this motion is random and hence does not favor a particular

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

147

direction. However, the repeated binding of mt-Hsp70 to the oscillating preprotein favors its forward movement into the matrix. Brownian motion combined with cycles of binding and release of mt-Hsp70 to the preprotein, accompanied by ATP hydrolysis, resembles a ratchet action and results in the complete entry of the preprotein into the matrix. 2. Force-Generated Motor In this model, mt-Hsp70 binds to an incoming preprotein and, through the action of ATP hydrolysis, the loosely folded domains of the preprotein on the cytosolic face of the mitochondria are pulled through the translocation channel. As observed with other Hsp7O homologues, proteolytic mapping experiments indicate that the conformations of the ADP-bound and the ATP-bound forms of mt-Hsp70 differ (von Ahsen et al., 1995). The repetitive structural changes of mt-Hsp70 in response to ATP hydrolysis are thought to constitute the mechanism for the pulling of a preprotein into the matrix. In order for mt-Hsp70 to exert such a force, it would have to be anchored to the inner mitochondrial membrane (Glick, 1995). Indeed, between 10 and 15% of mt-Hsp70 is membrane bound through its association with the inner membrane translocation component, Tim44 (Schneider et al., 1994; Rassow et al., 1994; Kronidou et al., 1994). This binding does not involve the substrate binding site of mt-Hsp70 (von Ahsen et al., 1995) and, accordingly, a complex can be isolated between Tim44, mt-Hsp70, and an incoming preprotein. The complex between Tim44 and mt-Hsp70, however, can be dissociated upon the addition of ATP (Kronidou et al., 1994; Rassow et al., 1994; Horst et al., 1995; von Ahsen et al., 1995). Recent evidence (Voos et al., 1996) indicates that mt-Hsp70 participates both as a Brownian ratchet and as an import motor. Yeast mitochondria containing a mutant form of mt-Hsp70 (sscl-2p) that does not bind Tim44 and is therefore defective in its pulling function were able to import an unfolded preprotein. In contrast, a preprotein that required unfolding in order to be translocated was unable to be imported into mitochondria of this mutant cell line. Thus, the action of mt-Hsp70 in preprotein import is dependent on the folded state of the preprotein, whereby partially folded preproteins require the pulling action of Tim44anchored mt-Hsp70, whereas unfolded preproteins appear to be independent of this association.

6 . Mitochondria1 GrpE and DnaJ Homologues The evolutionary connection between mitochondrial chaperones and bacterial chaperones is epitomized by mt-Hsp70, which shows a higher degree

148

MICHAEL T. RYAN E r AL.

of homology with DnaK than with other Hsp70 family members found in different subcellular locations of the same species (Craig et al., 1989; Webster et al., 1994). Given this, it is perhaps not surprising to find that the mitochondrial matrix also contains DnaJ and GrpE homologues that seem to perform evolutionally conserved functions. The predicted amino acid sequence of an open reading frame obtained during the random sequencing of the yeast genome showed 33% positional identity to E. coli DnaJ (Rowley et al., 1994). Subsequent biochemical analysis revealed that this DnaJ homologue was localized to the mitochondrial matrix. Mitochondria1 DnaJ (mt-DnaJ or Mdjlp) is heat inducible, and insertional inactivation of its gene resulted in loss of mitochondrial DNA and cell death at elevated temperatures (Rowley et al., 1994). Although cross-linking techniques have shown an association of mt-DnaJ with the translocation complex of the inner membrane (Kronidou et al., 1994), mt-DnaJ does not appear to be necessary for preprotein import. The analysis of a mt-DnaJ-deficient yeast strain revealed that, although their mitochondria could import preproteins, folding was impaired (Rowley et al., 1994). Many homologues of DnaJ or proteins with "J-domains" have been found. Although the mt-DnaJ member described previously may not be required for protein translocation, other as yet unidentified DnaJ homologues may perform such a function. For example, Rassow et al. (1994) reported that Tim44 contains limited amino acid sequence homology to a small region in DnaJ and this may be sufficient to provide Tim44 with DnaJ-like activity. A precedent for this has been found for the import of proteins into the endoplasmic reticulum, in which the ERlocated Hsp70 homologue BiP was found to be bound to an integral membrane receptor, Sec63, which contains a DnaJ-like motif (Gething and Sambrook, 1992). So far, no mammalian, mitochondrial homologue of DnaJ has been found. Like their bacterial counterparts, mt-Hsp70 and mt-GrpE associate in the absence of ATP. Yeast mt-GrpE was identified by its binding to Histagged mt-Hsp70 coupled to nickel beads (Bolliger et al., 1994), whereas mammalian mt-GrpE was identified biochemically through its affinity for immobilized DnaK (Naylor et al., 1995, 1996). In the absence of ATP, incoming preproteins can be isolated with the mt-Hsp70/mt-GrpE complex (Bolliger et al., 1994; Ikeda et al., 1994; Voos et al., 1994; Layloraya et al., 1994). The analysis of a series of yeast mt-GrpE mutants indicates that mtGrpE is essential for mitochondrial preprotein translocation (Layloraya et al., 1994; Westerman et al., 1995). Mt-GrpE exerts this function through dissociation of ADP from mt-Hsp70 bound to Tim44, a prerequisite for the mt-Hsp70 ATP motor to function optimally in preprotein translocation (Layloraya et al., 1995; Westerman et al., 1995).

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

149

C. mt-Hsp70 Reaction Cycle As mentioned previously, nucleotide-dependent conformational changes in the anchored mt-Hsp70 are thought to act in pulling preproteins into the matrix. However, the exact mechanism and the role of ATP hydrolysis in this process is not clear. Mt-Hsp70 binds to Tim44 in its ADP-bound or nucleotide-free state (von Ahsen et aZ., 1995). In the ADP-bound state, mtHsp70, like DnaK, is believed to bind and release substrate proteins slowly. The binding of mt-Hsp70 to Tim44 at the translocation channel conceivably favors binding of incoming preproteins. Nucleotide exchange catalyzed by mt-GrpE leads to binding of ATP to mt-Hsp70 and a subsequent conformational change pulls the preprotein into the matrix, leading in turn to the release of mt-Hsp70 from Tim44. Like DnaK, the soluble population of mt-Hsp70 would also depend on an ATPase activity, but in this case for its action as a Brownian ratchet and for the purpose of protein folding in the matrix.

VI. Protein Folding within the Matrix A. Matrix Located Chaperonins, Cpn6O and Cpnl 0 Mitochondria1 Cpn60 (Hsp60) and CpnlO (HsplO) are structurally and functionally related to the bacterial chaperonins GroEL and GroES. Cpn6O was first identified as a heat-inducible protein from the mitochondria of Tetrahyrnena thermophilu (McMullin and Hallberg, 1987). Antibodies raised against this protein cross-reacted with a mitochondrial protein of similar size from a number of organisms including yeast and humans (McMullin and Hallberg, 1988). Cpn60 was partially purified from HeLa cells as a protein that comigrated with a heat shock protein on two-dimensional gels (Mizzen ef al., 1989).Partial amino acid sequencing revealed its homology to GroEL. The isolation of the rat Cpn60 cDNA revealed that the encoded protein contained a 26 amino acid mitochondrial targeting signal and showed 49% sequence identity with GroEL (Peralta et al., 1990,1993) and 95% with other mammalian homologues (Jindal et al., 1989; Picketts et al., 1989). Like GroEL, Cpn6O from T. fherrnophila (McMullin and Hallberg, 1987), N. crussa (Hutchinson et al., 1989) and S. cerevisiue (McMullin and Hallberg, 1988) exists as a tetradecamer of 60 kDa subunits and is arranged into two stacked heptameric rings with a central cavity. Interestingly, however, electron microscopic and chromatographic analysis indicates that mammalian Cpn60 comprises a single toroid of seven subunits (Jindal et al., 1989; Picketts et al., 1989; Viitanen et ul., 1992b; Peralta et

150

MICHAEL T. RYAN ET AL.

al., 1993). This single-toroidal structure is also observed for the purified Cpn60 homologue of thermophilic bacteria Thermoanaerobacter brokii (Truscott et al., 1994). Mitochondria1 CpnlO was first identified and partially purified from bovine liver. This preparation could substitute for GroES in the refolding of chemically denatured Rubisco with GroEL (Lubben et al., 1990). Based on its comigration with a 10-kDa heat-inducible protein, CpnlO was later purified to homogeneity from rat liver mitochondria and sequenced (Hartman et al., 1992). Rat CpnlO exhibits 45% positional amino acid identity with GroES and promoted the in vitro refolding of chemically denatured OTC with GroEL (Hartman etal., 1992,1993).Based on a Rubisco refolding assay, a S. cerevisiae CpnlO homologue was subsequently identified and shown to be essential for cell viability (Rospert et al., 1993a,b). Recently, a simple and convenient affinity purification procedure for the isolation of CpnlO from a wide range of sources was developed (Ryan et al., 1995) and, with the isolation of a cDNA clone encoding rat CpnlO, it was shown that CpnlO is not proteotypically processed upon import (Ryan et al., 1994). By comparing the naturally produced N-acetylated CpnlO with nonacetylated recombinant CpnlO, it was established that N-acetylation has a marked effect on protease susceptibility (Ryan et al., 1995).

6 . CpnlO and Early Pregnancy Factor Using large quantities of human platelets as a source and a bioassay for early pregnancy factor (EPF), Cavanagh and Morton (1994) purified a protein that was found to be identical to mitochondria1 CpnlO. The EPF bioassay was based on the observation that immunosuppressive factors in antilymphocyte serum can inhibit the formation of rosettes between lymphocytes and red blood cells (Bach and Antoine, 1968). Moreover, a factor(s) that appeared in maternal serum within 24 h of fertilization increased this inhibition. EPF is proposed to suppress a maternal immune reaction to the developing fetus and hence is necessary for embryonic well-being (Cavanagh and Morton, 1996). The ability of CpnlO to perform in the rosette inhibition assay supports but does not prove the identification of EPF as CpnlO. However, the finding that EPF activity in serum could be depleted by passage through a GroEL affinity column provides further evidence for this connection (Cavanagh and Morton, 1994). The proposed dual role for CpnlO is far from understood and indicates an extracellular location for part of the CpnlO pool. The further characterization of mammalian CpnlO may provide some insights into its possible

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

151

function as EPF in addition to its well-established role as a molecular chaperone.

C. Chaperonins Are Required for Protein Folding Given their similarity to bacterial GroEL and GroES, it is not surprising to find that mitochondrial chaperonins are required for polypeptide folding. Direct evidence for the role of Cpn60 in this process comes from genetic studies using yeast mitochondrial import function (mif) mutants that are impaired in the assembly of preproteins but not in their translocation. The observed defects of the mif4 mutant were found to be due to a mutation in the nuclear located gene encoding Cpn60 (Cheng et af., 1989). This gene is essential for cell viability (Cheng et al., 1989). The requirement for Cpn60 in protein folding has also been illustrated biochemically. A chemically denatured, chimeric preprotein (Su9-DHFR) imported into ATP-depleted mitochondria could be trapped on Cpn60 in a protease-susceptible form (Ostermann et af., 1989). Addition of ATP to this complex released the preprotein from Cpn60 in a protease-resistant (i.e., folded) conformation. Using a genetic approach, Hallberg et af. (1993) arrived at a similar conclusion by demonstrating that depletion of Cpn60 activity did not impair preprotein import but led to the formation of protein aggregates. The importance of GroES in the folding of bacterial proteins with GroEL suggests that the highly conserved homologue, mitochondrial CpnlO, performs a similar function. Its role in protein folding was verified in yeast (Hohfeld and Hartl, 1994). A point mutation in the CpnlO gene reduced the binding of CpnlO to Cpn6O and resulted in a temperature-sensitive phenotype in which the folding of both mitochondrial aMPP and OTC was impaired at nonpermissive temperatures. The association of mammalian Cpn60 with CpnlO in the presence of K' ions and Mg-ATP and their ability to refold chemically denatured proteins in vitro indicate that the mechanisms of chaperonin action in protein folding are conserved between species (Viitanen et al., 1992b; Lubben et af.,1990; Hartman et af., 1992). The single-toroidal structure of mammalian Cpn60 may indicate, however, that the detailed mechanisms of protein folding may differ from those of other chaperonin homologues. If indeed Cpn60 is active as a heptamer, then CpnlO and the substrate polypeptide must bind to the same toroid as also suggested in recent GroEL-GroES models. However, such a mechanism is contradicted by studies with bacterial GroEL as a mutant form that exists as a single toroid, traps both GroES and polypeptides, but cannot efficiently support protein folding (Weissman et af., 1995,1996). A more likely scenario is therefore that two single toroids

152

MICHAEL T. RYAN ET AL.

of mammalian Cpn60 associate during protein folding in vivo. Indeed, in the presence of Mg-ATP and CpnlO, the individual T. brokii heptameric toroids assemble into a conventional double-toroidal structure (Todd et al., 1995).

D. Sequential Action of Matrix Chaperones in Protein Folding Langer et al. (1992b) proposed that the folding of some bacterial proteins is facilitated by the sequential action of molecular chaperones. Given the similarities between the bacterial and mitochondria1 chaperones, such a pathway is likely to exist in mitochondria. A time course study using coimmunoprecipitations to investigate the association of mt-Hsp70 and Cpn60 with newly imported preproteins in yeast revealed that,, after complexing with mt-Hsp70, some preproteins bound to Cpn60 (Manning-Krieg et al., 1991). At a point following this association, the preproteins were released in a protease-resistant conformation that indicated a folded, compact state. Interestingly, those proteins that associated with Cpn6O normally exist as oligomeric proteins. These experiments were repeated in a recent study using two artificial and two authentic preproteins, all of which were monomeric when folded (Rospert et al., 1996). In this case, only rhodanese was observed to associate transitionally with Cpn60. Furthermore, although the possible interaction of newly imported preproteins with mt-DnaJ was not investigated, only one of these four proteins (Cyclophilin 20) was found to be associated with the non-membrane-associated form of mt-Hsp70. This indicates that after preproteins are released into the matrix from the Tim44bound mt-Hsp70, folding may occur via a number of different pathways. In addition, recent studies have proposed that the matrix-located protein, Cyclophilin 20, is involved in the folding of some newly imported proteins through its peptidyl-prolyl cis-trans isomerase activity (Rassow et aL, 1995; Matouschek et al., 1995). The extent of molecular chaperone involvement in protein folding most likely depends on the conformational state of proteins and the physiological conditions of the mitochondria. The folding of newly imported proteins in the mitochondria is depicted in Fig. 3.

VII. Roles of Chaperones in Protein Degradation within the Mitochondrion A. Many Stress Proteins Are Proteases The common consequence of many stress conditions appears to be the accumulation of unfolded or malfolded proteins in the cell. As discussed

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

153

FIG. 3 Folding of imported proteins by matrix-located molecular chaperones. Following import into the matrix, the protein is folded into its proper conformation. This can be facilitated by the action of matrix-located chaperones such as the mt-Hsp70/mt-DnaJ/mt-GrpE and the chaperonin (Cpn60/Cpn10) teams. These teams may act sequentially depending on the conformation of the folding protein. Other molecular chaperones such as Cyclophilin 20 have also been implicated in the folding of some proteins.

previously, several stress-inducible proteins facilitate cell recovery by their action as molecular chaperones to refold damaged proteins into their native, functional state. However, should a polypeptide be unable to attain its native conformation, it is rapidly degraded by another set of stress-inducible proteins that act as proteases or participate in protease action (Parsell and Lindquist, 1993). The “refold or degrade” model also applies to the action of stress proteins that are constitutively expressed within the cell. The cooperative action of chaperones (DnaK/ DnaJ/GrpE and GroEL/GroES) in cellular processes,

154

MICHAEL T. RYAN E r AL.

such as stabilization of preexisting proteins against aggregation, folding of nascent polypeptides, assembly of multimeric proteins, and membrane translocation, includes the degradation of inactive proteins with abnormal conformations that are consequences of errors in transcription/translation, chaperone mishandling, or age-related denaturation. Indeed, abnormal proteins exhibit an increased half-life in E. coli rpoH mutants that contain reduced levels of molecular chaperones (Goff et al., 1984; Grossman et al., 1984) and in E. coli strains with mutations in individual molecular chaperone genes (Straus et al., 1988). Under both normal and stressed cellular conditions, it is now believed that the molecular chaperones aid in the degradation of abnormal proteins by presenting them to proteases (Hayes and Dice, 1996). This would effectively render abnormal polypeptides susceptible to proteases for their efficient degradation and thus prevent the formation of aggregates that may be harmful to cells. In the following sections, an overview of the current knowledge about the possible relationship between mitochondrial proteolysis machineries and chaperone teams will be presented. In support of the endosymbiont theory on the origins of mitochondria, the proteolytic mechanisms thus far discovered in mitochondria all have homologues that have been better characterized in E. coli. Therefore, examples of certain events in protein turnover from E. coli, which are just beginning to be elucidated in mitochondria, will be used to give an idea of the possible functions and new members that are to be found in mitochondria.

