Chapter 7 Transport of Proteins into Mitochondria

CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 24

Chapter 7

Transport of Proteins into Mitochondria GRAEME A . REID' Department of Biochemistry. Biocenter Universitv of Basel Basel, Switzerland

I. Introduction: Mitochondrial Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. An Overview of Mitochondrial Protein Import.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Precursor Polypeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Mitochondrial Import Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c. Translocation and Proctssing . . . . . . . . . . . ....................... D. Assembly of Imported Mitochondrial Proteins . . . . . . . . . . . . . . . . . . . . . . . 111. Are Proteins Transported IV. The Molecular Approach ........................................... A. Isolation and Charac Protein Import. . . . . . . . . . . . .................................. B. Isolation and Charac Mitochondrial Polypeptides . . . . ................................ V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .......................

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INTRODUCTION: MITOCHONDRIAL BlOGENESlS

The eukaryotic cell is physically and biochemically divided into several distinct compartments by the presence of intracellular membranes. The organelles delineated by these membranes perform specialized functions, and this specialization is reflected in their polypeptide compositions: most polypeptides are found exclusively in one particular cellular compartment. How is this highly organized distribution generated? A polypeptide translated in the cytosol must find its way to its ultimate destination, be that in the nucleus, the mitochondrion, the cytosol, or elsewhere. What sort of signals are used to control this traffic? 'Present address: Department of Microbiology, University of Edinburgh, Edinburgh, Scotland.

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NUCLEAR DNA FIG. 1 . Mitochondria1 biogenesis. A few polypeptides are encoded on mitochondrial DNA, but the majority are encoded on nuclear chromosomes, synthesized in the cytosol, and transported to their particular destination within the mitochondrion.

In this article we shall consider the particular case of mitochondrial biogenesis. Mitochondria contain a small, usually circular genome which has been the focus of much interest in recent years (Borst and Grivell, 1978; Tzagoloff et al., 1979; Dujon, 1981). The complete nucleotide sequence of mitochondrial DNA from man and some other mammals has been determined (Anderson et al., 1981, 1982; Bibb et al., 1981). The mitochondrial genome encodes only a small number of polypeptides (about a dozen in yeasts and mammals, more in higher plants). The majority of mitochondrial polypeptides (about 90% by mass in yeast) are encoded on nuclear chromosomes, translated in the extramitochondrial cytoplasm, and transported into the mitochondria (Schatz and Mason, 1974; Schatz, 1979; Neupert and Schatz, 1981). We would like to know how such a large group of different proteins is directed specifically to the mitochondrion. Further sorting must also take place since the mitochondrion is delimited by two membranes (inner and outer) enclosing two distinct aqueous compartments (matrix and intermembrane space). An imported protein must find its way to the correct intramitochondrial compartment (Fig. 1). The mitochondrial membranes present barriers to proteins: How are polypeptides transported across these barriers? And once internalized by the mitochondrion, these polypeptides must assume an active conformation, perhaps by interacting specifically with other polypeptides. Relatively little is known about this final assembly step. Research from many laboratories has contributed much to provide a general outline of the

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ways by which mitochondria import proteins; current research is broadly aimed at elucidating the molecular mechanisms involved.

II.

AN OVERVIEW OF MITOCHONDRIAL PROTEIN IMPORT

Nuclear-coded mitochondria1 polypeptides are synthesized in the cytoplasm as precursor forms, distinguishable from their mature counterparts. The transport process is initiated by specific binding to receptors on the mitochondrial surface, from which the precursors are translocated into or across the mitochondrial membranes. Most precursors then undergo covalent modification. The maturation pathway is completed by assembly of imported polypeptides into biologically active proteins.

A. Precursor Polypeptides The majority of imported mitochondrial proteins are synthesized as larger precursors. When yeast spheroplasts are pulse-labeled for a short time and then subjected to immunoprecipitation with antibodies against particular mitochondria1 proteins, one generally observes a polypeptide which migrates more slowly on SDS-polyacrylamide gel electrophoresis than does the mature polypeptide (Maccecchini et a / . , 1979a). This larger form disappears upon a subsequent chase because it is converted to the mature protein. Such larger precursors can also be found in vitro by isolating mRNA, translating it in a reticulocyte lysate in the presence of a radioactive amino acid, and again performing immunoprecipitation. The size difference between precursor and mature forms varies widely among mitochondrial proteins and can be as much as 10 kDa (see Hay et al., 1983, for an extensive list of larger precursors). In those cases which have been directly investigated, it has been shown that the extra mass in the precursor is due to an N-terminal polypeptide extension. It is likely that N-terminal extensions will be the rule, but whether modifications also occur at the C-terminus remains to be shown. Several imported mitochondrial polypetides are synthesized without N-terminal extensions. Among these are several proteins of the mitochondrial outer membrane (Freitag et a l . , 1982b; Gasser and Schatz, 1983) and a few from other compartments; these include a matrix protein, 2-isopropylmalate synthase (Gasser et al., 1982b; Hampsey et al., 1983), an inner membrane protein, adenine nucleotide translocator (Zimmermann and Neupert, 1980), and cytochrome c , a component of the intermembrane space (Korb and Neupert, 1978; Zimmermann et al., 1979). Since some proteins can reach their final location without N-

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terminal extensions, one may reasonably ask why it is that most imported proteins are made as larger precursors. The fact that the extensions are removed during or shortly after transport suggests that their role is limited to the protein’s biogenesis. It may, for example, be important in providing a “signal” that allows the precursor to be recognized by the mitochondrion. Such signals could be carried entirely within the mature protein sequence in those cases where no larger precursor is made, in a manner analogous to the noncleaved signal sequence of ovalburnin which directs ovalbumin to the endoplasmic reticulum (Meek et al., 1982). It appears that the extramitochondrial precursors of several mitochondria1 proteins are markedly different in conformation from their intramitochondrial mature counterparts. The proteclipid (subunit 9) of Neurospora ATP synthase is an extremely hydrophobic polypeptide, but it is made as a larger precursor in the cytosol. In this case the role of the N-terminal extension may be largely to confer solubility on the protein (Viebrock et al., 1982). A conformational difference between extra- and intramitochondrial forms has been demonstrated with a protein which apparently undergoes no covalent change upon transport into mitochondria. The extramitochondrial precursor of Neurospora adenine nucleotide translocator binds to hydroxyapatite, but it no longer binds after import into mitochondria (Zimmermann and Neupert, 1980). Here the initial translation product is covalently identical to the mature protein, but differs in tertiary structure and location. A very striking example of a conformational difference is seen with the precursor and mature forms of cytochrome c. Antisera raised against apocytochrome c react well with the apocytochrome but not with holocytochrome c. Non-crossreacting antibodies against holocytochrome c could also be raised (Korb and Neupert, 1978). Conformational differences between precursor and mature polypeptides are also suggested by intermolecular associations. Rat ornithine transcarbamylase is a trimeric enzyme with a sedimentation coefficient of 6 S . The in vitro synthesized precursor sediments at 14 S, although the precursor is only 3-4 kDa larger than the mature protein (Miura et al., 1981). It is not known whether the precursor forms large homooligomers or is associated with other polypeptides, nor is it known whether precursor aggregates are biologically important. Aggregates of precursors are found also for the adenine nucleotide translocator (Zirnmennann and Neupert, 1980) and for the a-and P-subunits of the F, component of yeast ATP synthase (A. S . Lewin, S. Ohta, and G . Schatz, unpublished data). The in vivo synthesized precursor of the P-subunit (56 kDa) behaves as a particle of 500 kDa on gel filtration. The mature a-and P-subunits are components of the same enzyme complex, but their precursors behave as distinct species-they can be separated by chromatography on DEAE-cellulose ( S . Ohta, unpublished observations).