6.Protein Degradation within the Mitochondrion Proteolysis plays a key role in the maintenance of mitochondrial functions by potentially regulating the availability of certain short-lived regulatory proteins, ensuring the proper stoichiometry of multiprotein complexes and removing abnormal proteins. Early investigations on the turnover of proteins within mitochondria assumed that the organelle is degraded within the lysosome, following the observation of whole mitochondria within autophagic vacuoles (autophagosomes) (Hare, 1990). However, later studies have shown that the average half-lives of proteins differ in various mitochondrial compartments and that different proteins are degraded at distinct rates within the same compartment (Hare, 1990), suggesting that a distinct and selective mitochondrial proteolytic system exists. This notion is in accord with recent observations of several ATP-dependent proteases in yeast and mammalian mitochondria with significant homology to characterized bacterial counterparts.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

155

C. Chaperone-Assisted Proteases of f. coli In E. coZi, the transcription of at least 20 heat shock genes requires an alternative RNA polymerase sigma factor (d2). Several of these heat shock genes encode proteases or proteins that assist in protease action, namely, La (Lon), ClpP, ClpA, ClpB, ClpX, FtsH (HflB), HflX, HflK, and HflC (Georgopoulos et al., 1994). 1. La (or Lon) Protease Most of the work performed on this family of heat shock proteins has concentrated on the La (Lon) protease, the product of the lon gene in E. coli (Gottesman and Maurizi, 1992; Goldberg, 1992). It is a homotetramer (90-kDa subunit molecular mass) and an ATP-dependent serine endoprotease that catalyzes the rate-limiting steps in the specific degradation of highly abnormal proteins. An intriguing feature of the La protease is its ability to form a complex in vivo with DnaK, GrpE, and a short-lived mutant protein, phoA61, shortly before its degradation (Sherman and Goldberg, 1992). The isolation of several mutants has helped characterize the function of this complex and the roles of each member. Deletion of the dnaK or Zon genes renders phoA61 less susceptible to proteolysis, implying that both DnaK and the La protease are required for its efficient degradation. In the dnaJ259 mutant, degradation of phoA61 was inhibited and less DnaK was found in the complex with phoA61, whereas in the grpE280 mutant phoA61 degradation was accelerated and more DnaK was found in the complex. DnaJ may promote the binding of phoA61 to DnaK through its ability to act as a molecular chaperone, or it may affect the dissociation of the complex. Hence, DnaJ may not be required as a stable component of the complex. It is believed that the association of GrpE with DnaK and phoA61 stabilizes this interaction to promote efficient proteolysis by the La protease. Interestingly, the dnaK756 mutation accelerates phoA61 degradation. Because the DnaK756 protein is defective in its release of bound polypeptide substrate (Liberek et al., 1991b), it is perhaps not surprising that phoA6l is strongly destabilized because DnaK756 would effectively increase the accessibility of phoA61 to the La protease. 2. Clp Proteases In lon deletion mutants, there is a residual proteolysis of abnormal proteins. This is largely due to another ATP-dependent protease, Clp (Ti), which shares a number of characteristics with the La protease (Gottesman and

156

MICHAEL T. RYAN E r AL.

Maurizi, 1992; Goldberg, 1992). The Clp protease is an ATP-dependent serine endoprotease that catalyses the rate-limiting steps in the specific degradation of highly abnormal proteins. Clp is a complex protease, composed of two distinct subunits of 81 kDa (ClpA) and 21 kDa (ClpP). The ClpA component, which contains the ATPase domain, is the regulatory unit and forms a hexamer, whereas ClpP is the proteolytic subunit and forms a tetradecamer of two heptameric rings. The arrangement of the ClpP subunits is similar to that of the inner (p-type) subunits of both the eukaryotic and archael proteasomes and is reminiscent of the sevenfold symmetric structures of the chaperonin GroEL (Kessel et af., 1995). The conservation of this structural organization in seemingly unrelated proteins may be advantageous for the processing of polypeptide substrates that make repeated contact with the various subunits of these multimeric proteins. Recently, several homologues of the ClpA subunit have been identified, namely, ClpB, ClpC, ClpX, and ClpY. Together, they now comprise the Clp family. Interestingly, both ClpA and ClpX can independently perform molecular chaperone functions (Wickner et al., 1994; Wawrzyndw et af., 1995) and can target different proteins for degradation by ClpP (Gottesman et af.,1993; Wojtkowiak et al., 1993). Therefore, selective degradation by the ClpP protease appears to be determined by its interaction with different regulatory ATPase subunits. Surprisingly,it has been shown that a short-lived fusion protein (CRAG) is degraded in vivo by the ClpP subunit, in an ATP-dependent process, independent of several ATPase subunits of the Clp protease (viz. ClpA, ClpB, and ClpX; Kandror et al., 1994). Furthermore, the degradation of CRAG by ClpP also involves the chaperonins GroEL and GroES, but apparently not protease La, and the association of CRAG with GroEL is the rate-limiting step. Because these conclusions were derived from the sole use of mutant backgrounds in these proteins, attempts were made to reconstitute the system in vitro. When 35S-labeledCRAG was incubated in the presence of ClpP or La and purified chaperonins GroEL and GroES, no degradation was observed, suggesting that additional components are involved in CRAG degradation in vivo.Recently, an additional component of this process was found to be the trigger factor (TF) protein (Kandror et al., 1995), recognized as being a general molecular chaperone despite the fact that its function in vivo is unclear (Crooke and Wickner, 1987). TF enhances the capacity of GroEL to bind CRAG and other unfolded proteins (fetuin and histone) and is the rate-limiting step in CRAG degradation. It appears that an initial association formed between TF and GroEL is essential to target CRAG to the complex and for presentation of CRAG to ClpP for degradation. In addition, TF has recently been shown to possess both a domain belonging to the FK506-binding protein family and peptidyl-prolyl isomerase activ-

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

157

ity (Callebaut and Mornon, 1995; Stoller et al., 1996). The peptidyl-prolyl isomerse activity of TF may be important for its ability to stimulate the binding of unfolded proteins to GroEL by isomerizing critical proline residues. Together, these findings could suggest an additional role of TF as a cohort chaperone for GroEL to promote its chaperone activity in processes such as mediated folding of nascent polypeptides, assembly of multimeric proteins, and membrane translocation.

3. FtsH (HflB) Protease In E. coli the heat shock response is regulated by the heat shock promoterspecific d2subunit of RNA polymerase. With the disappearance of stress, the d2subunit is degraded rapidly (with a half-life of about 1 min) by another ATP-dependent protease, FtsH (HflB) (Tomoyasu et al., 1995; Herman et al., 1995). FtsH is a member of a novel ATPase family, referred to as the AAA protein family, which is characterized by a highly conserved ATP binding site (Kunau et al., 1993). Members of the family have been found to be involved in several diverse cellular processes including control of the cell cycle, regulation of transcription, insertion of proteins into membranes, secretion of protein, biogenesis of organelles, and degradation of proteins. Two features distinguish the FtsH protease from the La and Clp proteases: It is a metalloprotease with a conserved zinc-binding motif (HEXXH) and it is active as an integral inner-membrane protein. However, like the protease La, FtsH appears to require the cooperative action of DnaK, DnaJ, and GrpE for presentation of d2for degradation (Tomoyasu et al., 1995). The exact mechanism of this cooperation is still unclear but it is believed that, in the absence of substrate, DnaK binds d2,thereby inhibiting its rebinding to the RNA polymerase apoenzyme. DnaJ and GrpE could then be envisaged to facilitate the presentation of d2to the FtsH protease for selective degradation.

D. Chaperone-Assisted Proteases within the Mitochondrion 1. Homologues of the La (Lon) Protease The existence of an ATP-dependent protease, resembling the protease La, within rat liver and bovine adrenal cortex mitochondria has been known for some time (Desautels and Goldberg, 1982; Watabe and Kimura, 1985a). The corresponding proteases have been purified from bovine adrenal glands (Watabe and Kimura, 1985b), rat liver, and yeast (KutejovB et al., 1993). Compared with the E. coli protease La (87 kDa), which is active as a homotetramer, both the rat (105 kDa) and yeast (120 kDa) proteases are

158

MICHAEL T. RYAN ET AL.

apparently active as homohexamers (Kutejovd et al., 1993). Recently, an expressed sequence tag corresponding to a partial human clone with considerably homology to the E. coli protease La has appeared in nucleotide data banks (Adams et al., 1992). This permitted the cloning of a cDNA and gene encoding full-length protease La homologues from human (Wang et al., 1993; Amerik et al., 1994) and yeast (Suzuki et al., 1994; Van Dyck et al., 1994). The human La protease precursor is synthesized with a transient signal peptide and is imported efficiently into isolated mitochondria, where it is processed within the matrix into its soluble, mature form. Immunofluorescence microscopy has revealed an exclusive mitochondrial distribution for this protein (Wang et al., 1994). Disruption of the yeast PZMZ gene, encoding the yeast La protease (Pimlp), results in an inability of cells to grow on nonfermentable carbon sources, a deficiency in respiration, extensive deletions in mtDNA, and accumulation of electron-dense inclusions that probably represent aggregated mitochondrial proteins (Suzuki et al., 1994; Van Dyck et af., 1994). Like its E. coli homologue, expression of the yeast gene is induced by heat shock (Van Dyck et af., 1994), implicating a role for this protease in the degradation of misfolded proteins. Indeed, Pimlp is required for the ATP-dependent and selective degradation of the j3-subunits of the general matrix peptidase and the F1-ATPase in vivo. Reminiscent of the situation for phoA6l degradation in E. coli, recent studies have shown that Pimlp, with the assistance of mt-Hsp70, mt-GrpE, and probably mt-DnaJ, can degrade two unstable proteins mistargeted to the mitochondrial matrix (Wagner et al., 1994). A question that remains unresolved is whether molecular chaperones are required for the efficient degradation of authentic mitochondrial proteins that denature naturally during the normal operation of the organelle. 2. Homologues of the Clp Family and ClpP Proteolytic Subunit

Members of the Clp family have been identified in every organism studied thus far (Squires and Squires, 1992). DNA sequences encoding Clp homologues have been obtained from at least 11 different organisms and the corresponding proteins can be found in several compartments of the eukaryotic cell, including the chloroplast. Recently, Hsp78, a yeast mitochondrial matrix protein, was identified as a second member of the Clp family (Leonhardt et al., 1993). Like cytosolic Hspl04, Hsp78 is closely related to the E. coli ClpB protein as indicated by the presence of two consensus ATP binding sites in its primary structure. Surprisingly, disruption of the HSP78 gene had no apparent phenotypic trait; hence, the function of Hsp78 remains obscure. Only after double mutants were made that combined a deletion of the HSP78 gene with temperature-sensitive mutations of the SSCZ (mt-

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

159

Hsp70) gene was a possible function for Hsp78 recognized (Schmitt et af., 1995; Moczko et af., 1995). In both studies of these double mutants, a loss of mtDNA was observed (Moczko et af., 1995), suggesting that at least one of these heat shock proteins is required to maintain a wild-type state of the mitochondrial genome. Furthermore, the double mutants had a strongly reduced mitochondrial membrane potential, explaining the observed defect in the rate of preprotein import (Moczko et af., 1995). Schmitt et af. (1995) suggested that Hsp78 can act as a molecular chaperone in mitochondrial protein import by preventing aggregation of malfolded proteins under conditions of impaired mt-Hsp70 function. However, Moczko et al. (1995) suggested that Hsp78 functions by maintaining mutant mt-Hsp70 in a soluble state, thereby regulating its activity, and that Hsp78 becomes more important in situations in which the activity of mt-Hsp70 is limiting. Nevertheless, both these studies suggest that a cooperation of mt-Hsp70 and Hsp78 is required in the maintenance of essential mitochondrial functions. Initial investigations into the presence of ClpP homologues in animal cells used antibodies to the E. cofi ClpP proteolytic subunit. Cross-reacting proteins with M , of 20-30 kDa were observed in bacteria, lower eukaryotes, and plants and animal cells, indicating a universal conservation of the protein (Maurizi et al., 1990). Recently, three overlapping human-expressed sequence tags with significant homology to the E. cofi ClpP amino acid sequence have been identified and allowed the cloning of a full-length human ClpP homologue (Bross et af., 1995). Northern blotting showed the presence of the ClpP transcript in several organs, whereas the cDNA sequence revealed a mitochondrial transient signal peptide. It will be interesting to see if the primary translation product is efficiently imported into mitochondria and whether it can assemble into an active tetradecamer to promote specific protein degradation in association with a homologue(s) of the Clp family, such as a human Hsp78 homologue. Furthermore, it will also be intriguing to see if the mitochondrial chaperonins can work in conjunction with the Clp protease to facilitate protein degradation. If this is found to be the case, it is likely that a mitochondrial homologue of trigger factor will also be present.

3. Homologues of the FtsH (HflB) Protease In yeast mitochondria, five members of the AAA protein family have been identified, including Bcslp, Msplp (Yta4p), Ymelp (Ytallp), Ytal2p, and YtalOp. One of these (Bcslp) appears to be an integral protein of the inner mitochondrial membrane, which is consistent with its inferred role in the assembly of the ubiquinol-cytochrome C reductase complex (Nobrega et af., 1992). Msplp (Yta4p) is an integral protein of the outer mitochondrial membrane and functions in intramitochondrial protein sorting (Nakai et

160

MICHAEL T. RYAN ET AL.

al., 1993). Ymelp (Ytallp) was identified as a mutant affecting the escape of mt-DNA to the nucleus (Thorsness et al., 1993). Ytal2p has several features that are compatible with its mitochondrial location, but thus far its functional role remains unresolved (Schnall et al., 1994). YtplOp is an integral protein of the inner mitochondrial membrane and is essential for respiration-dependent growth (Tauer el al., 1994). Recently, Ymelp (Ytallp), Ytal2p, and YtalOp have been observed to be highly homologous to the E. coli FtsH protease and together constitute a subfamily of AAA proteins that have a HEXXH motif, which is characteristic for a variety of metal-dependent endopeptidases. The presence of this motif suggests a direct role of this subfamily in mitochondrial ATP-dependent proteolysis. Indeed, deletion of the YTPlO gene severely reduces the rate at which several incomplete subunits of the ATP synthase or respiratory chain complex are degraded (Pajic et al., 1994). YtalOp does not affect the proteolysis of malfolded proteins in the mitochondrial matrix, suggesting its activity is specific for abnormal inner membrane-associated polypeptides and that there are independent proteolytic systems in the mitochondrial inner membrane and matrix compartment.

VIII. Regulation of Chaperone Expression A. Role of Chaperones from Mitochondria during Heat Shock Mitochondria1 molecular chaperones are induced by cell stresses such as heat shock and treatment of cells with amino acid analogues (Craig et al., 1987; McMullin and Hallberg, 1987; Mizzen et al., 1989; Hartman et al., 1992). As described previously, the role of molecular chaperones at such times of stress is to protect polypeptides from denaturation and aggregation or to assist in their proteolytic removal. For example, there was a significant increase in the number of polypeptides bound to Cpn60 in heat-shocked Neurospora cells compared to unstressed cells (Martin et al., 1992). Furthermore, an artificial preprotein imported into mif4 (Cpn60 mutant) cells at permissive temperatures was folded correctly but was found as an aggregate at nonpermissive temperatures (Martin et al., 1992). This suggests that Cpn60 activity is required not only for the folding of newly imported preproteins but also for maintaining protein function by stabilizing folded proteins at times of stress. Proteins can also be stabilized at high temperatures in vitro using bacterial chaperones (Hartman et al., 1993).

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

161

B. Stress Response A number of genes encoding molecular chaperones have now been isolated from both fungi and multicellular organisms. The mammalian genes encoding molecular chaperones resemble other genes in that most contain introns and possess typical regulatory elements that promote their constitutive synthesis. However, the majority of these genes contain a heat shock element (HSE) that enables their induction at times of heat shock and other stresses that compromise protein folding (Ananthan et al., 1986). The palindromic HSE is evolutionally conserved and contains a number of inverted 5-bp repeats with the consensus sequence nGAAn (Amin et al., 1988), to which the heat shock transcription factor (HSF) binds (Fernandes et al., 1994). HSFs are encoded by three genes in vertebrates with HSFl being the general stress-responsive factor (Wu et al., 1994). HSFl is synthesized constitutively and exists in an inactive form under normal physiological conditions. In response to heat shock and other cell stresses, HSFl is converted to a high-affinity DNA-binding state in a process that involves trimerization and possibly phosphorylation (Perisic et al., 1989; Westwood et af., 1991; Wu et al., 1994). HSFl may be regulated through its known interaction with Hsc70 (Fig. 4; Abravaya et al., 1992). In general, a minimum of three 5-bp units are required for HSF binding (nGAAnnTTCnnGAAn or n'ITCnnGAAnn'ITCn; Fernandes et al., 1994). However, Drosophila mefanogaster HSF can interact with two 5-bp motifs (Perisic et al., 1989). The mechanism by which the binding of HSF trimers to the HSE leads to the stimulation of transcription is unknown. The mas3 mutation in S. cerevisiae, which results in temperature-sensitive defects in the assembly of mitochondrially imported OTC, was mapped to the gene encoding yeast HSF (Smith and Yaffe, 1991). This finding provides further direct evidence that under stressed conditions the induction of mitochondria1 chaperones is necessary for maintaining proteins in an active state.

C. Organelle-Specific Stress Signaling Pathways In addition to the HSE, an additional stress-related promoter element, termed the unfolded protein response element (UPRE), has been found in the gene encoding the endoplasmic reticulum Hsp70 homologue, BiP. The UPRE was identified from observations that the presence of unfolded proteins in the endoplasmic reticulum led to the induction of BiP synthesis, whereas levels of other Hsp70 homologues remained constant (Normington et al., 1989). Further studies have defined a signaling pathway from the

162

MICHAEL T. RYAN ET AL.

FIG. 4 Regulation of stress-activated molecular chaperones. A simple model for the regulation of molecular chaperones during normal and stressed conditions is shown. An equilibrium between molecular chaperones and folded and unfolded proteins is achieved by the binding of Hsp70 to heat shock factor (HSF) under normal cellular conditions. Upon cell stress, Hsp70 molecules, which have a high affinity for unfolded polypeptides, release HSF. HSF monomers trimerize and become activated whereupon they are translocated to the nucleus and bind to the heat shock element (HSE) in the promoters of many chaperone genes. This results in an increase in transcription from these genes and an increase in chaperone synthesis that results in protein stabilization and refolding. The increase in synthesis also allows the rebinding of Hsp70 to HSF, resulting in its inactivation.

endoplasmic reticulum to the nucleus that detects and reports the level of unfolded proteins within the compartment and adjusts the synthesis of BiP and other proteins accordingly. An endoplasmic reticulum-located transmembrane kinase is thought to be integral in this process (Gething et al., 1994). A similar communication pathway is likely to exist between mitochondria and the nucleus. Although mitochondria1 Cpn60 and CpnlO are nuclear encoded, their rate of synthesis is also influenced by the physiological state

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

163

of the mitochondria. Martinus et al. (1996) found that rat hepatoma cells cultured in a po state, in which mitochondria are devoid of DNA, exhibited a selective stress response. An increase in the level of total insoluble mitochondrial protein was found in this cell line and this was accompanied by an increase in both the levels of Cpn60 and CpnlO and transcription of their corresponding mRNAs. The levels of mt-Hsp70 and cytosolic Hsp70 were unaffected, although transcription of the genes encoding these chaper-

FIG. 5 A stress-activated pathway exists between the mitochondria and the nucleus. A specific stress applied to the mitochondria results in the increased synthesis of mitochondrial chaperonins Cpn60 and CpnlO but not cytosolic Hsp70 or mt-Hsp70. This indicates that an as yet unidentified pathway exists between the mitochondria and nucleus that regulates chaperonin synthesis. This model predicts that a mitochondrial transmembrane signaling protein detects an increase in the levels of unfolded proteins in the mitochondria and subsequently activates a cytosolic factor(s). This in turn activates a mitochondrial stress transcription factor (MSTF) that binds to an element contained in the shared promoter region of the chaperonin genes. MSTF increases Cpn60 and CpnlO synthesis in order to repair the misfolded and stressinactivated proteins in the mitochondria.

164

MICHAEL T. RYAN ET AL.

ones could be separated or increased by heat shock (Martinus et aZ., 1996). Thus, these findings have implied the presence of a pathway between the mitochondria and nucleus (Fig. 5 ) that, according to the requirements of mitochondria, controls the expression of Cpn6O and CpnlO at the gene level but that is separate from the heat shock or general stress response. Components of the signaling pathway between mitochondria and nucleus are yet to be identified.

D. Organization of Chaperonin Genes The cooperation of the chaperonins Cpn60 and CpnlO requires a coordination of their synthesis. This is achieved in bacteria by the organization of the groEL and groES genes into an operon, which is regulated by a common promoter (Tilly et aZ., 1981). A feature of the groE operon is the presence of a heat shock promoter element that is activated at times of cell stress. In contrast, in S. cerevisiae the H S P 6 0 and C P N l O genes are separated and localized on chromosomes XI1 and XV, respectively (Garrels, 1995). In this case, these separate genes still maintain a degree of coregulation by containing the same cis-acting elements found in their separate promoters. Surprisingly,it was recently shown that the mammalian Cpn60 and CpnlO genes are joined head to head by a bidirectional promoter of approximately 340 bp (Fig. 6; Ryan et al., 1996). Furthermore, transfection analysis showed that the shared promoter region of these mammalian genes was sufficient to drive the simultaneous expression of two reporter genes joined to either end of the promoter. In addition, a single HSE containing four palindromic repeats in this promoter was able to increase the synthesis of both reporters under heat shock conditions. The arrangement of the mammalian chaperonin genes suggests the potential to provide the coordinated regulation of their products in a manner that is mechanistically distinct from, yet conceptually similar to, that employed by the bacterial groE operon. There are numerous reports on the presence of Cpn60 genes in mammalian genomes, although none have been shown to be functional. Indeed, many Cpn60 pseudogenes have been found (Verner et al., 1990; Pochon and Mach, 1996). Prior to the discovery of the coupled Cpn60 and CpnlO genes, Pochon and Mach (1996) reported on the sequencing of a partial human Cpn60 gene. The Cpn60 gene comprises approximately 10 kbp and contains 11 introns (Ryan et al., 1996). Interestingly, the Cpn60 gene, like the human Hsp70 and Hsp90 genes (Sorger and Pelham, 1987; Rebbe et aZ., 1989), contains an intron in the 5' untranslated region. The CpnlO gene is approximately 3 kbp and contains 3 introns, the first of which is directly 3' of the ATG codon specifying the initiating methionine of CpnlO.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

165

Bacteria

Yeast

lcPNs0

chmmX"

l Z P ; T x v

Mammals CPNGO CPNlO

FIG. 6 Schematic representation of the organization of the bacterial, yeast, and mammalian chaperonin genes. The groES (CpnlO) and groEL (Cpn60) genes are located on an operon in bacteria such as E. coli and contain a common promoter. In lower eukaryotes such as yeast, the two chaperonin genes are found in the nucleus and are located on separate chromosomes containing separate promoters. In mammals such as rat, the chaperonin genes are located in the nucleus and are linked head to head and contain common promoter elements such as a heat-shock element.

When rat genomic DNA is probed with a CpnlO cDNA there are many CpnlO-related sequences, as found for Cpn60. However, when genomic DNA is probed with intron-specific DNA only, a single copy is found in rat, suggesting that as for Cpn60, the multiple CpnlO copies in the mammalian genome represent processed pseudogenes (Ryan et al., 1996). The arrangement of the mammalian Cpn6O and CpnlO genes in a head-to-head configuration is unusual but not new. Of particular interest is the arrangement of the mouse gene encoding the major inducible Hsp70 (Hsp70.3) that is joined head to head to the testis-specific variant Hsc70t by a 600-bp region, presumably arising from a gene duplication event (Snoek et al., 1993). In this case, the Hsp70.3 gene contains a cis element within its coding region, upstream of the Hsc70t transcription start site, that silences expression of the testis-specific Hsp70 (Shimokawa and Fujimoto, 1996). In addition to having a functional HSE, the bidirectional promoter of Cpn60/CpnlO has a putative regulatory element through which the mitochondrial-specific stress response is regulated. Thus, the effects on transcription of mitochondria1 stress and heat shock were additive in po cells (Martinus et al., 1996), implying the presence of a separate regulatory element from the HSE. Although the identity of this element is not known, it is tempting to speculate that it will be analogous to the UPRE in the

166

MICHAEL T. RYAN ET AL.

BiP gene, through which the endoplasmic reticulum-specific stress is regulated. The organization of the chaperonin genes into a single functional unit also points to a requirement for the stoichiometric supply of Cpn60 and CpnlO.

IX. Chaperones and Disease A. Introduction There are many reports that molecular chaperones, particularly members of the chaperonin family, are highly antigenic and that there are high levels of anti-chaperone antibodies and reactive T cells in circulation in both infectious and autoimmune disease states. The presence of these antibodies and T cells has led to suggestions that molecular chaperones play a role in the development of diseases such as autoimmune conditions. However, much of the evidence is circumstantial and the interpretation of many findings is complicated by the use of antibodies whose specificity is poorly defined and by confusion about the identity of reacting species. For example, a number of proteins with a molecular size of approximately 60 kDa have been implicated in certain disease states. These 60-kDa species may well be Cpn6O or GroEL from an infecting bacterium or some unrelated protein. Thus, the identity of most species is based on size and on reactivity with a particular antibody, which in earlier work was often directed against bacterial antigens with overlapping specificity for mammalian counterparts. There has rarely been unequivocal confirmation of the identity of interacting species by protein sequencing. In the field of prion disease, it is becoming evident that the accumulation of insoluble aggregates may be caused by aberrant protein folding. Because it is the role of molecular chaperones to ensure proper protein folding, these molecules have been implicated in these diseases. Evidence for their involvement in prion disease will be discussed later. Despite the large amount of research on the causes of autoimmune diseases, in most cases the antigens and mechanisms involved in the initiation of these diseases are yet to be elucidated. However, in a number of these conditions, such as rheumatoid arthritis, insulin-dependent Diabetes Mellitus (IDDM), and multiple sclerosis, there are both elevated levels of molecular chaperones at the sites of autoaggression and high levels of circulating antibodies and T cells reactive against molecular chaperones. Because molecular chaperones have a ubiquitous distribution, found in essentially all tissues, it may be surprising that autoreactive cells of the immune system against molecular chaperones have not been deleted,

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

167

thereby eliminating the possibility of producing an immune response against endogenous molecular chaperones. Perhaps there are advantages in maintaining the capacity to respond to molecular chaperones to maintain an effective response against infections or for immunosurveillance against senescent or malignant cells that display molecular chaperones as markers of stress (Kaufmann, 1994).

B. Infection It is well established that certain infections give rise to immune responses against molecular chaperones (Kaufmann, 1990; Young, 1990 Kaufmann and Schoel, 1994). Patients infected with Mycobacterium leprae or M. tuberculosis have been found to have increased levels of T cells against Cpn60 and CpnlO (GroEL and GroES) (Emmrich et al., 1986; Young, 1990; Mitra et al., 1995).Studies of y8T cells derived from thymuses of newborn animals or spleens of naive adult mice show that these cells recognize mycobacterial Cpn60 (Born et al., 1990; O’Brien et al., 1992; Fu et al., 1993). A 17-residue epitope spanning amino acids 180-196 of Cpn60 from M. leprae was found to be immunodominant in mice with as many as 20% of the y6 T cells being directed against this epitope (O’Brien et al., 1992). These cells are able to recognize Cpn60 from species other than M. leprae, including Cpn6O from the host mouse (Born et al., 1990). Because these cells were detected experimentally by immortalizing them as T-cell hybridomas, it is possible that in the mouse these cells are present in a nonactive state. Nevertheless, it has been suggested that a subset of Cpn60-reactive lymphocytes may have evolved to patrol peripheral tissues for signs of excess production of this protein (O’Brien et al., 1991). In any case, the immunodominance of this chaperonin has resulted in an immune system that can respond rapidly to the presence of these molecules (Cohen and Young, 1991). Due to the high degree of homology between chaperones from different species, a strong armor against molecular chaperones that provides the body with a rapid and effective defense against infectious events is also associated with the risk of an autoreactive situation. C. Autoimmune Conditions

1. Immune Response to Chaperones An autoimmune reaction may occur if there is an inappropriate response against molecular chaperones. This may result from localized expression or presentation of these molecules or of unrelated cellular proteins carrying

168

MICHAEL T. RYAN ET AL.

homologous epitopes ( Cohen and Young, 1991). Indeed, a study of epitope homology between mycobacterial chaperones and antigenic targets in autoimmunity revealed similarities including that between an epitope of myelin basic protein and mammalian Cpn60 (Jones et al., 1993). The list of autoimmune diseases in which an immune response against molecular chaperones has been documented is quite large, although the strongest evidence for involvement of this response in disease comes from rheumatoid arthritis and animal models including adjuvant arthritis of Lewis rats and experimental IDDM in nonobese diabetic (NOD) mice. For example, T cells against Cpn60 have been found in patients with rheumatoid arthritis (Res et al., 1988; Holoshitz et al., 1989; Danieli et al., 1992; De Graeff-Meeder et al., 1995). Elevated levels of antibodies against Cpn60 were also found in this condition (Tsoulfa et al., 1989), although in another study the levels of antibodies were lower than controls (Lai et al., 1995). In addition, a study of T cells and antibodies against Cpn60 found that there was no significant differences between controls and patients with rheumatoid arthritis (Fischer et al., 1991). There are also many reports regarding the presence of elevated levels of T cells and antibodies against Cpn60 in patients with multiple sclerosis (Selmaj et al., 1991,1992;Wiicherpfenning, 1992; Birnbaum et al., 1993; Prabhakar et al., 1994).In this autoimmune disease, a reaction against Hsp70 has also been documented (Salvetti et al., 1992; Birnbaum et al., 1993; Brosnan et al., 1996). However, the finding of reactive T cells or antibodies per se is no evidence for their role in the immune disease and it is possible that this response is secondary to associated inflammation occurring at the site of damage. Certainly, factors released in response to inflammation, such as tumor necrosis factor and interferon-y, cause an increase in molecular chaperone expression (Ferm et al., 1992). 2. Can Chaperones Cause Autoimmune Disease? The best evidence for molecular chaperone involvement in autoimmmune disease comes from studies with NOD mice. Expression of mouse Cpn6O in transgenic NOD mice resulted in a substantial reduction in insulitis commonly found in these mice (Birk et a1.,1996). An analysis of T cells showed that the frequency of y8 T cells directed against the immunodominant 437-460 Cpn60 epitope was greatly reduced, although an overall increase in tolerance against Cpn60 was not observed. This led the authors to conclude that T cells specific for selected epitopes of Cpn60 are likely to be involved in islet cell destruction in NOD mice, although it should be noted that induction of tolerance to an entirely different protein, glutamic acid decarboxylase, has a similar effect on the onset of this disease (Tisch et al., 1993;). Vaccination with the immunodominant 437-460 epitope of

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

169

mycobacterial Cpn60 was able to block the development of diabetes in NOD mice and abrogate the ongoing disease (Elias and Cohen, 1995). On the other hand, immunization with this same epitope can induce insulitis in some strains of mice not prone to spontaneous development of diabetes (Elias et al., 1995), demonstrating the fine balance between tolerance and disease induction. In another experimental model of autoimmune disease, adjuvant arthritis of Lewis rats, disease could be aggravated by T cell clones specific for the 180-188 epitope of mycobacterial Cpn60 (van Eden et al., 1988), whereas these provided protection in other instances (Hogervorst et al., 1992). Furthermore, immunization with Cpn6O peptides provided protection against the development of this disease (van Eden et al., 1988; Billingham et al., 1990). In a very similar experimental model of arthritis, pristane-induced arthritis of mice, T cells reactive to mycobacterial Cpn60 were found even though the animals were immunized with pristane containing no M. tuberculosis (Barker et al., 1996). In this model, immunization with Cpn6O and Hsp70 was also able to protect against the development of the disease and administration of Cpn60 after induction of the disease reduced its severity (Ragno et al., 1996). Even in models in which autoimmune disease can be manipulated by the administration (or expression) of molecular chaperones, caution is needed in interpreting the results. Thus, in the case of adjuvant arthritis, T cells reactive against molecular chaperones are very low and in some cases hard to detect at all (Life et al., 1995). In addition, NOD mice are notoriously difficult to handle and experimental manipulations such as insertion and expression of transgenes may in themselves perturb the delicate immunological balances in this animal. 3. Levels of Chaperones in Autoimmune Lesions Increased levels of molecular chaperones have been found in autoimmune lesions. Thus, increased surface expression of Cpn60 at the site of inflammation has been reported in multiple sclerosis (Freedman et al., 1992) and increased levels of Hsp70 have been found on astrocytes at the site of lesions (Brosnan et al., 1996). In an experimental model of multiple sclerosis, experimental autoimmune encephalopathy, Cpn60 was found on oligodendrocytes and astrocytes at the site of chronic lesions but not elsewhere in the central nervous system (Gao et al., 1995). However, M. tuberculosis CpnlO was unable to provide protection, although a 12-kDa protein related to CpnlO was able to protect against the development of this condition (Ben-Nun et al., 1995). In the adjuvant arthritis model, Cpn60 was also detected in inflamed joints at above normal levels (Barker et al., 1996).

170

MICHAEL T. RYAN €T AL.

4. Surface Expression

As described previously, the most dominant chaperone implicated in autoimmune disease is Cpn60, a protein normally associated with the matrix of the mitochondrion. Although many of the reactive T cells are either produced in response to bacterial Cpn60 as a result of infection or are present in naive animals as a preexisting subset of y S T cells, there is some evidence for the localization of molecular chaperones on the surface of cells. Early reports that Cpn60 was present on the surface of monocytes, stressed macrophages, lymphoblastoid Daudi cells, and myeloid leukemia cells were obtained by flow cytometry (Fisch et aZ., 1990; WandWiirttenberger et al., 1991; Fitzgerald and Keast, 1994); however, this method does not unequivocally identify the surface antigen as Cpn6O. Cpn60 has also been detected on the surface of cells by surface labeling and immunoprecipitation (Fisch et aL, '1990; Kaur et aL, 1993) and by biotinylation (Gao et aL, 1995). Because the extra mitochondrial abundance of the protein is very low, large amounts of cells were needed for these procedures and there is a distinct possibility that some of the labeled Cpn60 was released from mitochondria during the experimental procedures. Using immunoelectron microscopy (Soltys and Gupta, 1996) with a variety of Cpn60-specific monoclonal and polyclonal antibodies to minimize nonspecific cross-reaction, low levels of Cpn6O have been found on the surface of cells. A curious finding in these studies was the localization of immunoreactive species within vesicle-like structures at the surface of cells. In addition to Cpn60, immunological techniques have also indicated an association of Hsp70 with the surface of cells (Ferrarini et aL, 1992; Heufelder et aL, 1992; Multhoff et aL, 1995). Perhaps this association is related to the loading and delivery of antigenic peptides and translocation of MHC molecules to the cell surface, a process in which both Hsp70 and Hsp96 have been implicated. As previously mentioned, it has been proposed that a population of CpnlO is found extracellularly where it may function as an early pregnancy factor (Cavanagh and Morton, 1994). Because molecular chaperones do not possess typical information for targeting to the plasma membrane, their putative appearance at the cell surface is puzzling. With respect to the mitochondrial chaperonins, it is possible that they are stable enough to survive the normal turnover of mitochondria and upon release travel to the cell surface in association with other proteins. Although there is no quantitative data on the half-life of chaperones, in vitro studies with CpnlO indicated that the naturally occurring, N-acetylated mammalian form is very stable (Ryan et al., 1995). Alternatively, the reactivity of anti-chaperone antibodies with the surface of cells may be due to protein turnover because there is a considerable body of evidence suggesting that peptides derived from molecular chaperones

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

171

are displayed on the cell surface in association with either MHC class I or class I1 molecules (Suto and Srivastava, 1995).