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B. Mitochondria1 Import Receptors Only a very specific subset of the proteins synthesized in the cytosol are imported by mitochondria, and proteins destined for mitochondria are not transported across or into other cellular membranes. There must, therefore, be an efficient sorting mechanism allowing interaction of mitochondrial protein precursors with the mitochondrion. If these precursors carry a specific “addressing signal,” there must be a receptor on the mitochondrial surface which recognizes that signal. The best-studied mitochondrial import receptor is that involved in the transport of cytochrome c (Hennig and Neupert, 1981; Hennig er ul., 1983). These detailed studies have exploited the fact that large amounts of precursor proteins can be prepared chemically. Cytochrome c does not undergo proteolytic cleavage upon import into mitochondria, but nevertheless is covalently modified. As with all c-type cytochromes, the mature protein contains a covalently attached heme group; the attachment apparently takes place in the intermembrane space. Thus, the cytochrome c precursor is equivalent to apocytochrome c, which can be prepared by chemical removal of the heme group from mature holocytochrome c. In addition, of course, radioactive apocytochrome c can be prepared in trace amounts by translating mRNA in a cell-free protein-synthesizing system. The apocytochrome c receptor has been detected by its ability to bind radioactively labeled precursor in an in vitro assay. The precursor was synthesized in vitro in a Neurosporu or rabbit reticulocyte cell-free extract in the presence of [35S]methionine,then incubated with isolated mitochondria. Under suitable conditions, the extramitochondrial apocytochrome c was converted to intramitochondrial holocytochrome c. In order to study the binding reaction in the absence of net transport, Hennig and Neupert (198 1) added deuterohemin (which blocks the attachment of heme to apocytochrome c) to the mitochondria before adding precursor proteins. After incubation of in vitro translation products with Neurosporu mitochondria in the presence of deuterohemin, about half of the apocytochrome c was found associated with reisolated mitochondria. Its submitochondrial location was probed by investigating its sensitivity to externally added protease. The labeled apocytochrome c was sensitive to added trypsin whereas endogenous cytochrome c was resistant; the mitochondrial outer membrane should prevent access of the trypsin to cytochrome c in the intermembrane space. Thus, in the presence of deuterohemin, apocytochrome c appears to accumulate on the outer surface of the mitochondrion. Are the sites to which apocytochrome c is bound (1) specific and ( 2 ) relevant to the import pathway? The answer to both questions appears to be yes. First, the apocytochrome c is very tightly bound to the mitochondria but can be released by addition of an excess of chemically prepared apocytochrome c, a finding indicating reversibility as well as specificity of the binding reaction. Second, when the

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inhibitory effect of deuterohemin was reversed by addition of an excess of protohemin, apocytochrome c which had been bound at the outer mitochondrial surface was transported into the mitochondria and converted to the holocytochrome (Hennig et al., 1983). Unlabeled, chemically prepared apocytochrome c has been shown also to compete with labeled, in vitro-synthesized apocytochrome c for import into mitochondria (Hennig et al., 1983). Under conditions where the appearance of labeled cytochrome c in the mitochondria was almost completely blocked, the transport of the adenine nucleotide translocator and of the ATP synthase subunit 9 was unaffected by addition of unlabeled apocytochrome c, a finding suggesting that the latter two proteins do not require the apocytochrome c receptor for entry into mitochondria (Zimmermann et al., 1981). The binding of precursors other than cytochrome c to receptors on the mitochondrial surface has been described (Zwizinski et al., 1983; Riezman et al., 1983b). As described in Section II,C, the transport of precursor proteins into or across the mitochondrial inner membrane requires a transmembrane electrochemical potential difference. When “energization” of the inner membrane is blocked by inhibitors of oxidative phosphorylation, precursor polypeptides become associated with the external surface of the mitochondrion. This binding is tight; precursors remain bound during thorough washing of the mitochondria. When these mitochondria are reenergized, the surface-bound precursor becomes internalized. These experiments suggest that the precursor-binding sites can be used for import into the mitochondria (Zwizinski et al., 1983; Riezman et a f . , 1983b). Furthermore, it appears that transport occurs directly from these sitesthe precursor does not dissociate from the mitochondrial surface before translocation. This was shown by importing the mitochondria-bound precursor of Neurospora adenine nucleotide translocator at various dilutions of the mitochondria. No effect of concentration was observed, indicating that import occurs directly from the bound state (Zwizinski er af., 1983). To look in more detail at the precursor binding reaction in the absence of net transport, Riezman et al. (1983b) developed a precursor binding assay with yeast mitochondrial outer membrane vesicles. The isolated vesicles were shown to be sealed and to have the same orientation as outer membrane in intact mitochondria (Riezman et al., 1983a); components normally exposed on the outer mitochondrial surface are also exposed on the outer face of the outer membrane vesicles. The isolated vesicles bind labeled, in vitro-synthesized precursors of mitochondrial proteins. The binding activity is specific to the outer membrane: isolated mitochondrial inner membrane has little or no capacity to bind cytochrome b, precursor. Binding is specific for those proteins destined to be transported into mitochondria: in vitro-synthesized glyceraldehyde-3-phosphatedehydrogenase and hexokinase, two cytosolic proteins, are not bound. Binding to outer membrane vesicles is specific for the precursor forms of mitochondrial proteins:

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proteolytically processed precursors do not bind. Moreover, mature cytochrome b, does not compete with precytochrome b, for binding. The binding activity must be, at least in part, governed by one or more polypeptides of the outer membrane. When the outer membrane vesicles are treated with trypsin under mild conditions, the ability of the vesicles to bind mitochondrial protein precursors is dramatically reduced. When intact mitochondria are similarly subjected to protease treatment, they lose the ability to import proteins. This correlation further suggests that these protease-sensitive binding sites are “import receptors.” One would like to know much more about the properties of this receptor. In particular, is this a general receptor for a large class of imported proteins or is its specificity more restricted? If this receptor is shared by many precursor polypeptides, one would expect that these precursors would compete for binding to the same sites. The necessary experiments have so far been largely impossible for technical reasons. To demonstrate competition one would first need to isolate large amounts of a purified precursor in its native state, as was possible in the special case of cytochrome c (see above). Those precursors which have Nterminal extensions are less readily purified, but work in this direction has begun (see Section IV,A,l). A remarkable feature of the mitochondrial protein import system is that the import receptors and other components of the transport machinery must themselves be imported from the cytoplasm. This is clearly so since rho- yeast, which are unable to synthesize proteins in the mitochondria, still import proteins into mitochondria: the necessary catalysts must therefore be present. It will be interesting to investigate the molecular details of how receptor precursors are recognized: Does a receptor recognize its own precursor?