D. Other Conditions It has been suggested that immune reactions involving Cpn60 may be involved in atherosclerosis. Cpn60 was detected on the surface of rat aortic endothelial cells after treatment with tumor necrosis factor or heat shock (Xu et al., 1994) and Cpn60-reactive antibodies isolated from patients with arthrosclerosis of carotid arteries were capable of lysing heat-stressed human endothelial cells in the presence of complement (Schett et al., 1995). Hsp7O and Hsp96 isolated from tumor cells have been used to vaccinate animals against cancer. Mice immunized with Hsp70 or Hsp96 from normal tissues were not protected, suggesting that protection was afforded by tumor peptides associated with the molecular chaperones (Srivastava, 1994). Because of the important role played by molecular chaperones in protein targeting and folding, genetic defects affecting chaperone synthesis and/or function may be expected to be fatal or to have serious consequences as indicated by their requirement for cell viability in fungi and bacteria. A patient who died 2 days after birth was found to have only 20% of normal Cpn60 levels (Agsteribbe et al., 1993). Concomitant with the defect, this patient had deficiencies in a range of mitochondrial enzyme activities and major changes in mitochondrial morphology.

E. Prion Diseases There are many conditions in which proteins come out of solution and form aggregates. Thus, the overexpression of recombinant proteins in E. coli often results in the formation of inclusion bodies consisting of a single species of protein. There are also many disease states characterized by the accumulation of protein aggregates such as amyloid deposits on prion particles. Aggregation of proteins is a result of off-pathway reactions occurring during protein folding. In considering these reactions, it is important to note that the free energy of protein folding is approximately 50 kJ/mol and is equivalent to just a few weak interactions in the protein. Thus, proteins have evolved to be flexible rather than stable and it does not take major perturbation in the physiological conditions within the cell to push a protein down the path of unfolding and aggregation. Because the native, folded conformation of a protein occupies a minimum free energy state, folding intermediates must have even lower stability than the folded state and

172

MICHAEL T. RYAN E r AL.

hence misfolding and off-pathway reactions are very likely to occur (Jaenicke, 1995). As discussed previously, folding of soluble proteins involves the hydrophobic collapse of side chains in the nascent polypeptide chain, the relocation of domains, and the association of subunits. At each of these stages, mistakes leading to aggregation may occur. In particular, mutations that can affect both the rate of folding and stability of the folded conformation may be responsible for protein aggregation (Wetzel, 1994). One of the roles of molecular chaperones is to inhibit off-pathway reactions either by stabilizing the transition states within the folding pathway or by unfolding misfolded species (Clarke and Lund, 1996). Thus, an inadequacy of molecular chaperone function may play a role in conditions in which insoluble aggregates form, such as amyloidosis or prion disease. However, other than the association found between Cpn60 and Syrian hamster prion protein (Edenhofer et al., 1996), although there is a substantial body of evidence that prion particles represent insoluble isoforms of normal cellular proteins (Prusiner, 1994), no direct evidence for the involvement of molecular chaperones in conditions of protein aggregation in animals has been described to date. The most substantial pointer for the participation of these molecules in the pathology of protein aggregation comes from yeast. There are two conditions in Xcereviseae that exhibit remarkable similarities to prion diseases found in animals. These conditions, [URE3] and [PSI'], are characterized by the accumulation of prion-like particles containing chromosomally encoded proteins (Wickner, 1994). The [URE3] trait enables yeast stains carrying an aspartate transcarbamylase mutation to grow on ureidosuccinate in the presence of ammonia. Ammonia normally suppresses the uptake of unreidosuccinate in these mutants but not in the [URE3] mutants. The [URE3] phenotype requires the expression of the URE2 gene, encoding Ure2p. Ure2p in [URE3] cells is a modified form of the Ure2p found in wild-type cells and it exhibits increased proteinase K resistance (Masison and Wickner, 1995). Overexpression of Ure2p in wild-type strains increased the frequency of the [URE3] phenotype 20- to 200-fold. The [PSI+]trait is characterized by increases in translational read-through of all three nonsense codons that suppress nonsense mutations (Lindquist et al., 1995). The [PSIt] phenotype is caused by an aggregated form of the nuclear-encoded protein Sup35, a subunit of the translation release factor involved in the termination of translation at nonsense codons (Patino et al., 1996). In the case of the [PSI+] trait, the involvement of a molecular chaperone has been well established (Patino et al., 1996). Thus, overexpression of Hspl04 (Clp protease) eliminated the [PSI+]phenotype, as did the decreased expression of Hspl04. Hspl04 was shown to interact with the amino-terminal domain of Sup35. The involvement of Hspl04 in the curing

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

173

of the [PSI+] phenotype is consistent with its known ability to solubilize heat-induced protein aggregates and its function in enabling organisms to survive severe stress (Parsell et af., 1994). With the accumulating evidence that prions can produce heritable changes in yeast phenotype and that the level of activity of molecular chaperones is critical in the formation of the infectious conformation, it will be interesting to see whether a similar situation will be found for animal disease states caused by aggregate formation.

X. Concluding Remarks Molecular chaperones have attracted considerable attention since Ritossa’s (1962) discovery of the heat shock response. The role they play in protein import into mitochondria of fungi is now known in considerable detail, although less is known about their involvement in multicellular organisms. The mechanism of import is likely to be similar, but there are likely to be unique requirements in cells that divide infrequently or not at all, such as adult neuronal cells, compared to unicellular eukaryotic cells that divide constantly. Similarly, the process of protein turnover may take on considerable importance in such long-living and nondividing cells. Because the oxidative phosphorylation pathway of mitochondria relies on both organelle and nuclear encoded proteins, a pathway of communication between the mitochondrion and the nucleus exists to ensure that the appropriate stoichiometry is achieved for components of oxidative phosphorylation. However, the mechanism of nuclear-mitochondria1 communications is still to be worked out in any detail. Within the crowded compartments of the cell, the importance of molecular chaperones is in minimizing a protein’s chance to stray into off-pathway folding reactions. However, the prevailing way in which chaperones contribute to the process of protein folding is still in dispute: There is still no resolution of whether they act only as molecular sponges to minimize the concentration of unfolded and partially folded intermediates or whether they also play a more active role in unfolding misfolded proteins. In addition to their role in protein targeting, folding, and proteolysis in the normal cell, molecular chaperones are also stress proteins. Their increased requirement during stress reflects the effects of stress on the folded conformation of proteins and the involvement of accumulated unfolded protein in the intiation of the stress response. Although the conditions of stress that induce molecular chaperones are varied, there are a number of pathological conditions in which tissue temperatures increase significantly, such as thermal stress in long-distance runners and malignant hyperpyrexia in individu-

174

MICHAEL T. RYAN E r AL.

als susceptible to halothane. In these conditions, there is major tissue damage caused by proteolysis and, in view of the connection between proteolysis and chaperone activity, an involvement of chaperones in these conditions may prove to be important. An area that particularly needs clarification and that should provide interesting information in the future is the role of chaperones in disease. Certainly, there are many reports of antibodies and T cells directed against molecular chaperones in a range of disease states, but whether they play a role in the onset and maintenance of disease is far from resolved. Finally, the all-pervading importance of molecular chaperones to the normal function of cells no doubt reflects the fact that biological function is ultimately dependent on protein structure and any molecule that contributes to the formation and maintenance of protein strcture must occupy a central position in cell biology.

Acknowledgments This work was supported in part by grants from the National Health and Medical Research Council of Australia and the Australian Research Council. MTR, DJN, and MSC are recipients of Australian Post Graduate Awards. We gratefully acknowledge the secretarial assistance of Yvette Gaffney.

References Abravaya, K., Myers, M. P., Murphy, S. P., and Morimoto, R. I. (1992). The human heat shock protein hsp70 interacts with HSF, the transcription factor which regulates heat shock gene transcription. Genes Dev. 6,1153-1164. Adams, M. D., Dubnick, M., Kerlavage, A. R., Moreno, R., Kelley, J. M., Utterback, T. R., Nagle, J. W., Fields, C., and Venter, J. C. (1992). Sequence identification of 2,375 human brain genes. Nature (London) 355,632-634. Ades, I. Z., and Butow, R. A. (1980). The products of mitochondria-bound cytoplasmic polysomes in yeast. J. Biol. Chem. 255,9918-9924. Agsteribbe, E., Huckriede, A., Veehuis, M., Ruiters. M. H. J., Niezen-Konig, K. E., Skjeldal, 0. H., Skullerud, K., Gupta, R. S., Hallberg, R., van Diggelen, 0. P., and Scholte, H. R. (1993). A fatal, systemic mitochondria1 disease with decreased mitochondrial enzyme activities, abnormal ultrastructure of the mitochondria and deficiency of heat shock protein 60. Biochem. Biophys. Res. Commun. 193(1), 146-154. Alconada, A., Kiibrich, M., Moczko, M., Honlinger, A., and Pfanner, N. (1995). The mitochondrial receptor complex: The small subunit Mom8blIsp6 supports association of receptors with the general insertion pore and transfer of preproteins. Mol. Cell Biol. 15,6196-6205. Amerik, A. Y., Petukhova, G. V., Grigorenko, V. G., Lykov, I. P., Yarovoi, S. V., Lipkin, V. M., and Gorbalenya, A. E. (1994). Cloning and sequence analysis of the cDNA for a human homolog of eubacterial ATP-dependent Lon proteases. FEBS Lett. 340,25-28.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

175

Amin, J., Ananthan, J., and Voellmy, R. (1988). Key features of heat shock regulatory elements. Mol. Cell. Biol. 8, 3761-3769. Ananthan, J., Goldberg, A. L., and Voellmy, R. (1986). Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 232, 522-524. Anfinsen, C. B. (1973). Principles that govern the folding of proteins chains. Science 181, 223-230. Ang, D., Chandrasekhar, G. N., Zylicz, M., and Georgopoulos, C. (1986). Escherichia coli grpE gene codes for heat shock protein B25.3, essential for both A DNA replication at all temperatures and host growth at high temperature. J. Bacteriol. 167, 25-29. Argan, C., Lusty, C. J., and Shore, G. C. (1983). Membrane and cytosoliccomponents affecting transport of the precursor for ornithine carbamyltransferase into mitochondria. J. Biol. Chem. 258,6667-6670. Atencio, D. P., and Yaffe, M. P. (1992). MASS, a yeast homolog of DnaJ involved in mitochondrial protein import. Mol. Cell. Biol. 12,283-291. Bach, J. F., and Antoine, B. (1968). In vitro detection of immunosuppressive activity of antilymphocyte sera. Nature (London) 217,658-659. Baker, K. P., Schaniel, A., Vestweber, D., and Schatz, G. (1990). A yeast mitochondrial outer membrane protein essential for protein import and cell viability. Nature (London) 348,605-609. Barker, R. N., Wells, A. D., Ghoraishian, M., Easterfield, A. J., Hitsumoto, Y., Elson, C. J., and Thompson, S. J. (1996). Expression of mammalian 60-kD heat shock protein in the joints of mice with pristane-induced arthritis. Clin. Exp. Immunol. 103, 83-88. Beckmann, R. P., Mizzen, L. A., and Welch, W. J. (1990). Interaction of Hsp 70 with newly synthesized proteins: Implications for protein folding and assembly. Science 248,850-854. Ben-Nun, A., Mendel, I., Sappler, G., and Kerlero de Rosbo, N. (1995). A 12-kDa protein of Mycobacterium tuberculosis protects mice against experimental autoimmune encephalomyelitis. J. Immunol. 154, 2939-2948. Berthold, J., Bauer, M. F., Schneider, H-C., Klaus, C., Dietmeier, K., Neupert, W., and Brunner, M. (1995). The MIM complex mediates preprotein translocation across the mitochondria] inner membrane and couples it to the mt-Hsp70/ATF'driving system. Cell 81,10851093. Billingham, M. E., Carney, S., Butler, R., and Colston, M. J. (1990). A mycobacterial 65-kD heat shock protein induces antigen specific suppression of adjuvant arthritis, but is not itself arthritogenic. J. Exp. Med. 171, 339. Birk, 0. S., Douek, D. C., Elias, D., Takacs, K., Dewchmand, H., Gur, S. L., Walker, M. D., van der Zee, R.,Cohen, I. R., and Altmann, D. (1996). A role of Hsp60 in autoimmune diabetes: Analysis in a transgenic model. Proc. Natl. Acad. Sci. USA 93, 1032-1037. Birnbaum, G., Kotilinek, L., and Albrecht, L. (1993). Spinal fluid lymphocytes from a subgroup of multiple sclerosis patients respond to mycobacterial antigens. Ann. Neurol. 34, 18-24. Blom, J., Kobrich, M., Rassow, J., Voos, W., Dekker, P. J. T., Maarse, A., Meijer, M., and Pfanner, N. (1993). The essential yeast protein MIM44 (encoded by MPZI) is involved in an early step of preprotein translocation across the mitochondria1 inner membrane. Mol. Cell. Biol. 13, 7364-7371. Blond-Elguindi, S., Cwiria, S. E., Dower, W. J., Lipshutz, R. J., Sprang, S. R., Sambrook, J. F., and Gething, M-J. H. (1993). Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP. Cell 75,717-728. Bochkareva, E. S., and Girshovich, A. S. (1992). A newly synthesized protein interacts with GroES on the surface of chaperonin GroEL. J. Biol. Chem. 267,25672-25675. Boisvert, D. C., Wang, J., Otwinowski, Z., Horwich, A. L., and Sigler, P. B. (1996). The 2.4 A crystal structure of the bacterial chaperonin GroEL complexed with ATPyS. Nature Struct. Biol. 3, 170-177.

176

MICHAEL T. RYAN E r AL.

Bolliger, L., Deloche, O., Glick, B. S., Georgopoulos, C., Jeno, P., Kronidou, N., Horst, M., Morishima, N., and Schatz, G. (1994). A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability. EMBO J. 13, 1998-2006. Bolliger, L., Junne, T., Schatz, G., and Lithgow, T. (1995). Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria. EMBO J. 14,6318-6326. Bomer, U., Pfanner, N., and Dietmeier, K. (1996). Identification of a third yeast mitochondrial Tom protein with tetratrico peptide repeats. FEBS Lett. 382, 153-158. Bork, P., Sander, C., and Valencia, A. (1992). An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. Proc. Natl. Acad. Sci. USA 89,7290-7294. Born, W., Hall, L., Dallas, A., Boymel, J., Shinnick, T., Young, D., Brennan, P., and O’Brien, R. (1990). Recognition of a peptide by heat shock reactive y8 T lymphocytes. Science 249, 67-69. Braig, K., Simon, M., Furuya, F., Hainfeld, J. F., and Horwich, A. L. (1993). A polypeptide bound by the chaperonin groEL is localized within a central cavity. Proc. Natl. Acad. Sci. USA 90,3978-3982. Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C., Joacimiak, A., Horwich, A. L., aad Sigler, P. B. (1994). The crystal structure of the bacterial chaperonin GroEL at 2.8 A. Nature (London) 371,578-586. Braig, K., Adams, P. D., and Brunger, A. T. (1995). Conformational variability in the refined structure of the chaperonin GroEL at 2.8 A resolution. Nature Struct. Biol. 2, 1083-1093. Brosnan, C. F., Battistini, L., Gao, Y. L., Raine, C. S., and Aquino, D. A. (1996). Heat shock proteins and multiple sclerosis: A review. J. Neuropathol. Exp. Neurol. 55, 389-402. Bross, P., Andresen, B. S., Knudsen, I., Kruse, T. A., and Gregersen, N. (I 995). Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene. FEBS Lett. 377,249-252. Bruch, M. D., and Hoyt, D. W. (1992). Conformational analysis of a mitochondrial presequence derived from the F,-ATPase P-subunit by CD and NMR spectroscopy. Biochim. Biophys. Acta 1159, 81-93. Buchberger, A., Schroder, H., Buttner, M., Valencia, A., and Bukau, B. (1994). A conserved loop in the ATPase domain of the DnaK chaperone is essential for stable binding of GrpE. Struct. Biol. 1, 95-101. Buchberger, A., Theyssen, H., Schroder, H., McCarty, J. S., Virgallita, G., Milkereit, P., Reinstein, J., and Bukau, B. (1995). Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication. J. Biol. Chem. 270, 16903-1 6910. Burston, S. G., Ranson, N. A., and Clarke, A. R. (1995). The origins and consequences of asymmetry in the chaperonin reaction cycle. J. Mol. Biol. 249, 138-152. Callebaut, I., and Mornon, J-P. (1995). Trigger factor, one of the Escherichia coli chaperone proteins, is an original member of the FKBP family. FEBS Letr. 374, 211-215. Caplan, A. J., and Douglas, M. G. (1991). Characterization of YDJ1: A yeast homologue of the bacterial dnaJ protein. J. Cell Biol. 114, 609-621. Caplan, A. J., Tsai, J., Casey, P. J., and Douglas, M. G. (1992a). Farnesylation of YDJlp is required for function at elevated growth temperatures in Saccharomyces cerevisiae. J. Biol. Chem. 267,18890-18895. Caplan, A. J., Cyr, D. M., and Douglas, M. G. (1992b). YDJlp facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism. Cell 71,1143-1155. Cavanagh, A., and Morton, H. (1994). The purification of early-pregnancy factor to homogeneity from human platelets and identification as chaperonin 10. Eur. J. Biochem. 222,551-560. Cavanagh, A. C., and Morton, H. (1996). EPF: Embryo detector and molecular chaperone. Today’s Life Sci., May, 24-27.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

177

Chappell, T. G., Konforti, B. B., Schmidt, S. L., and Rothman, J. E. (1987). The ATPase core of a clathrin uncoating protein. J. Biol. Chem. 262, 746-750. Cheetham, M. E., Jackson, A. P., and Anderton, B. H. (1994). Regulation of 70-Da heatshock-protein ATPase activity and substrate binding by human DnaJ-like proteins, HSJla and HSJlb. Eur. J. Biochern. 226, 99-107. Chen, S., Roseman, A. M., Hunter, A. S., Wood, S. P., Burston, S. G., Ranson, N. A,, Clarke, A. R., and Saibil, H. R. (1994). Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryo-electron microscopy. Nature (London) 371, 261-264. Chen, W.-J., and Douglas, M. G. (1987). Phosphodiester bond cleavage outside mitochondria is required for the completion of protein import into the mitochondrial matrix. Cell 49, 651-658. Cheng, M. Y., Hartl, F.-U., Martin, J., Pollock, R. A,, Kalousek, F., Neupert, W., Hallberg, E. M., Hallberg, R. L., and Horwich, A. L. (1989). Mitochondria1 heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature (London) 337,620-625. Christopher, J. A., and Baldwin, T. 0. (1996). Implications of N and C-terminal proximity for protein folding. J. Mol. Biol. 257, 175-187. Clarke, A. R., and Lund, P. A. (1996). Kinetic and energetic aspects of chaperonin function. In “The Chaperonins” (R. J. Ellis, Ed.), pp. 167-212. Academic Press, San Diego. Cohen, I. R., and Young, D. B. (1991). Autoimmunity, microbial immunity and the imunological homunculus. Immunol. Today 12,105-110. Corrales, F. J., and Fersht, A. R. (1996). Toward a mechanism for GroEL-GroES chaperone activity: An ATPase-gated and -pulsed folding and annealing cage. Proc. Natl. Acad. Sci. USA 93,4509-4512. Craig, E. A., and Jacobsen, K. (1985). Mutations in cognate gene of Saccharomyces cerevisiae HSP70 result in reduced growth rates at low temperatures. Mol. Cell. B i d . 5, 3517-3524. Craig, E. A,, Kramer, J., and Kosic-Smithers, J. (1987). SSCI, a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae, is essential for growth. Proc. Natl. Acad. Sci. USA 84,4156-4160. Craig, E. A., Kramer, J., Shilling, J., Werner-Washburne, M., Holmes, S., Kosic-Smithers, J., and Nicolet, C. M. (1989). SSCI, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein. Mol. Cell. Biol. 9, 3000-3008. Crooke, E., and Wickner, W. (1987). Trigger factor: A soluble protein that folds pro-OmpA into a membrane-assembly-competent form. Proc. Natl. Acad. Sci. USA 84,5216-5220. Cyr, D. M., Lu, X., and Douglas, M. G. (1992). Regulation of HSWO function by a eukaryotic DnaJ homolog. J. Biol. Chem. 267,20927-20931. Cyr, D. M., Langer, T., and Douglas, M. G. (1994). DnaJ-like proteins: Molecular chaperones and specific regulators of Hsp70. Trends Biochem. Sci. 19, 177-181. Danieli, M. G., Candela, M., Ricciatti, A. M., Reginelli, R., Danieli, G., Cohen, I. R., and Gabrielli, A. (1992). Antibodies to mycobacterial 65kDa heat shock protein in systemic sclerosis (scleroderma). J. Autoimmunity 5,443-452. De Graeff-Meeder, E. R., van Eden, W., Rijkers, G. T., Prakken, B. J., Kuis, W., VoorhorstOgink, E. M., van der Zee, R., Schuurman, H. J., Helders, P. J. M., and Zegers, B. J. (1995). Juvenile chronic arthritis: T cell reactivity to human hsp60 in patients with a favorable course of arthritis. J. Clin. Invest. 95, 934-940. Dekker, P. J. T., Keil, P., Rassow, J., Maarse, A. C., Pfanner, N., and Meijer, M. (1993). Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane. FEBS Lett. 330,66-70. Desautels, M., and Goldberg, A. L. (1982). Demonstration of an ATP-dependent, Vanadatesensitive endoprotease in the matrix of rat liver mitochondria. J. Biol. Chem. 257, 1167311679.

178

MICHAEL T. RYAN E r AL.

Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A., and Schekman, R. (1988). A subfamily of stress proteins facilitates translocation of secretory and mitochondria1precursor polypeptides. Nature (London) 332, 800-805. Edenhofer, F., Rieger, R., Famulok, M., Wendler, W., Weiss, S., and Winnaker, E. L. (1996). Prion protein PrPc interacts with molecular chaperones of the HSP60 family. J. Virol. 70,4724-4728. Eilers, M., and Schatz, G. (1986). Binding of a specific ligand inhibits import of a purified precursor into mitochondria. Nature (London) 322,228-232. Elias, D., and Cohen, I. R. (1995). Treatment of autoimmune diabetes and insulitis in NOD mice with heat shock protein 60 peptide p277. Diabetes 44,1132-1138. Elias, D., Reshef, T., Birk, 0. S., van der Zee, R., Walker, M. D., and Cohen, I. R. (1990). Vaccination against autoimmune mouse diabetes with a T-cell epitope of the human 65-kDa heat shock protein. Proc. Natl. Acad. Sci. USA 88,3088-3091. Elias, D., Marcus, H., Reshef, T., Ablamunits, V., and Cohen, I. R. (1995). Induction of diabetes in standard mice by immunization with the p277 peptide of a 60-kDa heat shock protein. Eur. J. Immunol. 25, 2851-2857. Ellis, J., and Hartl, F.-U. (1996). Protein folding in the cell: Competing models of chaperonin function. FASEB J. 10, 20-26. Ellis, R. J. (1987). Proteins as molecular chaperones. Nature (London) 328,378-379. Ellis, R. J. (1996). Chaperonins: Introductory perspective. In “The Chaperonins” (R. J. Ellis, Ed.), pp. 2-25. Academic Press, San Diego. Emmrich, F., Thole, J., van Embden, J., and Kaufmann, S. H. E. (1986). A recombinant 64 kDa protein of Mycobacterium bovis BCG specifically stimulates human T4 clones reactive to mycobacterial antigens. J. Exp. Med. 163, 1024-1029. Emtage, J. L. T., and Jensen, R. E. (1993). MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway. J. Cell Biol. 122,1003-1012. Endo, T., Shimada, I., Roise, D., and Inagaki, F. (1989). N-terminal half of a mitochondrial presequence takes a helical conformation when bound to dodecylphosphocholine micelles: A proton nuclear magnetic resonance study. J. Biochem. 106,396-400. Fayet, O., Ziegelhoffer, T., and Georgopoulos, C. (1989). The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J. Bacteriol. 171, 1379-1385. Fenton, W. A., Kashi, Y., Furtak, K., and Horwich, A. L. (1994). Residues in chaperonin GroEL required for polypeptide binding and release. Nature (London) 371, 614-619. Ferm, M. T., Sbderstrom, K., Jindal, S., Grbnberg, A., Ivanyi, J., Young, R., and Kiessling, R. (1992). Induction of human hsp60 expression in monocytic cell lines. Int. Immunol. 4,305-311. Femandes, M., O’Brien, T., and Lis, J. T. (1994). Structure and regulation of heat shock gene promoters. In “The Biology of Heat Shock Proteins and Molecular Chaperones” (R. I. Morimoto, A. Tissibres, and C. Geogopoulos, Eds.), pp. 375-393 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Ferrarini, M., Heltai, S., Zocchi, M. R., and Rugarli, C. (1992). Unusual expression and localization of heat-shock proteins in human tumour cells. Int. J. Cancer 51, 613-619. Fisch, P., Malkovsky, M., Kovats, S., Sturm, E., Braakman, E., Klein, B. S., Voss, S. D., Morrissey, L. W., de Mars, R., Welch, W. J., Bolhuis, R. L. H., and Sondel, P. M. (1990). Recognition by human V,9NA2 T cells of a GroEL homolog on Daudi Burkitt’s lymphoma cells. Science 250, 1269-1273. Fischer, H. P., Charrock, C. E. M., Colston, M. J., and Panayi, G.S. (1991). Limiting dilution analysis proliferative T cell responses to mycobacterial65-kDa heat shock protein fails to show significant frequency differences between synovial fluid and peripheral blood of patients with rheumatoid arthritis. Eur. J. Immunol.21, 2937-2941.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

179

Fitzgerald, M., and Keast, D. (1994). Fab fragments from the monoclonal antibody ML30 bind to treated human myeloid leukemia cells. FASEB J. 8, 259-261. Flaherty, K. M., DeLuca-Flaherty, C., and McKay, D. B. (1990). Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature (London) 346,623-628. Flynn, G. C., Pohl, J., Flocco, M. T., and Rothman, J. E. (1991). Peptide-binding specificity of the molecular chaperone BiP. Nature (London) 353,726-730. Freedman, M. S., Buu, N. N., Ruijs, T. C. J., Williams, K., and Antel, J. P. (1992). Differential expression of heat shock proteins by human glial cells. J. Neuroimmunol. 41, 231-238. Frydman, J., Nimmesgern, E., Ohtsuka, K., and Hartl, F. U. (1994). Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature (London) 370,111-117. Fu, Y-X., Cranfill, R., Vollmer, M., van der Zee, R., O’Brien, R. L., and Born, W. (1993). In vivo response of murine yA T cells to a heat shock protein-derived peptide. Proc. Narl. Acud. Sci. USA 90,322-326. Gamer, J . , Multhaup, G., Tomoyasu, T., McCarty, J. S., Riidiger, S., Schonfeld, H-J., Schirra, C., Bujard, H., and Bukau, B. (1996). EMBO J. 15,607-617. Gao, Y. L., Brosnan, C. F., and Raine, C. S. (1995). Experimental autoimmune encephalomyelitis: Qualitative and semiquantitative differences in heat shock protein 60 expression in the central nervous system. J. Immunol. 154(7), 3548-3556. Garrels, J. I. (1995). YPD-A database for the proteins of Succhuromyces cerevisiue. Nucleic Acid Res. 24, 46-49. Gavel, Y., and von Heijne, G. (1990). Cleavage-sitemotifs in mitochondrial targeting peptides. Protein Eng. 4, 33-37. Georgopoulos, C., Ang, D., Liberek, K., and Zylicz, M. (1990). Properties of the Escherichiu coli heat shock proteins and their role in bacteriophage h growth. In “Stress Proteins in Biology and Medicine” (R. I. Morimoto, A. Tissii?res, and C. Georgopoulos, Eds.), pp. 191-221. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Georgopoulos, C., Liberek, K., Zylicz, M., and Ang, D. (1994). Properties of heat shock proteins of Escherichia coli and the autoregulation of the heat shock response. In “The Biology of Heat Shock Proteins and Molecular Chaperones” (R. I. Morimoto, A. Tissieres, and C. Georgopoulos, Eds.), pp. 209-249. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Gething, MA., and Sambrook, J. (1992). Protein folding in the cell. Nature (London) 355, 33-45. Gething, M-J., Blond-Elguindi, S., Mori, K., and Sambrook, J. F. (1994). Structure, function, and regulation of the endoplasmic reticulum chaperone, BiP. In “The Biology of Heat Shock Proteins and Molecular Chaperones” (R. I. Morimoto, A. Tissikres, and C. Geogopoulos, Eds.), pp. 111-135. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Glick, B. (1995). Can HSP70 proteins act as force-generating motors? Cell 80, 11-14. Glick, B., Wachter, C., and Schatz, G. (1991). Protein import into mitochondria: Two systems acting in tandem. Trends Cell Biol. 1, 99-103. Glick, B., Beasley, E. M., and Schatz, G. (1992). Protein sorting in mitochondria. Trends Biochem. Sci. 17,453-459. Goff, S. A,, Casson, L. P., and Goldberg, A. L. (1984). Heat shock regulatory gene hrpR influences rates of protein degradation and expression of the Ion gene in Escherichiu coli. Proc. Nurl. Acad. Sci. USA 81, 6647-6651. Goldberg, A. L. (1992). The mechanism and function of ATP-dependent proteases in bacterial and animal cells. Eur. J. Biochem. 203, 9-23. Goping, I. S., Millar, D. G., and Shore, G. C. (1995). Identificationof the human mitochondrial protein import receptor, huMas20p. Complementation of Amus20 in yeast. FEBS Lerr. 373,4550.

180

MICHAEL T. RYAN E r AL.

Gottesman, S., and Maurizi, M. R. (1992). Regulation by proteolysis: Energy-dependent proteases and their targets. Microbiol. Rev. 56,592-621. Gottesman, S . , Clark, W. P., De Crecy-Lagard, V., and Maurizi, M. R. (1993). ClpX, an alternative subunit for the AlT-dependent Clp protease of Escherichia coli. J. Biol. Chem. 268,22618-22626. Gragerov, A., Zeng, L., Zhao, X., Burkholder, W., and Gottesman, M. E. (1994). Specificity of DnaK-peptide binding. J. Mol. Biol. 235, 848-854. Gratzer, S., Lithgow, T., Bauer, R. E., Lamping, E., Paltauf, F., Kohlwein, S. D., Haucke, V., Junne, T., Schatz, G., and Horst, M. (1995). Mas37p, a novel receptor subunit for protein import into mitochondria. J. Cell Biol. U9, 25-34. Gray, M. W. (1989). The evolutionary origins of organelles. Trends Genet. 5, 294-299. Grossman, A. D., Erickson, J. W., and Gross, C. A. (1984). The htpR gene product of E. coli is a sigma factor for Heat-shock proteins. Cell 38, 383-390. Hachiya, N., Alam, R., Sakasegawa, Y., Sakaguchi, M., Mihara, K., and Omura, T. (1993). A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins. EMBO J. 12,1579-1586. Hachiya, N., Komiya, T., Alam, R., Iwahashi, J., Sakaguchi, M., Omura, T., and Mihara, K. (1994). MSF, a novel cytoplasmic chaperone which functions in precursor targeting to mitochondria. EMBO J. 13, 5146-5154. Hachiya, N., Mihara, K., Suda, K., Horst, M., Schatz, G., and Lithgow, T. (1995). Reconstitution of the initial steps of mitochondrial protein import. Nature (London) 376, 705-709. Hackenbrock, C. R. (1968). Chemical and physical fixation of isolated mitochondria in lowenergy and high-energy states. Biochemistry 61,598-605. Hallberg, E. M., Shu, Y., and Hallberg, R. L. (1993). Loss of mitochondrial hsp60 function: Nonequivalent effects on matrix-targeted and intermembrane-targeted proteins. Mol. Cell. Biol. 13,3050-3057. Hammen, P. K., Gorenstein, D. G., and Weiner, H. (1994). Structure of the signal sequences for two mitochondrial matrix proteins that are not proteolytically processed upon import. Biochemistry 33, 8610-8617. Hansen, W. J., Lingappa, V. R., and Welch, W. J. (1994). Complex environment of nascent polypeptide chains. J. Biol. Chem. 269,26610-26613. Hanson, B., Nuttall, S., and Hoogenraad, N. (1996). A receptor for the import of proteins into human mitochondria. Eur. J. Biochem. 235,750-753. Hare, J. F. (1990). Mechanisms of membrane protein turnover. Biochem. Biophys. Acta 1031971-90. Hartl, F.-U. (1994). Secrets of a double-doughnut. Nature (London) 371, 557-559. Hartl, F.-U. (1996). Molecular chaperones in cellular protein folding. Nature (London) 381,571-580. Hartl, F-U., Pfanner, N., Nicholson, D. W., and Neupert, W. (1989). Mitochondria1 protein import. Biochim. Biophys. Acta 988, 1-45. Hartman, D. J., Hoogenraad, N. J., Condron, R., and H@j,P. B. (1992). Identification of a mammalian 10-kDa heat shock protein, a mitochondrial chaperonin 10 homologue essential for assisted folding of trimeric ornithine transcarbamoylase in vitro. Proc. Natl. Acud. Sci. USA 89,3394-3398. Hartman, D. J., Surin, B. P., Dixon, N. E., Hoogenraad, N. J., and Hcij, P. B. (1993). Substoichiometric amounts of the molecular chaperones GroEL and GroES prevent thermal denaturation and aggregation of mammalian mitochondrial malate dehydrogenase in vitro. Proc. Natl. Acad. Sci. USA 90,2276-2280. Hattori, H., Liu, Y.-C., Tohnai, I., Ueda, M., Kaneda, T., Kobayashi, T., Tanabe, K., and Ohtsuka, K. (1992). Intracellular localization and partial amino acid sequence of a stressinducible 40-kDa protein in HeLa cells. Cell Struct. Funct. 17, 77-86.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