C. Translocation and Processing Once bound to a receptor site on the mitochondrial surface, precursor polypeptides must be transported into or across the mitochondrial membranes. Since the mitochondrion is composed of four distinct compartments, it is perhaps not surprising that different import pathways exist, though we do not yet know how many, nor how they are organized. We can, at the moment, consider proteins imported to the inner membrane and matrix as one group and outer membrane proteins as another; apparently at least two routes are used to transport proteins to the intermembrane space. 1. ENERGY-DEPENDENT IMPORT Transport of proteins into or across the mitochondrial inner membrane is energy dependent. This was indicated by Nelson and Schatz (1979), who pulse-

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labeled yeast spheroplasts in the presence and absence of various energy poisons. They then analyzed the radioactive polypeptides by immunoprecipitation and gel electrophoresis. In the absence of inhibitors, radioactivity appeared in the mature forms of the a-,(3-, and y-subunits of the F,-ATPase and two subunits of the cytochrome bc, complex (ubiquino1:cytochrome c reductase). When CCCP, an uncoupler of oxidative phosphorylation, was present during the labeling, radioactivity remained in the larger precursor forms of these polypeptides. Precursors accumulated in the presence of CCCP are outside the mitochondrion (Reid and Schatz, 1982b). The nature of the energy dependence of polypeptide import has been investigated in more detail using an in vitro transport assay. These experiments involved synthesis of precursor polypeptides in vitro in a reticulocyte lysate in the presence of [35S]methionine, followed by incubation of the labeled precursors with isolated mitochondria. After the incubation, mitochondria were reisolated from the suspension by centrifugation, and polypeptides in the supernatant and in the mitochondrial pellet were examined by immunoprecipitation and gel electrophoresis. Thus, it could be determined whether particular polypeptides were present as their mature or larger precursor forms. To determine whether these polypeptides were inside or outside the mitochondria, their sensitivity to externally added protease was examined; polypeptides internalized by mitochondria should be inaccessible to the protease because of the physical barrier imposed by the membranes. The import of the P-subunit of F,-ATPase was shown to be completely dependent on the presence of ATP or a substrate for respiration (Gasser et al., 1982a). In the presence of such an “energy source,” import of ornithine transcarbamylase into rat liver mitochondria (Mori et af., 1981b; Kolansky et af., 1982), the adenine nucleotide translocator, ATP synthase subunit 9 (Schleyer et af.,1982), and four subunits of the cytochrome bc, complex (Teintze et al., 1982) into Neurospora mitochondria, and several polypeptides into yeast mitochondria (Gasser et al., 1982a) could be demonstrated. In all cases import was blocked by uncouplers of oxidative phosphorylation. Uncouplers dissipate the electrochemical gradient of protons across the mitochondrial membrane, but a secondary effect of this is to stimulate ATP hydrolysis by the reverse reaction of the H -translocating ATP synthase, thereby depleting the mitochondrial matrix of ATP. One would like to know whether ATP drives transport directly or whether a transmembrane electrochemical potential is required. By investigating in vitro import in the presence of various inhibitors, Schleyer et al. (1982) and Gasser et af. (1982a) were able to answer this question. Gasser et af. (1982a) synthesized precursors in a reticulocyte lysate, then separated the precursors and other proteins from small molecules (including ATP and respiratory substrates) by gel filtration. These precursor polypeptides were then incubated with isolated yeast mitochondria in the presence of KCN (to inhibit endogenous respiration) and ATP. Under these condi+

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tions a large fraction of the precursor to the P-subunit of F,-ATPase was transported into the mitochondria. This import was entirely dependent on added ATP and could be blocked by addition of either carboxyatractyloside or oligomycin. Carboxyatractyloside inhibits the adenine nucleotide translocator, thus blocking the entry of added ATP into the mitochondria. Oligomycin inhibits the proton-translocating ATPase. The inhibition of protein import by these compounds indicates that the added ATP must enter the mitochondrion and be hydrolyzed by the F,F,-ATPase. Since oligomycin inhibits hydrolysis of ATP by the ATPase, it should increase the ATP concentration in the mitochondrial matrix. The fact that oligomycin blocks protein import already suggests that ATP is not the direct source of energy for translocation. Indeed, the inhibitory effect of oligomycin could be overcome by presenting the mitochondria with a substrate for respiration, thus restoring a transmembrane electrochemical gradient. Whether supported by respiration or by ATP hydrolysis, the ability of mitochondria to import proteins always correlated with conditions where the electrochemical potential gradient would be expected to be relatively large, regardless of the ATP concentration in the matrix. Schleyer et ul. (1982) reached the same conclusion when investigating the transport of proteins into Neurosporu mitochondria. Protein import was blocked by a combination of oligomycin and antimycin, an inhibitor of electron transfer through the cytochrome bc, complex. Under these conditions, both respiration and ATP hydrolysis would be inhibited. Protein import was restored by addition of ascorbate and tetramethylphenylenediamine(TMPD), thereby allowing reduction of cytochrome c and the subsequent regeneration of a proton electrochemical potential gradient by the activity of cytochrome c oxidase. The ATP concentration should be unaffected by the presence of ascorbate and TMPD, but one would expect a significant difference in the electrochemical potential gradient in the presence and absence of this substrate combination. Thus, it appears that import of proteins to the mitochondrial matrix and inner membrane is dependent on an electrochemical gradient across the inner membrane, contrary to the initial suggestion of Nelson and Schatz (1979) that molecular ATP is the energy source. This suggestion was made partly because a mitochondrial petite (rho-) yeast mutant, which lacks a functional ATP synthase and respiratory chain, was able to import proteins in the absence but not in the presence of bongkrekate, an inhibitor of adenine nucleotide transport. In the presence of this compound ATP cannot enter the mitochondria. It was assumed that no significant electrochemical potential gradient across the mitochondrial inner membrane could be generated in this mutant since proton translocation by respiration and by ATP hydrolysis are absent. However, the movement of adenine nucleotides across the mitochondrial inner membrane is itself an electrogenic process and could thus generate a significant potential difference, albeit probably only about one-fourth of that found in normal respiring mitochondria. Despite the smaller potential

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difference, rho- mitochondria can still import proteins, a finding suggesting that a smaller electrochemical potential difference across the inner membrane is required for protein transport than for ATP synthesis. Several findings show that translocation rather than proteolytic processing of precursors is the energy-dependent step in protein import. First, the import of proteins without larger precursors into the mitochondrial matrix (e.g., 2-isopropylmalate synthase; Gasser et al., 1982b; Hampsey et al., 1983) or the inner membrane (e.g., adenine nucleotide translocator; Schleyer et al., 1982) requires energy, though obviously no proteolytic processing occurs. Second, processing is still catalyzed by a partially purified matrix protease (cf. below). Third, the transport and processing of cytochrome c peroxidase are temporally separated (cf. below; Reid et al., 1982); the initial transport step is blocked by CCCP, whereas subsequent processing is not. The transport of cytochrome c into mitochondria does not require a transmembrane electrochemical potential gradient (Zimmermann et al., 1981). Cytochrome c is a component of the intermembrane space and presumably the mitochondrial inner membrane is not directly involved in the import of this protein. The insertion of proteins into the mitochondrial outer membrane similarly lacks an energy requirement (see Section II,C,4). Thus, the requirement for an energized inner membrane is found only for those proteins which are transported into or across this membrane. As described above, we now know that the transport of proteins into the mitochondrial matrix and inner membrane requires an “energized” inner membrane, but we do not know what the electrochemical potential gradient is needed for. The effect may be essentially electrophoretic, with transport being initiated by the movement of a cluster of positively charged residues of a precursor polypeptide toward the more electronegative mitochondrial matrix. It appears that the N-terminal regions of those precursors so far investigated are predominantly basic in nature (see Section IV,B). Alternatively, the energy requirement may be less direct-indeed we do not know whether energy is actually consumed during transport. The electrochemical gradient could conceivably be required to maintain a state compatible with translocation, perhaps involving the membrane lipid conformation, as suggested by Schatz and Butow (1983), or protein conformation. It will be difficult to analyze how energy facilitates protein transport using whole mitochondria. More detailed analyses may eventually be possible with reconstituted components of the import machinery. OF IMPORTED PRECURSORS 2. PROTEOLYTIC PROCESSING

Those mitochondrial polypeptides initially synthesized as larger precursors must at some stage during their maturation be processed to their mature size. Pulse-labeling and pulse-chase labeling experiments indicated that this is gener-

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ally a rapid process: the half-life of the precursor of the P-subunit of F,-ATPase in yeast is about 0.5 minute (Reid and Schatz, 1982b) and rat carbamylphosphate synthase precursor disappears with a half-life of approximately 2 minutes (Raymond and Shore, 1981). This half-life reflects the overall process of transport and porteolytic cleavage. Is the precursor first transported and then cleaved, or the other way around? Since the processing protease is found in the mitochondria1 matrix (Bohni et al., 1980), at least part of the precursor must be translocated before cleavage takes place. Proteins translocated across the endoplasmic reticulum and the Escherichia coli plasma membrane can be proteolytically processed while they are being transported; since transport in these cases is at least partly cotranslational, the existence of processed nascent polypeptides showed this temporal relationship quite clearly. It is not known whether imported mitochondrial proteins can be processed during their translocation. If processing takes place after completion of translocation, one might expect to find intramitochondrial precursors. In all but one case these were not detectable, so processing must at least occur very soon after transport (Reid and Schatz, 1982b). The exception to this rule is cytochrome c peroxidase, which is processed over a period of many minutes following its import into mitochondria (Reid et al., 1982). This temporal separation of the transport and processing steps clearly demonstrates that they are not obligately coupled: Proteolytic cleavage is not required for translocation. This is also shown by the fact that several imported precursors have no N-terminal extension. 3. IMPORTOF PROTEINS TO THE INTERMEMBRANE SPACE: TWO-STEPPROCESSING

Proteins destined for the mitochondrial matrix clearly must traverse two membranes during import, but to reach the intermembrane space it would appear that only the outer membrane must be crossed. Surprisingly, the transport of at least some proteins to the intermembrane space is rather more complicated than initially imagined. Cytochrome b, and cytochrome c peroxidase are soluble proteins of the yeast mitochondrial intermembrane space (Daum et al., 1982a). Cytochrome c , is attached to the mitochondrial inner membrane as a component of the cytochrome bc, complex, but its bulk protrudes into the intermembrane space, where it interacts with cytochrome c (Li et al., 1981). It may be considered a component of the intermembrane space, particularly as its import shares many features with the import of cytochrome b, and cytochrome c peroxidase. Each of these three proteins is initially made outside the mitochondrion as a larger precursor (Gasser er al., 1982b; Maccecchini et al., 1979b; Nelson and Schatz, 1979) and subsequently imported into the mitochondrion. The import of each of these polypeptides was found to be energy dependent. Pulse-labeling of yeast spheroplasts in the presence of CCCP resulted in accumulation of label in

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the precursor form of cytochrome c, (Nelson and Schatz, 1979). Similarly, the maturation of cytochrome b, and cytochrome c peroxidase was blocked by CCCP in intact yeast cells (Reid et al., 1982). The energy requirement for the import of cytochrome b, has also been investigated in vitro (Gasser et al., 1982a,b; Daum et al., 1982b) and, as with proteins transported to the matrix and inner membrane, an electrochemical potential gradient across the inner membrane is needed. Thus, the inner membrane apparently has a functional role in the transport of proteins to the intermembrane space. When yeast proteins synthesized in a reticulocyte lysate are incubated with the partially purified processing protease from the mitochondrial matrix (Section IV,A), the precursors of mitochondrial matrix and inner membrane proteins are processed to the corresponding mature polypeptides (Bohni et al., 1983; Cerletti et al., 1983). This protease does not digest any nonmitochondrial protein tested. It converts the cytochrome b, precursor (68 kDa), not to the mature size (58 kDa) but to an intermediate-size form (64 ka). This could, of course, be an in vitro artifact; indeed it was not initially expected that cytochrome b, precursor on its way from the cytosol to the intermembrane space would ever become accessible to a protease in the mitochondrial matrix. That the intermediate form is biologically significant has been shown by pulse-labeling of intact yeast cells. The cells were pulse-labeled with [35S]methioninein the presence of 20 p M CCCP, under which conditions import of cytochrome b, is blocked and all the pulse-labeled cytochrome b, is in the precursor form. When CCCP is inactivated with 2mercaptoethanol (Kaback et al., 1974) and the cells are then chased with unlabeled methionine, the accumulated cytochrome b, precursor is transported into the mitochondria. During this import, the precursor is first converted to the intermediate form and then to the mature form of the cytochrome (Fig. 2; Reid et al., 1982). The intermediate form of cytochrome b, is also found during in vitro import into mitochondria, and again it behaves as a kinetic intermediate between the precursor and mature forms (Daum et al., 1982b). The precursor of cytochrome b, is synthesized as a soluble polypeptide outside the mitochondrion (Reid and Schatz, 1982a), and the mature polypeptide is a soluble component of the intermembrane space (Daum et al., 1982a; Reid et al., 1982). The intermediate, in contrast, is firmly attached to the mitochondrial inner membrane, apparently with most of its bulk exposed to the intermembrane space. Since the precursor is cleaved on the matrix side of the inner membrane, the intermediate generated by this cleavage is presumably transmembranous. The same location was found for pulse-labeled cytochrome c peroxidase precursor which had already been imported to the mitochondrion (Reid et al., 1982). This precursor undergoes very slow processing by the matrix protease, and no intermediate form was observed in these experiments, although cytochrome c peroxidase probably does share the two-step processing pathway with cytochrome b, (see Section IV,B).

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FIG. 2. Cytochrome b2 precursor is converted first to an intermediate form, then to mature cytochrome b2 in intact yeast cells. Labeled cytochrome b2 was accumulated by pulse-labeling yeast cells in the presence of CCCP. The uncoupler was inactivated by addition of 2-mercaptoethanol, and the maturation of cytochrome b2 was examined after various periods of chase (indicated above each lane, in minutes). The immunoprecipitated cytochrome b2 was analyzed by SDS-polyacrylamide gel electrophoresis (Reid er ul.. 1982). Lanes P and M contain precursor and mature cytochrome h Z , respectively,

The import and maturation of cytochrome c 1 follows a pathway similar to that of cytochrome b,. The in vitro-synthesized precursor is processed to an intermediate-size form by the matrix-located protease (Gasser et al., 1982b; Ohashi et al., 1982) and the orientation of the cytochrome c 1 intermediate is the same as that found for the cytochrome b, intermediate: attached to the inner membrane with its bulk protruding into the intermembrane space (Ohashi et al., 1982). During its maturation, cytochrome c, undergoes covalent attachment of heme. Ohashi er a / . (1982) investigated when this reaction takes place in relation to the proteolytic maturation steps. This was achieved using a heme-deficient yeast mutant, lacking 5-aminolevulinate synthase. When this mutant was pulse-labeled with [35S]methioninein the absence of heme precursors and cytochrome c 1 was subsequently immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis, it was found that the intermediate polypeptide was accumulated. When these pulse-labeled cells were chased with unlabeled methionine in the presence of 5-aminolevulinic acid, the accumulated intermediate form was converted to mature cytochrome c1. Thus, the second processing step in cytochrome c, maturation is dependent on the availability of heme. The two-step processing pathway involved in the maturation of cytochrome b, and cytochrome cI in yeast is summarized in Fig. 3. Cytochrome c1 has also been shown to be imported into

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pre-ryt

b2

(,

-naow&v pre-ryt

CYTOPLASM

*c

MATRIX

IM

FIG.3. Suggested pathway for the maturation of cytochrome b2 and cytochrome cI in yeast. The zigzag line signifies the N-terminal peptide extension of the precursors and the arrows signify proteolytic cleavages. Noncovalently and covalently bound heme are represented by an open halfcircle and a box, respectively. The first proteolytic cleavage of each precursor generates a new Nterminus (N’). Cleavage of the membrane-bound intermediate is presumed to occur near the outer face of the inner membrane and generates the N-terminus of the mature protein (N”). This second cleavage releases cytochrome b2 in a soluble form into the intermembrane space. In contrast, cytochrome cI remains attached to the inner membrane by its hydrophobic C-terminus (Wakabayashi et al., 1980).