181

Haucke, V., Lithgow, T., Rospert, S.,Hahne, K., and Schatz, G. (1995). The yeast mitochondrial protein import receptor Mas2Op binds precursor proteins through electrostatic interaction with the positively charged presequence. J. Biol. Chem. 270,5565-5570. Haucke, V., Horst, M., Schatz, G., and Lithgow, T. (1996). The Mas2Op and Mas70p subunits of the protein import receptor of yeast mitochondria interact via the tetratricopeptide repeat motif in Mas20p: Evidence for a single hetero-oligomeric receptor. EMBO J. 15,1231-1237. Hayer-Hartl, M. K., Martin, J., and Hartl, F. U. (1995). Asymmetrical interaction of GroEL and GroES in the ATPase cycle of assisted protein folding. Science 269, 836-841. Hayes, S. A., and Dice, J. F. (1996). Roles of molecular chaperones in protein degradation. J. Cell Biol. 132, 255-258. Hemmingsen, S. M., Woolford, C., van der Vies, S. M., Tilly, K., Dennis, D. T., Georgopoulos, C. P., Hendrix, R. W., and Ellis, R. J. (1988). Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature (London) 333,330-334. Hendrick, J. P., Langer, T., Davis, T. A., Hartl, F. U., and Wiedmann, M. (1993). Control of folding and membrane translocation by binding of the chaperone DnaJ to nascent polypeptides. Proc. Natl. Acad. Sci. USA 90, 10216-10220. Hentschel, C. C., and Birnstiel, M. L. (1981). The organisation and expression of histone gene families. Cell 25, 301-313. Herman, C . ,ThCvenet, D., D’Ari, R., and Bouloc, P. (1995). Degradation of d2, the heat shock regulator in Escherichia coli, is governed by HflB. Proc. Natl. Acad. Sci. USA 92,3516-3520. Heufelder, A. E., Wenzel, B. E., and Bahn, R. S. (1992). Cell surface localization of a 72 kilodalton heat shock protein in retroocular fibroblasts from patients with Graves’ opthalmopathy. J. Clin. Endocrinol. Metab. 74, 732-736. Hindersson, P., Hoiby, N., and Bangsborg, J. (1991). Sequence analysis of the Legionella micdadei groELS operon. FEMS Microbiol. Lett. 77, 31-38. Hogervorst, E. J. M., Wagenaar, J. P. A., Boog, C. J. P., van der Zee, R., van Ernbden, J. D. A., and van Eden, W. (1992). Adjuvant arthritis and immunity to the mycobacterial 65 kDa heat shock protein. In?. Zmmunol. 4, 719-724. Hohfeld, J., and Hartl, F. U. (1994). Role of chaperonin cofactor hsplO in protein folding and sorting in yeast mitochondria. J. Cell Biol. 126, 305-315. Hohfeld, J., Minami, Y., and Hartl, F. U. (1995). Hip, a novel cochaperone involved in the eukaryotic hsc70hsp40 reaction cycle. Cell 83, 589-598. Holoshitz, J., Koning, F., Coligan, J. E., DeBruyn, J., and Strober, S. (1989). Isolation of CD4- CD8-mycobacteriurn-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid. Nature (London) 339, 226-229. Honlinger, A., Kiibrich, M., Moczko, M., Gartner, F., Mallet, L., Bussereau, F., Eckerskorn, C., Lottspeich, F., Dietmeier, K., Jacquet, M., and Pfanner, N. (1995). The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins. Mol. Cell Biol. 15, 3382-3389. Honlinger, A., Bomer, U., Alconada, A., Eckerskorn, C., Lottspeich, F., Dietmeier, K., and Pfanner, N. (1996). Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import. EMBO J. 15, 212552137, Horst, M., Hilfiker-Rothenfluh, S., Oppliger, W., and Schatz, G. (1995). Dynamic interaction of the protein translocation systems in the inner and outer membranes of yeast mitochondria. EMBO J . 14,2293-2297. Honvich, A. L., Brooks Low, K., Fenton, W. A., Hirshfield, I. N., and Furtak, K. (1993). Folding in vivo of bacterial cytoplasmic proteins: Role of GroEL. Cell 74, 909-917. Hunt, J. F., Weever, A. J., Landry, S. J., Gierasch, L., and Deisenhofer, J. (1996). The crystal structure of the GroES co-chaperonin at 2.8 A resolution. Nature (London) 379, 37-45. Hutchinson, E. G., Tichelaar, W., Hofhaus, G., Weiss, H., and Leonard, K. R. (1989). Identification and electron microscopic analysis of a chaperonin oligomer from Neurospora crassa mitochondria. EMBO J. 8, 1485-1490.

182

MICHAEL T. RYAN €T AL.

Ikeda, E., Yoshida, S., Mitsuzawa,H., Uno, I., and Toh-e, A. (1994). YGEZ is a yeast homologue of Eschericia coli grpE and is required for maintenance of mitochondrial functions. FEBS Lett. 339, 265-268. Jaenicke, R. (1995). Folding and association versus misfolding and aggregation of proteins. Philos. Trans. R. SOC.London. B 348,97-105. Jarvis, J. A., Ryan, M. T., Hoogenraad, N. J., Craik, D. J., and Haj, P. B. (1995). Solution structure of the acetylated and noncleavable targeting signal of rat Chaperonin 10. J. Biol. Chem. 270,1323-1331. Jensen, R. E., and Yaffe, M. P. (1988). Import of proteins into yeast mitochondria: The nuclear MAS2 gene encodes a component of the processing protease that is homologous to the MASZ-encoded subunit. EMBO J. 7,3863-3871. Jindal, S., Dudani, A. K., Singh, B., Harley, C. B., and Gupta, R. S. (1989). Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen. Mol. Cell. Biol. 9,2279-2283. Jones, D. B., Coulson, F. W., and Duff, G. W. (1993). Sequence homologies between hsp60 and autoantigens. Immunol. Today 14,115-118. Kalousek, F., Neupert, W., Omura, T., Schatz, G., and Schmitz, U. K. (1993). Uniform nomenclature for the mitochondrial peptidases cleaving precursors of mitochondrial proteins. Trends Biochem. Sci. 18, 249. Kandror, O., Busconi, L., Sherman, M., and Goldberg, A. L. (1994). Rapid degradation of an abnormal protein in Eschericia coli involves the chaperones GroEL and GroES. J. Biol. Chem. 269,23575-23582. Kandror, O., Sherman, M., Rhode, M., and Goldberg, A. L. (1995). Trigger factor is involved in GroEL-dependent protein degradation in Escherichia coli and promotes binding of GroEL to unfolded proteins. EMBO J. 14, 6021-6027. Kang, P.-J., Ostermann, J., Shilling, J., Neupert, W., Craig, E. A., and Pfanner, N. (1990). Requirement for hsp7O in the mitochondrial matrix for translocation and folding of precursor proteins. Nature (London) 348,137-143. Karslake, C., Piotto, M. E., Pak, Y. K., Weiner, H., and Gorenstein, D. G. (1990). 2D NMR and structural model for a mitochondrial signal peptide bound to a micelle. Biochemistry 29, 9872-9878. Kaufmann, S. H. E. (1990). Heat shock proteins and the immune response. Zmmunol Today 11,129-136. Kaufmann, S . H. E. (1994). Heat shock proteins and autoimmunity: A critical appraisal. Znt. Arch. Allergy Immunol.103, 317-322. Kaufmann, S. H. E., and Schoel, B. (1994). Heat shock proteins as antigens in immunity against infection and self. The biology of heat shock proteins. Curr. Topics Microbiol. Immunol. 167,191-205. Kaur, I., Voss, S. D., Gupta, R. S., Schell, K., Fisch, P., and Sondel, P. M. (1993). Human peripheral yA T cells recognize hsp60 molecules on Daudi Burkitt’s lymphoma cells. J. Zmmunol. 150,2046-2055. Kellems, R. E., and Butow, R. A. (1972). Cytoplasmic-type 80 S ribosomes associated with yeast mitochondria. J. Biol. Chem. 247, 8043-8050. Kessel, M., Maurizi, M. R., Kim, B., Kocsis, E., Trus, B. L., Singh, S. K., and Steven, A. C. (1995). Homology in the structural organisation between E. coli ClpAP protease and the Eukaryotic 26 S proteasome. J. Mol. Biol. 250,587-594. Kiebler, M., Pfaller, R., Siillner, T., Griffiths, G., Horstman, H., Pfanner, N., and Neupert, W. (1990). Identification of a mitochondrial receptor complex required for recognition and membrane insertion of precursor proteins. Nature (London) 348,610-616. Kiebler, M., Keil, P., Schneider, H., van der Klei, I. J., Pfanner, N., and Neupert, W. (1993). The mitochondrial receptor complex: A central role of MOM22 in mediating preprotein transfer from receptors to the general insertion pore. Cell 74, 483-492.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

183

Komiya, T., Sakaguchi, M., and Mihara, K. (1996). Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria. EMBO J. 15,399-407. Kozak, M. (1989). The scanning model for translation, an update. J. Cell Biol. 108,229-241. Kronidou, N. G., Oppliger, W., Bolliger, L., Hannavy, K., Glick, B. S., Schatz, G., and Horst, M. (1994). Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane. Proc. Natl. Acad. Sci. USA 91, 12818-12822. Kubrich, M., Keil, P., Rassow, J., Dekker, P. J. T., Blom, J., Meijer, M., and Pfanner, N. (1994). The polytopic mitochondrial inner membrane proteins MIM17 and MIM23 operate at the same preprotein import site. FEBS Lett. 349, 222-228. Kubrich, M., Dietmeier, K., and Pfanner, N. (1995). Genetic and biochemical dissection of the mitochondrial protein-import machinery. Curr. Genet. 27,393-403. Kunau, W. H., Beyer, A,, Franken, T., Gotte, K., Marzioch, M., Saidowsky, J., SkaletzRorowski, A., and Wiebel, F. F. (1993). Two complementary approaches to study peroxisome biogenesis in Saccharomyces cerevisiae. Biochimie 75,"209-224. Kutejovi, E., Durcova, G., Surovkova, E., and Kuzela, S. (1993). Yeast mitochondrial ATPdependent protease: Purification and comparison with the homologous rat enzyme and the bacterial ATP-dependent protease La. FEBS Lett. 329, 47-50. Lai, N-S., Lan, J-L., Yu, C-L., and Lin, R-H. (1995). Antibody to Mycobacterium tuberculosis 65kDa heat shock protein in patients with rheumatoid arthritis-A survey of antigen-specific antibody isotypes and subclasses in an endemic area of previous tuberculosis infection. Ann. Rheum. Dis. 54, 225-228. Landry, S. J., and Gierasch, L. M. (1991). The chaperonin GroEL binds a polypeptide in and a-helical conformation. Biochemistry 30, 7359-7362. Landry, S. J., Jordan, R., McMacken, R., and Gierasch, L. M. (1992). Different conformations for the same polypeptide bound to chaperones DnaK and GroEL. Nature (London) 355, 455-457. Landry, S. J., Zeilstra-Ryalls, J., Fayet, O., Georgopoulos, C., and Gierasch, L. M. (1993). Characterization of a functionally important domain of GroES. Nature (London) 364, 255-258. Langer, T., Pfeifer, G., Martin, J., Baumeister, W., and H a d , F.-U. (1992a). Chaperoninmediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. EMBO J. 11,4757-4765. Langer, T., Lu, C . , Echols, H., Flanagan, J., Hayer, M. K., and Hartl, F.-U. (1992b). Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature (London) 356,683-689. Laskey, R. A., Honda, B. M., Mills, A. D., and Finch, J. T. (1978). Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA. Nature (London) 275,416-420. Layloraya, S., Gambill, B. D., and Craig, E. A. (1994). A role for a eukaryotic GrpE-related protein, Mgelp, in protein translocation. Proc. Natl. Acad. Sci. USA 91,6481-6485. Layloraya, S., Dekker, P. J. T., Voos, W., Craig, E. A,, and Pfanner, N.(1995). Mitochondria1 GrpE modulates the function of matrix hsp70 in translocation and maturation of preproteins. Mol. Cell. Biol. 15,7098-7105. Lee, F.-J. S., Lin, L.-W., and Smith, J. A. (1989). N" acetylation is required for normal growth and mating of Saccharomyces cerevisiae. J. Bacteriol. 171, 5795-5802. Legname, G., Fossati, G., Gromo, G., Monzini, N.,Marcucci, F., and Modena, D. (1995). Expression in Eschericia coli, purification and functional activity of recombinant human chaperonin 10. FEBS Lett. 361,211-214. Leonhardt, S. A., Fearon, K., Danese, P. N.,and Manson, T. L. (1993). HSP78 encodes a yeast mitochondrial heat shock protein in the Clp family of ATP-dependent protease. Mol. Cell. Biol. 13, 6304-6313.

184

MICHAEL T. RYAN ET AL.

Lesser, G. J., and Rose, G. D. (1990). Hydrophobicity of amino acid subgroups in proteins. Proteins Struct. Funct. Genet. 8, 6-13. Leustek, T., Dalie, B., Amir-Shapka, D., Brot, N., and Weissbach, H. (1989). A member of the hsp70 family is localized in mitochondria and resembles Escherichia coli DnaK. Proc. Natl. Acad. Sci. USA 86, 7805-7808. Liberek, K., Marszalek, J., Ang, D., and Georgopoulos, C. (1991a). Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc. Natl. Acad. Sci. USA 88, 2874-2878. Liberek, K., Skowyra, D., Zylicz, M., Johnson, C., and Georgopoulos, C. (1991b). The Escherichia coli DnaK chaperone protein, the Hsp70 eukaryotic equivalent, changes its conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein. J. Biol. Chem. 266, 14491-14496. Life, P., Hassell, A,, Williams, K., Young, S., Bacon, P., Southwood, T., and Gaston, J. S. (1993). Responses to Gram negative enteric bacterial antigens by synovial T cells from patients with juvenile chronic arthritis: Recognition of heat shock protein HSP60. J. Rheumatol. 20,1388-1396. Lill, R., Stuart, R. A., Drygas, M. E., Nargang, F. E., and Neupert, W. (1992). Import of cytochrome c heme lyase into mitochondria: A novel pathway into the intermembrane space. EMBO J. 11,449-456. Lindquist, S., Patino, M. M., Chernoff, Y. O., Kowal, A. S., Singer, M. A., Liebman, S. W., Lee, K. H., and Blake, T. (1995). The role of HSP104 in stress tolerance and [PSI+] propagation in Saccharomyces cerevisiae Cold Spring Harbor Symp Quant. Biol. 60,451-460. Linton, J. P., Yen, J.-Y. J, Selby, E., Chen, Z . , Chinsky, J. M., Liu, K., Kellens, R. E., and Crouse, G. F. (1989). Dual bidirectional promoters at the mouse dhfr locus: Cloning and characterisation of two mRNA classes of the divergently transcribed REP-1 gene. Mol. Cell. Biol. 9, 3058-3072 Lithgow, T., Haj, P. B., and Hoogenraad, N. J. (1993a). Do cytosolic factors prevent promiscuity at the membrane surface? FEBS Lett. 329, 1-4. Lithgow, T., Ryan, M., Anderson, R., Haj, P. B., and Hoogenraad, N. J. (1993b). A constitutive form of heat-shock protein 70 is located in the outer membranes of mitochondria from rat liver. FEBS Lett. 332,277-281. Lithgow, T., Junne, T., Suda, K., Gratzer, S., and Schatz, G. (1994). The mitochondrial outer membrane protein Mas22p is essential for protein import and viability of yeast. Proc. Nail Acad. Sci USA 91,11973-11977. Lithgow, T., Glick, S., and Schatz, G. (1995). The protein import receptor of mitochondria. Trends Biochem. Sci. 20, 98-101. Lorimer, G. H. (1996). A quantitative assessment of the role of the chaperonin proteins in protein folding in vitro. FASEB J. 10, 5-9. Lubben, T. H., Gatenby, A. A., Donaldson, G. K., Lorimer, G. H., and Viitanen, P. V. (1990). Identification of a groES-like chaperonin in mitochondria that facilitates protein folding. Proc. Natl. Acad. Sci. USA 87, 7683-7687. Luke, M. M., Sutton, A., and Amdt, K. T. (1991). Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins. J. Cell Biol. 114, 623-638. Maarse, A. C., Blom, J., Grivell, L. A., and Meijer, M. (1992). MPZl, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria. EMBO J. 11,3619-3628. Maarse, A. C., Blom, J., Keil, P., Pfanner, N., and Meijer, M. (1994). Identification of the essential yeast protein MIM17, an integral mitochondrial inner membrane protein involved in protein import. FEES Lett. 349, 215-221. MacLachlan, L. K., Harris, P. I., Reid, D. G., White, J., Chapman, D., Lucy, J. A., and Austen, B. M. (1994). A spectroscopic study of the mitochondrial transit peptide of rat malate dehydrogenase. Biochem J. 303,657-662.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