Neurospora mitochondria by a two-step pathway (Teintze et al., 1982). In vitro import of cytochrome c, has not been demonstrated in yeast, probably because the precursor is relatively unstable (Reid and Schatz, 1982a), but the corresponding Neurosporu polypeptide is imported into mitochondria in an energy-dependent manner (Teintze et ul., 1982). The import of cytochrome c, is not blocked

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by an excess of apocytochrome c. indicating that these two hemoproteins are transported by different pathways. Little is known about the second proteolytic step in the two-step processing pathway. It is not clear whether a single enzyme catalyzes the conversion of each intermediate to the corresponding mature polypeptide, though this at present would be the most attractive possibility. No specific inhibitors of the second processing step have been found despite extensive searching, though the activity is sensitive to the detergent digitonin (Daum eta/. , 1982b). It has been suggested from in vitro experiments that the conversion of intermediate to mature cytochrome b, does not require an energized mitochondrial inner membrane (Daum et al., 1982b), but apparently the conversion of cytochrome c , intermediate to mature protein was inhibited by CCCP in Neurospora cells. The significance of these possibly contradictory results is not clear. A two-step processing pathway has also been proposed for a mitochondrial matrix protein (Mori et al., 1980), but recent findings suggest that the observed intermediate-size polypeptide may be an artifact. Rat liver ornithine transcarbamylase (OTC) is synthesized as a larger precursor (Conboy et al., 1979) and can be imported into mitochondria in vitro. Incubation of in vitro-synthesized precursor with isolated mitochondria leads to the formation, not only of a mature-size polypeptide, but also a form of OTC intermediate in size between the precursor and mature polypeptides (Mori et a/., 1980, 1981b; Morita et al., 1982a,b; Conboy and Rosenberg, 1981; Kraus et al., 1981; Kolansky et al., 1982). The conversion of the intermediate-size form of OTC to the mature protein has not, however, been demonstrated. Of some concern is the inability to detect the intermediate-size form of OTC in pulse-chase labeled, intact hepatocytes (Mori et al., 1981a; Morita et al., 1982b), suggesting the possibility that this polypeptide is an in virro artifact. Indeed, at least some of the intermediate-size form is found outside the mitochondria after incubation with in vitrosynthesized precursors (Kolansky et al., 1982), though the processing protease is found in the mitochondrial matrix (Mori et al., 1980; Miura et al., 1982a). The conversion of OTC precursor to the intermediate-size and to the maturesize polypeptide is sensitive to 1,lO-phenanthroline and other chelators of divalent metal ions, but it has recently been suggested that different enzymes are responsible for each of these proteolytic cleavages (Conboy et al., 1982). The conversion of precursor to mature OTC was found to be greatly enhanced by addition of Zn2 or Co2 , and the enzyme catalyzing this reaction was identified as a component of the mitochondrial matrix. This protease is probably analogous to that initially described by Bohni et al. (1980). The activity generating the intermediate-size polypeptide had a somewhat different submitochondrial distribution and metal-ion requirement; whether its activity is physiologically significant remains to be shown. This latter enzyme has been purified (Miura el al., 1982a) and found not to process the precursor of carbamoyl-phosphate +

+

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synthase I which, like OTC, is imported to the mitochondrial matrix. The significance of these findings is not yet clear.

4. BIOCENESISOF THE MITOCHONDRIAL OUTERMEMBRANE In contrast to our extensive knowledge of the functions of the mitochondrial matrix and inner membrane, we know rather little of the biochemistry of the outer membrane. Some enzyme activities, such as kynurenine hydroxylase, are associated with this membrane and a pore function has recently been ascribed to a major polypeptide component (Zalman et af.,1980). However, the functions of most of the major polypeptides are currently unknown, and they are therefore described by their apparent size according to their mobility on polyacrylamide gels. It is clear, though, that the outer membrane must be important in the communication of mitochondria with the rest of the cell: it must contain receptors for proteins imported by mitochondria and possibly also components which interact with the cytoskeleton. The insertion of proteins into the mitochondrial outer membrane, unlike the transport of proteins into or across the inner membrane, does not require an electrochemical potential difference across the inner membrane, nor does it require ATP. The major polypeptides of the outer membrane [with one possible exception (Shore et a f . , 1981)] appear not to undergo proteolytic processingthey are synthesized without transient N-terminal extensions. These features indicate that the mitochondrial outer membrane proteins are transported by a pathway rather different from that (or those) involved in transport to the interior compartments of the mitochondrion. The mitochondrial outer membrane is permeable to solutes of molecular mass up to 2000-8000 (Pfaff et al., 1968; Colombini, 1979). This pore activity has been shown to reside with the most predominant band in Coomassie blue-stained SDS-polyacrylamide gels of outer membranes. This polypeptide has a molecular weight of 30,000 in rat and mung bean mitochondria and 3 1,000 in Neurospora (Zalman et al., 1980; Freitag et al., 1982a). The major polypeptide of yeast mitochondrial outer membrane has a molecular weight of 29,000 and presumably this, too, is the pore protein, which has been termed porin by analogy to a group of proteins which are found in the outer membranes of gram-negative bacteria and similarly act as nonspecific pores (Osborn and Wu, 1980). The biosynthesis of this polypeptide and its insertion into the outer membrane have recently been investigated. Mitochondria1 porin is synthesized almost exclusively on free polysomes, i.e., not on membrane-bound polysomes (Freitag et al., 1982b; Suissa and Schatz, 1982). It thus appears that it is inserted into the outer membrane posttranslationally. The initial translation product of porin mRNA has the same mobility on SDS-polyacrylamide gels as the mature polypeptide (Freitag et al., 1982b; Mihara et al., 1982; Gasser and Schatz, 1983). The N-terminal meth-

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ionine residue remains with porin after its insertion into the membrane. Thus, no proteolytic processing appears to be necessary for the biogenesis of this protein, nor apparently for at least three other outer membrane polypeptides (Gasser and Schatz, 1983). When Neurospora or yeast proteins were synthesized in a reticulocyte lysate in the presence of [35S]methionineand incubated with mitochondria in the absence of further protein synthesis, most of the radioactive porin was found associated with the reisolated mitochondria. Whereas the soluble precursor was sensitive to added proteases, the porin associated with the mitochondria was resistant to digestion with trypsin or proteinase K (Freitag et al., 1982b; Mihara et af., 1982; Gasser and Schatz, 1983). The authentic, endogenous porin in isolated mitochondria is similarly protease-resistant. The in vitro-synthesized porin inserted not only into isolated mitochondria but also into isolated outer membrane which was essentially free of inner membrane components (Gasser and Schatz, 1983). Again the membrane-associated porin became protease resistant, a result suggesting that it had inserted into the outer membrane. The insertion of porin into isolated outer membrane vesicles indicates that its translocation is not dependent on a bulk potential across the inner membrane; no such potential can be generated across the outer membrane since small ions can readily diffuse through the pore. In agreement with this conclusion, the insertion of porin into mitochondria is not inhibited by CCCP or valinomycin under conditions where transport to the matrix and inner membrane are blocked (Freitag et al., 1982b; Gasser and Schatz, 1983). When porin is imported into Neurospora mitochondria in v i m at 25"C, essentially all of the labeled precursor quickly becomes associated with the mitochondria and immediately becomes resistant to protease. When the incubation is carried out at 4"C, protease resistance develops relatively slowly so that at early time points a large fraction of the porin associated with the mitochondria is in a protease-sensitive conformation; presumably it has not yet inserted into the outer membrane (Freitag et al., 1982b). The sites to which porin initially binds are probably different from those involved in binding and import of cytochrome b, and the P-subunit of F,-ATPase (Gasser and Schatz, 1983; Riezman et al., 1983b). When mitochondria are mildly treated with trypsin, they lose the ability to bind and import these two polypeptides, but the insertion of porin is unaffected. The binding sites for porin must, however, be specific to the mitochondria] outer membrane: whereas in vitro-synthesized porin will insert posttranslationally into isolated outer membrane, it does not insert into isolated endoplasmic reticulum membranes. This result contradicts the suggestion that the mitochondrial outer membrane is formed by differentiation of the endoplasmic reticulum (Shore, 1979). This membrane-specific, posttranslational insertion was also demonstrated with a 7O-kDa, a 45-kDa, and a 14-kDa polypeptide of the yeast mitochondria1 outer membrane (Gasser and Schatz, 1983). It was not possible to

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show whether these proteins inserted correctly into the outer membrane, and this remains a major drawback in interpreting the above findings. To overcome this, detailed comparison with the orientation of the in vivo-synthesized polypeptides would be required, but little is currently known of the architecture of the outer membrane. In contrast to the findings described above, Shore et al. (198 1) reported that a 35-kDa polypeptide from rat liver mitochondria outer membrane is intially made as a slightly larger (35.5 kDa) precursor. It is not known whether this apparent difference in size is due to N-terminal processing; the results of Gasser and Schatz (1983) would suggest that this is unlikely. This 35.5-kDa polypeptide was shown to be synthesized on free polysomes and was posttranslationally imported into mitochondria in vitro (Shore et al., 1981).

D. Assembly of Imported Mitochondria1 Proteins Once imported into the mitochondrion and proteolytically or otherwise matured, a polypeptide must assume its active conformation. In many cases this involves specific interactions with other subunits, either homologous or heterologous. The assembly of a miltisubunit enzyme, such as cytochrome c oxidase, might be expected to follow a defined pathway, in which case assembly intermediates containing some but not all subunits might be formed. The techniques for detecting such intermediates have not yet been adequately developed. It is possible to investigate assembly in the absence of mitochondria1 protein synthesis either in a rho- yeast strain (Schatz, 1968) or by using specific inhibitors (e.g., de Jong et al., 1979), but such studies have not revealed specific assembly intermediates. By using the methods of in vitro mutagenesis, it should now be possible to alter a specific subunit of a complex such that assembly is no longer completed. One could then determine whether the remaining subunits become associated. Furthermore, one could isolate second-site revertants which allow assembly of the mutated polypeptide. Such reversions could be due to compensating mutations in another subunit of the complex, thus indicating an interaction between the subunits at some step in assembly; or one may perhaps find mutations in other components required for assembly: we do not know whether assembly requires catalysts. The molecular details of the assembly process will no doubt be examined in v i m , but a major difficulty so far has been to demonstrate that polypeptides imported into mitochondria in vitro become assembled into biologically active units, attaining all the characteristics of the corresponding protein in the living cell. Three approaches have been taken to determine whether this is indeed the case. The questions posed by these investigations are as follows: Does an imported protein reach the correct submitochondrial location? Does it assume a three-

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dimensional structure comparable to that of the normal protein'? Does it become biologically active'? After import of in vitro-synthesized precursors into isolated mitochondria, Gasser et ul. (1982b) examined the submitochondrial distribution of various proteins. Imported cytochrome b, was found in the intermembrane space and not in the matrix, while 2-isopropylmalate synthase was found in the matrix fraction, not in the intermembrane space. These results reflect the distribution of the unlabeled mature enzymes within the mitochondrion. However, a disproportionately large amount of the labeled, imported polypeptides was isolated with the mitochondria1 membranes, for reasons which remain to be clarified. The membrane-bound forms may represent intermediate steps on the maturation pathways of these proteins (Gasser et ul., 1982b). Imported membrane proteins were found exclusively in the membrane fraction. Do in vitro-imported polypeptides assume the same conformation as the in vivo-synthesized protein? Evidence that they do indeed has come from studies of two enzymes. The adenine nucleotide translocator of Neurosporu is specifically inhibited by carboxyatractyloside, which binds tightly to the protein. When the inhibitor is present, the translocator protein does not bind to hydroxyapatite, whereas the soluble precursor does. Upon import into isolated mitochondria, however, the in virro-synthesized translocator no longer binds to hydroxyapatite in the presence of carboxyatractyloside, a finding suggesting that it has acquired the ability to bind this inhibitor (Schleyer and Neupert, 1984). The imported and processed form of ornithine transcarbamylase, but not the precursor, binds a transition state analog of carbamoyl phosphate, as does the active enzyme, thus indicating that the active conformation has been reached. Furthermore, the imported protein comigrates with the active trimeric mature protein on a gel filtration column (L. Rosenberg, personal communication). It has been suggested that in virro import of rat carbamoyl-phosphate synthase (Campbell et ul., 1982) and yeast phenylalanyl-tRNA synthase (Diatewa and Stahl, 1981) leads to new enzymatic activity. Further experimentation is required to clarify this point, the main problems being to find a system with a sufficiently low background activity and to import sufficient precursor to generate detectable enzyme activity.

111. ARE PROTEINS TRANSPORTED INTO MITOCHONDRIA COTRANSLATIONALLY OR POSTTRANSLATIONALLY?

In a series of reports, Kellems, Allison, and Butow (Kellems and Butow, 1972, 1974; Kellems et ul.. 1974, 1975) described cytoplasmic-type, 80 S ribosomes bound to the surface of yeast mitochondria. These bound ribosomes

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were observed in spheroplasts and remained attached to mitochondria during isolation of the organelle. The interaction between these bound polysomes and the mitochondrial surface showed features remarkably similar to the binding of ribosomes to the rough endoplasmic reticulum. In particular, the ribosomes could be released from the mitochondria by a combination of puromycin and concentrated salt, but by neither condition alone. This finding strongly indicates that the ribosome-membrane binding is mediated by nascent polypeptides, as is the case with ribosomes bound to the endoplasmic reticulum. On the basis of these results it was proposed that transport of proteins into mitochondria is a cotranslational process: the mitochondria-bound ribosomes were considered to be directly involved in the translocation of nascent mitochondrial polypeptides across the membrane(s). It has since been clearly demonstrated that there is no obligate coupling of protein translocation across mitochondrial membranes to protein synthesis. Mitochondrial polypeptides can be synthesized in vitro by translating purified RNA in a homologous (Harmey et a l . , 1977) or heterologous (Maccecchini et a l . , 1979a) protein synthesizing system, and the finished precursor polypeptides can subsequently be imported by isolated mitochondria. The import reaction is unaffected by cycloheximide, a potent inhibitor of polypeptide chain elongation on 80 S ribosomes. Such experiments show unambiguously that polypeptides can be imported into mitochondria posttranslationally, at least in vitro. Posttranslational import has also been described in vivo in yeast. The import of most mitochondrial proteins is energy dependent and can be conveniently inhibited by CCCP, an uncoupler of oxidative phosphorylation. Yeast spheroplasts pulse-labeled with [35S]methioninein the presence of CCCP accumulate labeled precursors of mitochondrial proteins-no significant conversion to the mature proteins is observed (Nelson and Schatz, 1979). On subcellular fractionation, these labeled precursors were found outside the mitochondria (Reid and Schatz, 1982b). When the effects of CCCP were abolished by addition of 2-mercaptoethanol, the extramitochondrial precursor of the P-subunit of F, -ATPase was imported into the mitochondria and converted to the mature protein. The chase was performed in the presence of an excess of unlabeled methionine, so that labeled precursor was synthesized only during the pulse. Again the transport and maturation of this polypeptide was not inhibited by cycloheximide. These experiments demonstrated that mitochondrial protein import can occur posttranslationally even in vivo. The experiments described above relate to nonphysiological conditions, but we would like to know the sigificance of posttranslational transport into the mitochondria in a growing cell. If mitochondrial protein import is indeed posttranslational, one might expect to find pools of extramitochondrial precursors awaiting import. Such pools were first detected by double isotope pulse-chase experiments in Neurosporu (Hallermayer et a l . , 1977). These experiments dem-