185

Mande, S. C., Mehra, V., Bloom, B. R., and Hol, W. G. J. (1996). Structure of the heat shock protein chaperonin-10 of Mycobacterium leprae. Science 271, 203-207. Manning-Krieg, U. C., Scherer, P. E., and Schatz, G. (1991). Sequential action of mitochondrial chaperones in protein import into the matrix. EMBO J. 10, 3273-3280. Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L., and Hartl, F.-U. (1991). Chaperonin-mediated protein folding at the surface of groEL through a ‘molten globule’like intermediate. Nature (London) 352, 36-42. Martin, J., Horwich, A. L., and Hartl, F.-U. (1992). Prevention of protein denaturation under heat stress by the chaperonin hsp60. Science 258, 995-998. Martinus, R. D., Garth, G . P., Webster, T. L., Cartwright, P., Naylor, D. J., Hoj, P. B., and Hoogenraad, N. J. (1996). Selective induction of mitochondrial chaperones in response to loss of the mitochondrial genome. Eur. J. Biochem. 240, 98-103. Masison, D. C., and Wickner, R. B. (1995). Prion-inducing domain of yeast Ure2p and protease resistance of Ure2p in prion-containing cells. Science 270, 93-95. Matouschek, A., Rospert, S., Schmid, K., Click, B. S., and Schatz, G. (1995). Cyclophilin catalyzes protein folding in yeast mitochondria. Proc. Natl. Acad. Sci. USA 92,6319-6323. Maurizi, M. R., Clark, W. P., Kim, S-H., and Gottesman, S. Clp P represents a unique family of serine proteases. J. Biol. Chem. 265, 12546-12552. Mayer, A., Lill, R., and Neupert, W. (1993). Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria. J. Cell Biol. 121, 1233-1243. Mayer, A., Nargang, F. E., Neupert, W., and Lill, R. (1995). MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19. EMBO J. 14,4204-4211. Mayhew, M., da Silva, A. C. R., Martin, J., Erdjument-Bromage, H., Tempst, P., and Hartl, F. U. (1996). Protein folding in the central cavity of the GroEL-GroES chaperonin complex. Nature (London) 379,420-426. McMullin, T. W., and Hallberg, R. L. (1987). A normal mitochondrial protein is selectively synthesized and accumulated during heat shock in Tetrahymena thermophila. Mol. Cell. Biol. 7,4414-4423. McMullin, T. W., and Hallberg, R. L. (1988). A highly evolutionarily conserved mitochondrial protein is structurally related to the protein encoded by the Escherichia coli groEL gene. Mol. Cell. Biol. 8, 371-380. Mihara, K., and Omura, T. (1996). Cytoplasmic chaperones in precursor targeting to mitochondria: The role of MSF and hsp70. Trends Cell B i d . 6, 104-108. Mitra, P. K., Mehra, N. K., Maiti, T. K., Banerjee, A., Taneja, V., Rajalingam, R., Ahuja, R. K., and Bhattacharya, B. C. (1995). Cd4+ T-cell responses to recombinant hsp65 and hspl8 of M . leprae and their trypsin digested fragments in leprosy-Diversity in HLA-DR restriction. Int. J. Leprosy 63(4), 518-528. Miura, S., Mori, M., and Tatibana, M. (1983). Transport of ornithine carbamoyltransferase precursor into mitochondria: Stimulation by potassium ion, magnesium ion and a reticulocyte cytosolic protein(s). J. Biol. Chem. 258, 6671-6674. Mizzen, L. A., Chang, C., Garrels, J. I., and Welch, W. J . (1989). Identification, characterization and purification of two mammalian stress proteins present in mitochondria, grp 75, a member of the hsp 70 family and hsp 58, a homolog of the bacterial groEL protein. J. Biol. Chem. 264,20664-20675. Moczko, M., Dietmeier, K., Sollner, T., Segui, B., Steger, H. F., Neupert, W., and Pfanner, N. (1992). Identification of the mitochondrial receptor complex in Saccharomyces cerevisiae. FEES Lett. 310, 265-268. Moczko, M., Ehmann, B., Gartner, F., Honlinger, A., Schafer, E., and Pfanner, N. (1994). Deletion of the receptor MOM19 strongly impairs import of cleavable preproteins into Saccharomyces cerevisiae mitochondria. J. Biol. Chem. 269, 9045-9051. Moczko, M., Schonfisch, B., Voos, W., Pfanner, N., and Rassow, J. (1995). The mitochondrial ClpB homolog Hsp78 cooperates with matrix Hsp70 in the maintenance of mitochondrial function. 1. Mol. Biol. 254, 538-543.

186

MICHAEL T. RYAN ET AL.

Multhoff, G., Botzler, C., Wiesnet, M., Muller, E., Meier, T., Wilmanns, W., and Issels, R. D. (1995). A stress-inducible 72kDa heat shock protein (Hsp72) is expressed on the surface of human tumor cells, but not on normal cells. Int. J. Cancer 61, 272-279. Murakami, K., and Mori, M. (1990). Purified presequence binding factor (PBF) forms an import-competent complex with a purified mitochondrial precursor protein. EMBO J. 9, 3201-3208. Murakami, H., Pain, D., and Blobel, G. (1988a). 70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria. J. Cell Biol. 107,2051-2057. Murakami, K., Amaya, Y., Takiguchi, M., Ebina, Y., and Mori, M. (1988b). Reconstitution of mitochondrial protein transport with purified ornithine carbamoyltransferase precursor expressed in Escherichia coli. J. Biol. Chem. 263, 18437-18442. Murakami, K., Tokunaga, F., Iwanaga, S., and Mori, M. (1990). Presequence does not prevent folding of a purified mitochondrial precursor protein and is essential for association with a reticulocyte cytosolic factor(s). J. Biochem. 108, 207-214. Murakami, K., Tanase, S., Morino, Y., and Mori, M. (1992). Presequence binding factordependent and -independent import of proteins into mitochondria. J. Biol. Chem. 267, 13119-13122. Nakai, M., Endo, T., Hase, T., and Matsubara, H. (1993). Intramitochondrial protein sorting. J. Biol. Chem. 268,24262-24269. Naylor, D. J., Ryan, M. T., Condron, R., Hoogenraad, N. J., and Hej, P. B. (1995). Affinity purification and identification of GrpE homologues from mammalian mitochondria. Biochim. Biophys. Acta l248,75-79. Naylor, D. J., Hoogenraad, N. J., and Haj, P. B. (1996). Isolation and characterisation of a cDNA encoding rat mitochondrial GrpE, a stress inducible chaperone of ubiquitous appearance in mammalian organs. FEBS Lett. 396, 181-188. Nelson, R. J., Ziegelhoffer, T., Nicolet, C., Werner-Washburne, M., and Craig, E. A. (1992). The translation machinery and 70 kd heat shock protein cooperate in protein synthesis. Cell 71, 97-105. Neupert, W., Hartl, F-U., Craig, E. A., and Pfanner, N. (1990). How do polypeptides cross the mitochondrial membranes? Cell 63,447-450. Nobrega, F. G., Nobrega, M. P., and Tzagoloff, A. (1992). BCSI, a novel gene required for the expression of functional Rieske iron-sulphur protein in Saccharomyces cerevisiae. EMBO J. 11,3821-3829. Normington, K., Kohno, K., Kozutsumi, Y . , Gething, M.-J., and Sambrook, J. (1989). S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell 57, 1223-1236. O’Brien, R. L., Happ, M. P., Dallas, A., Cranfill, R., Hall, L., Lang, J., Fu, Y-X., Kubo, R., and Born, W. (1991). Recognition of a single Hsp60 epitope by an entire subset of y6 T lymphocytes. Immunol. Rev. 121, 155-170. O’Brien, R. L., Fu, Y-X., Cranfill, R., Dallas, A., Ellis, C., Reardon, C., Lang, J., Carding, S. R., Kubro, R., and Born, W. (1992). Heat shock protein hsp60-reactive y6 cells: A large, diversified T-lymphocyte subset with highly focused specificity. Proc. Natl. Acad. Sci. USA 89,4348-4352. Ohba, M., and Schatz, G. (1987). Disruption of the outer membrane restores protein import to trypsin-treated yeast mitochondria. EMBO J. 6, 2109-2115. Ohta, S., and Schatz, G. (1984). A purified precursor polypeptide requires a cytosolic protein fraction for import into mitochondria. EMBO J. 3,651-657. Okazaki, A., Ikura, T., Nikaido, K., and Kuwajima, K. (1994). The chaperonin GroEL does not recognise apo-a-lactalbumin in the molten globule state. Struct. Biol. 1,439-445. Ostermann, J., Horwich, A. L., Neupert, W., and Hartl, F.-U. (1989). Protein folding in mitochondria requires complex formation with hsp60 and ATF’ hydrolysis. Nature (London) 341, 125-130.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

187

Ostermann, J., Voos, W., Kang, P. J., Craig, E. A., Neupert, W., and Pfanner, N. (1990). Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix. FEBS Lett. 277, 281-284. Pajic, A., Tauer, R., Feldmann, H., Neupert, W., and Langer, T. (1994). YtalOp is required for the ATP-dependent degradation of polypeptides in the inner membrane of mitochondria. FEBS Lett. 353,201-206. Palleros, D. R., Welch, W. J., and Fink, A. L. (1991). Interaction of hsp7O with unfolded proteins: Effects of temperature and nucleotides on the kinetics of binding. Proc. Natl. Acad. Sci. USA 88,5719-5723. Parsell, D. A., and Lindquist, S. (1993). The function of heat-shock proteins in stress tolerance: Degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27, 437-496. Parsell, D. A., Kowal, A. S., Singer, M. A,, and Linquist, S. (1994). Protein disaggregation mediated by heat-shock protein HsplM. Nature (London) 372,475-478. Patino, M. M., Liu, J-J., Glover, J. R., and Lindquist, S. (1996). Support for the pnon hypothesis for inheritance of a phenotypic trait in yeast. Science 273, 622-626. Pchelintseva, O., Pak, Y. K., and Weiner, H. (1995). Identification of protein-receptor components required for the import of prealdehyde dehydrogenase into rat liver mitochondria. Arch. Biochem. Biophys. 323, 54-62. Pelham, H. R. B. (1986). Speculations on the functions of the major heat shock and glucoseregulated proteins. Cell 46,959-961. Peralta, D., Hartman, D. J., McIntosh, A. M., Hoogenraad, N. J., and H@j,P. B. (1990). cDNA and deduced amino acid sequence of rat liver prehsp60 (chaperonin-60). Nucleic Acids Res. 18, 7162. Peralta, D., Lithgow, T., Hoogenraad, N. J., and H@j,P. B. (1993). Prechaperonin 60 and preornithine transcarbamylase share components of the import apparatus but have distinct maturation pathways in rat liver mitochondria. Eur. J. Biochem. 211, 881-889. Peralta, D., Hartman, D. J., Hoogenraad, N. J., and H@j,P. B. (1994). Generation of a stable folding intermediate which can be rescued by the chaperonins GroEL and GroES. FEBS Lett. 339, 45-49. Perisic, O., Xiao, H., and Lis, J. T. (1989). Stable binding of Drosophila heat shock factor to head-head and tail-tail repeats of a conserved 5 bp recognition unit. Cell 59,797-806. Perryman, R. A., Mooney, B., and Harmey, M. A. (1995). Identification of a 42-kDa plant mitochondrial outer membrane protein, MOM42, involved in the import of precursor proteins into plant mitochondria. Arch. Biochem. Biophys 316, 659-664. Pfanner, N., and Meijer, M. (1995). Pulling in the proteins. Curr. Biol. 5, 132-135. Pfanner, N., Hartl, F.-U., and Neupert, W. (1988). Import of proteins into mitochondria: A multi-step process. Eur. J. Biochem. 175, 205-212. Pfanner, N., Rassow, J., Wienhues, U., Hergesberg, C., Sollner, T., Becker, K., and Neupert, W. (1990). Contact sites between inner and outer membranes: Structure and role in protein translocation into the mitochondria. Biochirn. Biophys. Acta 1018,239-242. Pfanner, N., Rassow, J., van der Klei, I. J., and Neupert, W. (1992). A dynamic model of the mitochondrial protein import machinery. Cell 68, 999-1002. Pfanner, N., Craig, E., and Meijer, M. (1994). The protein import machinery of the mitochondrial inner membrane. Trends Biochem. Sci. 19, 368-372. Pfanner, N., Douglas, M. G., Endo, T., Hoogenraad, N. J., Jensen, R. E., Meijer, M., Neupert, W., Schatz, G., Schmitz, U. K., and Shore, G. C. (1996). Uniform nomenclature for the protein transport machinery of the mitochondrial membranes. Trends Biochem. Sci. 21, 51-52. Picketts, D. J., Mayanil, C. S. K., and Gupta, R. S. (1989). Molecular cloning of a Chinese hamster mitochondrial protein related to the “chaperonin” family of bacterial and plant proteins. J. Biol. Chem. 264, 12001-12008.

188

MICHAEL T. RYAN ET AL.

Pochon, N. A., and Mach, B. (1996). Genetic complexity of the human hsp 60 gene. Int. Imrnun. 8,221-230. Prabhakar, S., Kurien, E., Gupta, R. S., Zielinski, S.. and Freedman, M. S. (1994). Heat shock protein immunoreactivity in CSF: Correlation with oligoclonal banding and demyelinating disease. Neurology 44,1644-1648. Prusiner, S. B. (1994). Biology and genetics of prion diseases. Annu. Rev. Microbiol. 48, 655-686. Ptitsyn, 0 .B. (1981). Protein folding: General physical model. FEBS Left. 131, 197-202. Ragno, S., Winrow, V. R., Mascagni, P., Lucietto, P., DiPierro, F., Morris, C. J., and Blake, D. P. (1996). A synthetic 10-kD heat shock protein (hsplO) from Mycobacterium tuberculosis modulates adjuvant arthritis. Clin. Exp. Immunol. 103, 384-390. Randall, S. K., and Shore, G. C. (1989). Import of a mutant mitochondrial precursor fails to respond to stimulation by a cytosolic factor. FEES Lett. 250,561-564. Rassow, J., Maarse, A. C., Krainer, E., Kiibrich, M.. Miiller, H., Meijer, M., Craig, E. A., and Pfanner, N. (1994). Mitochondria1 protein import: Biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44. J. Cell Biol. 127,15471556. Rassow, J., Mohrs, K., Koidl, S., Barthelmess, I. B., Pfanner, N., and Tropschug, M. (1995). Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones Hsp70 and Hsp60. Mol. Cell Biol. 15,2654-2662. Rebbe, N. F., Hickman, W. S., Ley, T. J., Stafford, D. W., and Hickman, S. (1989). Nucleotide sequence and regulation of a human 90-kDa heat shock protein gene. J. Biol. Chem. 264, 15006-15011. Reid, G. A., and Schatz, G . (1982). Import of proteins into mitochondria: Yeast cells grown in the presence of carbonyl cyanide m-chlorophenylhydrazone accumulate massive amounts of some mitochondrial precursor polypeptides. J. Biol. Chem. 257, 13056-13061. Res, P. C. M, Schaar, C. G., Breedveld, F. C., van Eden, W., van Embden, J. D. A., Cohen, I. R., and de Vries, R. R. P. (1988). Synovial fluid T cell reactivity against 65kDa heat shock protein of Mycobacteria in early chronic arthritis. Lancet, 478-480. Richarme, G., and Kohiyama, M. (1994). Amino acid specificity of the Eschericia coli chaperone GroEL (heat shock protein 60). J. Biol. Chem. 269,7095-7098. Ritossa, F. (1962). A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 18, 571-573. Rospert, S., Glick, B. S., Jeno, P., Schatz, G., Todd, M. J., Lorimer, G . H., and Viitanen, P. V. (1993a). Identification and functional analysis of chaperonin 10, the groES homolog from yeast mitochondria. Proc. Natl. Acad. Sci. USA 90, 10967-10971. Rospert, S., Junne, T., Glick, B. S., and Schatz, G . (1993b). Cloning and disruption of the gene encoding yeast mitochondrial chaperonin 10, the homolog of E. coli groES. FEES Lett. 335, 358-360. Rospert, S., Looser R., Dubaquie, Y., Matouschek, A., Glick, B. S., and Schatz, G. (1996). Hsp60-independent protein folding in the matrix of yeast mitochondria. EMBO J. 15, 764-774. Rowley, N., Prip-Buus, C.,Westermann, B., Brown, C., Schwarz, E., Barrell, B., and Neupert, W. (1994). Mdjlp, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding. Cell 77, 249-259. Ryan, M. T., Hoogenraad, N. J., and H 4 , P. B. (1994). Isolation of a cDNA clone specifying rat chaperonin 10, a stress inducible mitochondrial matrix protein synthesised without a cleavable presequence. FEBS Lett. 337, 152-156. Ryan, M. T., Naylor, D. J., Hoogenraad, N. J., and Hoj, P. B. (1995). Affinity purification, overexpression and characterization of Chaperonin 10 homologues synthesized with and without N-terminal acetylation. J. Biol. Chem. 270, 22037-22043.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

189

Ryan, M. T., Herd, S. M., Sberna, G., Samuel, M. S., Hoogenraad, N. J., and Hoj, P. B. (1996). The genes encoding mammalian Chaperonin 60 and Chaperonin 10 are linked heat-to-head and share a bidirectional promoter. Gene, in press. Sadis, S., and Hightower, L. E. (1992). Unfolded proteins stimulate molecular chaperone hsc70 ATPase by accelerating ADP/ATP exchange. Biochemistry 31, 9406-9412. Salvetti, M., Butinelli, C., Ristori, G., Carbonari, M., Cherchi, M., Fiorelli, M., Grazia Grasso, M., Toma, L., and Pozzilli, C. (1992). T-lymphocyte reactivity to the recombinant mycobacterial 65 and 70kDa heat shock proteins in multiple sclerosis. J. Autoimmunity 5, 691-702. Scherer, P. E., Krieg, U. C., Hwang, S. T., Vestweber, D., and Schatz, G. (1990). A precursor protein partly translocated into yeast mitochondria is bound to a 70 kd mitochondrial stress protein. EMBO J . 9,4315-4322 Scherer, P. E., Manning-Krieg, U. C., Jeno, P., Schatz, G., and Horst, M. (1992). Identification of a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane. Proc. Natl. Acad. Sci. USA 89, 11930-11934. Schett, G . , Xu, Q., Amberger, A., Van der Zee, R., Reheis, H., Willeit, J., and Wick, G. (1995). Autoantibodies against heat shock protein 60 mediate endothelial cytotoxicity. J. Clin.Invest. 96, 2569-2577. Schleyer, M., and Neupert, W. (1985). Transport of proteins into mitochondria: Translocational intermediates spanning contact sites between outer and inner membranes. Cell 43,339-350. Schleyer, M., Schmidt, B., and Neupert, W. (1982). Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria. Eur. J. Biochem. 125,109-116. Schlossman, D. M., Schmid, S. L., Braell, W. A., and Rothman, J. E. (1984). An enzyme that removes clathrin coats: Purification of an uncoating ATPase. J. Cell B i d 99, 723-733. Schmid, D., Baici, A,, Gehring, H., and Christen, P. (1994). Kinetics of molecular chaperone action. Science 263, 971-973. Schmidt, M., and Buchner, J. (1992). Interaction of GroE with an all-P-protein. J. B i d . Chem. 267, 16829-16833. Schmitt, M., Neupert, W., and Langer, T. (1995). Hsp78, a Clp homologue within mitochondria, can substitute for chaperone functions of mt-hsp70. EMBO J. 14, 3434-3444. Schnall, R., Mannhaupt, G., Stucka, R., Tauer, R., Ehnle, S., Schwarzlose, C., Vetter, I., and Feldmann, H. (1994). Identification of a set of yeast genes coding for a novel family of putative ATPases with high similarity to constituents of the 26s protease complex. Yeast 10, 1141-1155. Schneider, H.-C., Berthold, J., Bauer, M. F., Dietmeier, K., Guiard, B., Brunner, M., and Neupert, W. (1994). Mitochondria1 Hsp70/MIM44 complex facilitates protein import. Science 254, 1659-1662. Schroder, H., Langer, T., Hartl, F.-U., and Bukau, B. (1993). DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage. EMBO J. 12, 4137-4144. Seckler, R., and Jaenicke, R. (1992). Protein folding and protein refolding. FASEB J. 6,25452552. Segui-Real, B., Kispal, G., Lill, R., and Neupert, W. (1993). Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes: Passage of preproteins through the intermembrane space. EMBO J. 12,2211-2218. Seki, N., Moczko, M., Nagase, T., Zufall, N., Ehmann, B., Dietmeier, K., Schafer, E., Nomura, N., and Pfanner, N. (1995). A human homolog of the mitochondrial protein import receptor Mom19 can assemble with the yeast mitochondrial receptor complex. FEBS Lett. 375, 307-310. Selmaj, K., Brosnan, C. F., and Raine, C. S. (1991). Colocalization of lymphocytes bearing yS T-cell receptor and heat shock protein hsp 65+ oligodendrocytes in multiple sclerosis. Proc. Natl. Acad. Sci. USA. 88, 6452-6456.