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onstrated that the appearance of pulse-labeled proteins in the mitochondria lagged behind the labeling of proteins in other cellular fractions. Incorporation of radioactive amino acids into transported proteins was rapidly stopped by addition of cycloheximide, but labeled proteins continued to appear in the mitochondrial fraction, a finding indicating transport of previously synthesized polypeptides into the organelle. In similar experiments with yeast, Ades and Butow (1980a) were unable to detect a lag in the labeling of mitochondrial proteins, a result suggesting the absence of large extramitochondrial pools of polypeptides awaiting transport. This finding does not exclude the possible existence of such pools but does define an upper limit for the pool size under the experimental conditions employed. The sensitivity of the experiments of Hallermayer et al. (1977) and of Ades and Butow (1980a) was limited by the fact that the radioactivity in immunoprecipitable mitochondrial proteins was not determined separately for precursor polypeptides and their mature forms. By fractionation of pulse-labeled yeast, it could indeed be demonstrated that extramitochondrial pools of precursors exist (Reid and Schatz, 1982b). Interestingly the pool size depends upon physiological conditions; this may at least partly explain the inability of Ades and Butow (1980a) to detect such pools. One might expect the size of an extramitochondrial precursor pool to depend on the rate of precursor synthesis and on the rate of precursor transport into mitochondria. By lowering the rate of protein synthesis with cycloheximide during pulse-labeling of intact yeast cells, the pool of the precursor to the P-subunit of F,-ATPase was lowered as well (Reid and Schatz, 1982b). Since mitochondrial protein import is probably unaffected by cycloheximide, the fewer precursor molecules being synthesized should spend less time in the cytosol. If mitochondrial protein import is indeed posttranslational in vivo, what is the function of mitochondria-bound 80 S ribosomes'? That they may have a function was suggested by analysis of the polypeptides being synthesized on these polysomes. Ades and Butow (1980b) examined the synthesis of the three largest subunits of F,-ATPase in a readout system where the nascent chains on polysomes are synthesized to completion. Each was found to be preferentially made on mitochondria-bound polysomes compared with unattached polysomes. Suissa and Schatz (1982) isolated mRNA from these two polysome populations and analyzed their in vitro translation products. The mRNAs for many mitochondrial polypeptides were enriched in the mitochondria-bound polysomes compared to the mRNAs for cytosolic proteins. These results show that the interaction between polysome and mitochondrion is specific and, as described above, that it may be mediated by nascent chains. Thus, the mitochondrial surface can recognize a specific subset of nascent polypeptides, namely, those destined to be imported into mitochondria. However, not all mitochondrial polypeptides are preferentially synthesized on these isolated mitochondria-bound polysomes, and even where enrichment is

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greatest, at most 60% of the total mRNA for a mitochondrial polypeptide is found associated with bound polysomes (Suissa and Schatz, 1982). Indeed, some imported mitochondrial proteins are synthesized essentially exclusively on unattached polysomes. Thus, mitochondria-bound polysomes cannot account for the bulk of mitochondrial protein import. The conditions used to observe and to isolate mitochondria-bound polysomes would in fact tend to maximize their apparent significance: to prevent completion of nascent chains, with the consequent dissociation of polysomes, protein synthesis is usually frozen by addition of cyclohexirnide. This may well effect a redistribution of polysomes since nascent mitochondrial polypeptides will have time to bind to the mitochondrial surface without their synthesis being completed. Thus, the amount of mitochondria-bound polysomes found by this procedure might be much higher than that existing in growing cells. If the nascent chains on mitochondria-bound polysomes bind to a functional receptor on the mitochondrial surface, as suggested by the apparent specificity of the interaction, then these nascent chains should be en route to the mitochondria. Ades and Butow (1980b) examined the fate of these polypeptides upon completion of polypeptide chain elongation and found that mitochondrial proteins did indeed become sequestered within the mitochondria. These experiments do not, however, distinguish whether the nascent polypeptides are discharged directly into the mitochondria, or whether translocation only occurs once the polypeptide chain has been completed. In summary, cotranslational import of proteins into mitochondria is suggested by the properties of mitochondria-bound polysomes, but there is no direct evidence that nascent polypeptides can be translocated across the mitochondrial membranes. On the other hand, there is a wealth of evidence supporting a posttranslational import pathway into mitochondria, both in vitro and in vivo. Import may be exclusively posttranslational, but at the moment it is impossible to exclude that cotranslational and posttranslational import coexist, their relative importance perhaps depending on physiological factors.

IV. THE MOLECULAR APPROACH The previous sections of this article have dealt largely with the phenomenology of mitochondrial protein import: I have described what happens, but now we want to know how it happens. For this we need to look at the properties of the molecules involved in the transport process: the extramitochondrial precursor polypeptides, their receptors on the mitochondrial surface, the translocation machinery, processing enzymes, and possibly other components with as yet unidentified functions.

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A. Isolation and Characterization of the Molecules Involved in Mitochondria1 Protein Import 1. PRECURSORS OF MITOCHONDRIAL POLYPEPTIDES

Imported mitochondrial polypeptides are synthesized in the cytosol, a sea of proteins from which they must be fished out by the mitochondria. What structural features of the precursor polypeptides are recognized by the mitochondrial import machinery'? Do different precursors share common structural features'? How different is the structure of a precursor from that of its mature counterpart'? These are some of the questions which cun only be answered by a molecular analysis of precursor polypeptides. Such studies will require the isolation of milligram amounts of precursor polypeptide, but precursors are normally only found in very small amounts. They are usually seen, either when synthesized in vitro or in pulse-labeled cells, only by virtue of incorporation of radioactive amino acids into the polypeptide chain. This problem of low abundance has been overcome in two ways: one a rather special case, cytochrome c; the other more generally applicable. Cytochrome c is made without a polypeptide extension. The only covalent difference between precursor and mature cytochrome c is the attachment of a heme group to the latter. This heme group can be removed by chemical cleavage to yield the apocytochrome, which behaves as expected of cytochrome c precursor (see Section 11,B).Since mature cytochrome c can be readily purified in large amounts, this provides a plentiful source of a pure precursor polypeptide. Cytochrome c, however, is not a typical imported mitochondrial polypeptide: apart from having no N-terminal extension, it is imported via a receptor not shared by other mitochondrial proteins tested so far and its import does not require an energized inner membrane (Zimmermann et al., 1981). The approach used to prepare cytochrome L' precursor is not generally applicable to other proteins, such as those whose maturation involves proteolytic processing. Can one find a situation where large amounts of precursor polypeptide are synthesized and accumulated'? In yeast, at least, the answer is yes. By growing yeast in the presence of CCCP (an uncoupler of oxidative phosphorylation), the energy-dependent import of proteins into mitochondria is blocked, but protein synthesis and growth continue; as a result, precursor polypeptides accumulate outside the mitochondria (Reid and Schatz, 1982a,b). The extent of accumulation is dependent on the stability of the precursor polypeptide in the yeast cytoplasm. The precursor to cytochrome c , disappears with a half-life of 10 minutes, whereas some precursors accumulate in large amounts. After growth for several hours in the presence of CCCP, rlzo- yeast contains as much precursor of the P-subunit of F,-ATPase as the corresponding mature polypeptide