190

MICHAEL T. RYAN ET AL.

Selmaj, K., Brosnan, C.F., and Raine, C.S. (1992). Expression of heat shock protein-65 by oligodendrocytes in vivo and in vitro. Implications for multiple sclerosis. Neurology 42, 795-800. Sheffield, W. P., Shore, G. C., and Randall, S. K. (1990). Mitochondria1 precursor protein: Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation and import competence. J. Eiol. Chern. 265, 11069-11076. Sherman, M. Y., and Goldberg, A. L. (1992). Involvement of the chaperone dnaK in the rapid degradation of a mutant protein in Escherichia coli. EMEO J. 11,71-77. Shimokawa, T., and Fujimoto, H. (1996). Identification of a transciptional silencer in the protein coding region of the mouse major inducible Hsp70 gene. Eiochem. Eiophys. Res. Cornrnun. 221,843-848. Simon, S . M., Peskin, C. S., and Oster, G. F. (1992). What drives the translocation of proteins? Proc. Natl. Acad. Sci. USA 89, 3770-3774. Smith, B. J., and Yaffe, M. P. (1991). A mutation in the yeast heat-shock factor gene causes temperature-sensitive defects in both mitochondrial protein import and the cell cycle. Mol. Cell Biol. 11,2647-2655. Snoek, M., Jansen, M., Olavesen, M. G., Campbell, R. D., Teuscher, C., and van Vugt, H. (1993). Three Hsp70 genes are located in the C4-H-2D region: Possible candidates for the Orch-1 locus. Genornics 15, 350-356. Soltys, B. J., and Gupta, R. S. (1996). Immunoelectron microscopic localization of the 60kDa heat shock chaperonin protein (Hsp60) in mammalian cells. Exp. Cell Res. 222,16-27. Sorger, P. K., and Pelham, H. R. B. (1987). Cloning and expression of a gene encoding hsc73, the major hsp70-like protein in unstressed rat cells. EMEO J. 6, 993-998. Squires, C., and Squires, C. L. (1992). The Clp proteins: Proteolysis regulators or molecular chaperones? J. Bacteriol. 174, 1081-1085. Srivastava, P. K. (1994). Heat shock proteins in immune response to cancer: The fourth paradigm. Experientia 50,1054-1060. Stoller, G., Tradler, T., Rticknagel, P., Rahfeld, Jens-U., and Fischer, G. (1996). An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidylprolyl cishrans isomerase activity. FEBS Lett 384, 117-122. Straus, D., Walter, W., and Gross, C. A. (1988). Escherichia coli heat shock gene mutants are defective in proteolysis. Genes Dev. 2, 1851-1858. Suto, R., and Srivastava, P. K. (1995). A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science 269,1585-1588. Suzuki, C. K., Suda, K., Wang, N., and Schatz, G. (1994). Requirement for the yeast gene LON in intramitochondrialproteolysisand maintenance of respiration. Science 264,273-276. Szabo, A., Langer, T., Schroder, H., Flanagan, J., Bukau, B., and Hartl, F. U. (1994). The ATP hydrolysis-dependent reaction cycle of the Eschericia coli Hsp70 system-DnaK,DnaJ, and GrpE. Proc Natl Acad. Sci. USA 91,10345-10349. Szabo, A., Korzun, R., Hartl, F. U., and Flanagan, J. (1996). A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates. EMBO J. 15,408-417. Tauer, R., Mannhaupt, G., Schnall, R., Pajic, A., Langer, T., and Feldmann, H. (1994). YtalOp, a member of a novel ATPase family in yeast, is essential for mitochondrial function. FEBS Lett. 353, 197-200. Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D. G. (1993). Import, processing, and two-dimensional NMR structure of a linker-deleted signal peptide of rat liver mitochondrial aldehyde dehydrogenase. J. Biol. Chem. 268,19906-19914. Thorsness, P. E., White, K. H., and Fox, T. D. (1993). Inactivation of YMEI, a member of the FTSH-SEC18-PASI-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharom,yces cerevisiae. Mol. Cell. Biol. 13,54185426.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

191

Tilly, K., Murialdo, H., and Georgopoulos, C. (1981). Identification of a second Escherichia coli groE gene whose product is necessary for bacteriophage morphogenesis. Proc. Natl. Acad. Sci. USA 78, 1629-1633. Tisch, K., Yang, X-D., Singer, S. M., Liblau, R. S., Fugger, L., and McDevitt, H. 0. (1993). Immune responses to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature (London) 366,72-75. Todd, M. J., Viitanen, P. V., and Lorimer, G. H. (1993). Hydrolysis of adenosine 5’-triphosphate by Eschericia coli GroEL Effects of GroES and potassium ion. Biochemistry 32,8560-8567. Todd, M. J., Viitanen, P. V., and Lorimer, G. H. (1994). Dynamics of the chaperonin ATPase cycle: Implications for facilitated folding. Science 265, 659-666. Todd, M. J., Walke, S., Lorimer, G., Truscott, K., and Scopes, R. K. (1995). The single-ring Thermoanaerobacter brokii Chaperonin 60 (Tbr-EL,) dimerizes to Tbr-EL,,.Tbr-ES, under protein folding conditions. Biochemistry 34, 14932-14941. Todd, M. J., Lorimer, G. H., and Thirumalai, D. (1996). Chaperonin-facilitated protein folding: Optimisation of rate and yield by an iterative annealing mechanism. Proc. Natl. Acad. Sci. USA 93,4030-4035. Tomoyasu, T., Gamer, J., Bukau, B., Kanemori, M., Mori, H., Rutman, A. J., Oppenheim, A. B., Yura, T., Yamanaka, K., Niki, H., Hirage, S., and Ogura, T. (1995). Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor d2.EMBO J. 14, 2551-2560. Truscott, K. N., Hoj, P. B., and Scopes, R. K. (1994). Purification and characterisation of chaperonin 60 and chaperonin 10 from the anaerobic thermophile Thermoanaerobacter brokii. Eur. J. Biochem. 222, 277-284. Tsoulfa, G., Pook, G. A. W., van Embden, J. D. A,, Young, D. B., Mehlert, A., Isenberg, D. A., Hay, F. C., and Lydyard, P. M. (1989). Raised serum IgG and IgA antibodies to mycobacterial antigens in rheumatoid arthritis. Ann. Rheum. Dis. 48,118-123. Ungermann, C., Neupert, W., and Cyr, D. M. (1994). The role of Hsp70 in conferring unidirectionality on protein translocation into mitochondria. Science 266, 1250-1253. Van Dyck, L., Pearce, D. A., and Sherman, F. (1994). PIMl encodes a mitochondrial ATPdependent protease that is required for mitochondrial function in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 269, 238-242. van Eden, W., Thole, J. E. R., van der Zee, E., Noordzij, A,, van Embden, J. D. A., Hensen, E. J., and Cohen, I. R. (1988). Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis. Nature (London) 331, 171-173. Verner, K. (1993). Co-translational protein import into mitochondria: An alternative view. Trends Biochem. Sci. 18, 366-371. Vestweber, D., Brunner, J., Baker, A., and Schatz, G. (1989). A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site. Nature (London)341,205-209. Viitanen, P. V., Gatenby, A. A,, and Lorimer, G. H. (1992a). Purified chaperonin 60 (groEL) interacts with the nonnative states of a multitude of Escherichia coli proteins. Protein Sci. 1, 363-369. Viitanen, P. V., Lorimer, G. H., Seetharam, R., Gupta, R. S., Oppenheim, J., Thomas, J. O., and Cowan, N. J. (1992b). Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring. J. Biol. Chem. 267, 695-698. von Ahsen, O., Voos, W., Henninger, H., and Pfanner, N. (1995). The mitochondrial protein import machinery: Role of ATP in dissociation of the Hsp7OMim44 complex. J. Biol. Chem. 270,29848-29853. von Heijne, G. (1986). Mitochondria1 targeting sequences form amphiphilic helices. EMBO J. 5,1335-1342. Voos, W., Gambill, D. B., Layloraya, S., Ang, D., Craig, E. A., and Pfanner, N. (1994). Mitochondrial GrpE is present in a complex with hsp7O and preproteins in transit across membranes. Mol. Cell. Biol. 14. 6627-6634.

192

MICHAEL T. RYAN E r AL.

Voos, W., von Ahsen, O., Miiller, H., Guiard, B., Rassow, J., and Pfanner, N. (1996). Differential requirement for the mitochondrial hsp70-Tim44 complex in unfolding and translocation of preproteins. EMBO J. 15,2668-2677. Wagner, I., Ark, H., van Dyck, L., Langer, T., and Neupert, W. (1994). Molecular chaperones cooperate with PIMl protease in the degradation of misfolded proteins in mitochondria. EMBO J. 13,5135-5145. Waltner, M., and Weiner, H. (1995). Conversion of a non processed mitochondrial precursor protein into one that is processed by the mitochondrial processing peptidase. J. Biol. Chem. 270, 26311-26317. Wand-Wiirttenberger, A., Schoel, B., Ivanyi, J., and Kaufmann, S.H.E. (1991). Surface expression by mononuclear phagocytes of an epitope shared with mycobacerial heat shock protein 60. Eur. J. Immunol. 21, 1089-1092. Wang, N., Gottesman, S., Willingham, M. C., Gottesman, M. M., and Maurizi, M. R. (1993). A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease. Proc. Natl. Acad. Sci. USA 90, 11247-11251. Wang, N., Maurizi, M. R., Emmert-Buck, L., and Gottesman, M. M. (1994). Synthesis, processing, and localisation of human Lon protease. J. Biol. Chem. 269, 29308-29313. Watabe, S., and Kimura, T. (1985a). ATP-dependent protease in bovine adrenal cortex. J. Biol. Chem. 260, 5511-5517. Watabe, S., and Kimura, T. (1985b). Adrenal cortex mitochondrial enzyme with ATPdependent protease and protein-dependent ATPase activities. J. Biol. Chem. 260, 1449814504. Wawrzynbw, A., and Zylicz, M. (1995). Divergent effects of ATP on the binding of the DnaK and DnaJ chaperones to each other, or to their various native and denatured protein substrates. J. Biol. Chem. 270, 19300-19306. Wawrzynbw, A., Wojtkowiak, D., Marszalek, J., Banecki, B., Jonsen, M., Graves, B., Georgopoulos, C., and Zylicz, M. (1995). The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone. EMBO J. 14, 1867-1877. Webster, T. J., Naylor, D. J., Hartman, D. J., Haj, P. B., and Hoogenraad, N. J. (1994). cDNA cloning and efficient mitochondrial import of pre-mtHSP70 from rat liver. DNA Cell Biol. l3,1213-1220. Weissman, J. S., Kashi, Y., Fenton, W. A., and Horwich, A. L. (1994). GroEL-mediated protein folding proceeds by multiple rounds of binding and release of nonnative forms. Cell 78, 693-702. Weissman, J. S., Hohl, C. M., Kovalenko, O., Kashi, Y., Chen, S., Braig, K., Saibil, H. R., Fenton, W. A., and Horwich, A. L. (1995). Mechanism of GroEL-action: Productive release of polypeptide from a sequestered position under GroES. Cell 83, 577-587. Weissman, J. S., Rye, H. S., Fenton, W. A., Beechem, J. M., and Horwich, A. L. (1996). Characterisation of the active intermediate of a GroEL-GroES-mediated protein folding reaction. Cell 84, 481-490. Welch, W. J. (1990). The mammalian stress response: Cell physiology and biochemistry of stress proteins. In “Stress Proteins in Biology and Medicine” (R. I., Morimoto, A. Tissikres, and C. Georgopoulos, Eds.), pp. 223-278. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Welch, W. J., and Feramisco, J. R. (1985). Rapid purification of mammalian 70,000-dalton stress proteins: Affinity of the proteins for nucleotides. Mol. Cell. Biol. 5, 1229-1237. Werner-Washburne, M., Stone, D. E. and Craig, E. A. (1987). Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae. Mol. Cell. Biol. 7, 2568-2577. Westerman, B., Pripp-Bum, C., Neupert, W., and Schwarz, E. (1995). The role of the GrpE homologue, Mgelp, in mediating protein import and protein folding in mitochondria. EMBO J. 14, 3452-3460.

MOLECULAR CHAPERONES IN PROTEIN IMPORT AND FOLDING

193

Westwood, J. T., Clos, J., and Wu, C. (1991). Stress-induced oligomerization and chromosomal relocalization of heat-shock factor. Nature (London) 353, 822-827. Wetzel, I. R. (1 994). Mutations and off-pathway aggregation of proteins. Trends Biotechnol. 12, 193-198. Wickner, R. B. (1994). [URE3] as an altered URE2 protein: Evidence for a prion analog in Saccharamyces cerevisiae. Science 264,566-569. Wickner, S., Gottesman, S., Skowyra, D., Hoskins, J., McKenney, K., and Maurizi, M. R. (1994). A molecular chaperone, ClpA functions like DnaK and DnaJ. Proc. Natl. Acad. Sci. USA 91,12218-12222. Wiedmann, B., Sakai, H., Davis, T. A., and Wiedmann, M. (1994). A protein complex required for signal-sequence-specific sorting and translocation. Nature (London) 370, 434-440. Witte, C., Jensen, R. E., Yaffe, M. P., and Schatz, G. (1988). M A S I , a gene essential for yeast mitochondria1 assembly, encodes a subunit of the mitochondria1 processing protease. EMBO J. 7, 1439-1447. Wojtkowiak, D., Georgopoulos, C., and Zylicz, M. (1993). Isolation and characterisation of ClpX, a new ATP-dependent specificity component of the Clp protease of Escherichia coli. J. Biol. Chem. 268,22609-22617. Wu, C., Clos, J., Giorgi, G., Haroun, R. I., Kim, S-J., Rabindran, S. K., Westwood, J. T., Wisniewski, J., and Yim, G. (1994). Structure and regulation of heat shock transcription factor. In “The Biology of Heat Shock Proteins and Molecular Chaperones” (R. I., Morirnoto, A. Tissittres, and C. Georgopoulos, Eds.), pp. 395-416. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Wiicherpfenning, K. W., Newcombe, J., Li, H., Keddy, L., Cuzner, M. L., and Hafler, D. A. (1992). y/S T-cell reperoire in acute multiple sclerosis lesions. Proc. Natl. Acad. Sci. USA 89,4588-4592. Xu, Q., Schett, G., Seitz, C. S., Hu, Y., Gupta, R. S., and Wick, G . (1994). Surface staining and cytotoxic activity of heat-shock protein 60 antibody in stressed aortic endothelical cells. Circ. Res. 75, 1078-1085. Yang, M., Jensen, R. E., Yaffe, M. P., Oppliger, W., and Schatz, G. (1988). Import of proteins into yeast mitochondria: The purified matrix processing protease contains two subunits which are encoded by the nuclear MAS1 and MAS2 genes. EMBO J. 7,3857-3862. Young, R.A. (1990). Stress proteins and immunology. Annu. Rev. Zmmunol. 8,401-420. Zhong, T., and Amdt, K. T. (1993). The yeast SISl protein, a DnaJ homolog, is required for the initiation of translation. Cell 73, 1175-1186. Zhu, X., Zhao, X., Burkholder, W. F., Gragerov, A,, Ogata, C. M., Gottesman, M. E., and Hendrickson, W. A. (1996). Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272, 1606-1614. Ziegelhoffer, T., Lopez-Buesa, P., and Craig, E. A. (1995). The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydjlp and peptide substrates. J. Biol. Chem. 270, 10412-10419. Zylicz, M., and Georgopoulos, C. (1984). Purification and properties of the Eschericia coli dnaK replication protein. J. Biol. Chem. 259, 8820-8825.