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(about 150 pg/g cell protein; Reid and Schatz, 1982a). Isolation of this precursor from such cells requires approximately 7000-fold purification; as discussed below, this has proved possible. The stability of some accumulated yeast precursors contrasts with the rapid degradation of the precursor of the mitochondrial matrix enzyme carbamoyl-phosphate synthase when import is blocked in rat liver explants (Raymond and Shore, 1981). The precursor of mitochondrial aspartate aminotransferase is similarly unstable in chick fibroblasts (Jaussi et al., 1982). When the F,-ATPase @-subunit is accumulated in the yeast cytoplasm, it remains competent to be imported into mitochondria and processed to the mature protein upon removal of the import block. S. Ohta has purified several hundred micrograms of the F,-ATPase @-subunit precursor in a denatured form from CCCP-treated rho- yeast; at least some of the denatured precursor can then be renatured such that it regains the ability to be transported into mitochondria and become proteolytically matured (Ohta and Schatz, 1984). Thus, the import of this precursor into isolated mitochondria may be studied in the absence of other precursor polypeptides, and it may be determined whether extramitochondrial factors, perhaps present in reticulocyte lysate, are also important in directing the precursor to its intramitochondrial destination. 2. IMPORTRECEPTORS It has been shown that mitochondrial protein import requires protease-sensitive components on the mitochondrial surface (Section II,B), but these receptor-like polypeptides have not yet been identified. Identification could perhaps be achieved by solubilizing and separating the components of the mitochondrial outer membrane, reconstituting them into phospholipid vesicles, and determining which proteins are able to bind precursors. The first steps in this direction have been taken, and the results suggest the feasibility of this approach. Riezman et af. (1983b) solubilized yeast mitochondrial outer membrane vesicles with the nonionic detergent octyl polyoxyethylene. The solubilized material was reconstituted into vesicles by removing the detergent by dialysis, and these vesicles were shown to retain cytochrome b, precursor binding activity comparable to that of the original outer membranes. The constituted vesicles contained many polypeptides; it remains to be determined which of these is (or are) responsible for the binding activity. 3. PROCESSING PROTEASES At least two proteases are involved in the maturation of imported mitochondrial proteins. The protease(s) catalyzing the second step of cytochrome b,, cytochrome c peroxidase, and cytochrome c , maturation has proved difficult to examine. No specific inhibitors are known, and its activity is lost in the presence of detergent (Daum et af., 1982b). Fortunately, we know somewhat more about

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another protease which appears to be responsible for the single-step maturation of many precursors and which also catalyzes the first cleavage step of those proteins imported to the intermembrane space by a two-step mechanism (Section II,C,3). This protease is a soluble component of the mitochondria1 matrix in yeast, rat, and maize (Bohni et a/., 1980, 1983; McAda and Douglas, 1982; Mori et al., 1980; Miura et al., 1982a). Its activity is maximal at neutral pH, is insensitive to serine protease inhibitors, but is inhibited by chelators of divalent metal ions such as 1 ,lo-phenanthroline, EDTA, and GTP. This inhibition could be at least partially reversed by addition of an excess of Zn2+ or Co2 (Bohni et af., 1983; Conboy e t a / . , 1982) or Mn2+ (McAda and Douglas, 1982). It is not known which of these cations is normally present in the active enzyme. The protease has been partially purified from yeast mitochondria (McAda and Douglas, 1982; Bohni et a/., 1983). It behaves on gel filtration as a molecule with a molecular weight of 110,000 to 115,000. Complete purification was not achieved, but McAda and Douglas (1982) suggested that the activity of the protease, judged by its ability to convert F, -ATPase P-subunit precursor to mature form, correlated best with the presence of a band with an apparent molecular weight of 59,000 on SDS-polyacrylamide gel electrophoresis, though no such band was detected in the purest preparations of Bohni et al. (1983). Bohni et a / . (1983) demonstrated that this enzyme is responsible for the cleavage of several larger precursors of imported mitochondria1 polypeptides; it may, in fact, cleave all precursors with N-terminal extensions. In this respect the enzyme may be considered to have a broad specificity, though the structural features around the cleavage sites of different precursors are not known, but presumably share some recognizable characteristics. In other respects the protease exhibits a remarkably high degree of specificity. It does not cleave nonmitochondrial proteins, nor does it process denatured precursors. This conformational requirement indicates that the protease does not simply recognize a particular amino acid sequence, rather some three-dimensional domain of the precursor. Since the partially purified protease also cleaves in vitro-synthesized precursors in the absence of mitochondria, it appears that the conformation of at least the Nterminal precursor regions are similar in solution and during (or soon after) translocation into the matrix. The matrix-located enzyme appears to be an endoprotease since partially processed intermediates are not normally observed. A clear demonstration that this is the case might be achieved by detection of the intact N-terminal peptide after processing, but this has not yet been done. It seems then that a single cleavage is generally involved in processing, but the maturation of the proteolipid (subunit 9) of Neurospora ATP synthase may occur in two discrete steps; since each of these steps is sensitive to 1,lO-phenanthroline (unlike two step-processing of intermembrane space enzymes where the second step is insensitive to chelators), they are perhaps catalyzed by the same enzyme (W. Neupert, personal commu+

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nication). The processing of in vim-synthesized precursors by the matrix protease accurately reflects their maturation in vivo in that the correct N-terminus is generated, as determined by analysis of the N-terminal amino acid sequence (Cerletti et al., 1983). Whereas the matrix-located protease processes the precursors of matrix and inner membrane proteins to the mature forms, it generates an intermediate in the maturation pathway of some proteins of the intermembrane space (Gasser et al., 1982a; Bohni et al., 1983; see Section II,C,3). The protease is present in mitochondria of rho- yeast which lack mitochondrial protein synthesis; it must, therefore, be made extramitochondrially. It is not known whether it is made as a larger precursor, in which case it would presumably cleave its own precursor. 4. CYTOSOLIC SOLUBLE FACTORS Recent studies have shown that in vitro transport of proteins into mitochondria can be stimulated by a soluble factor which is present in reticulocyte lysates and in the yeast cytosol (Ohta and Schatz, 1984; Argan et al., 1983; Miura et al., 1983). The yeast soluble factor is a protein with an apparent molecular weight of 40,000 as judged by gel filtration (Ohta and Schatz, 1984). The role of this factor in protein transport is unknown, but its purification will greatly enhance investigation of its structure and function.

B. Isolation and Characterization of the Nuclear Genes Encoding Mitochondria1 Polypeptides Recently developed methods for isolating and manipulating genes have provided a new tool with which to elucidate the molecular features of the import process. Many nuclear genes encoding mitochondrial polypeptides have been isolated, and some partially characterized. What can these genes tell us about the precursor polypeptides encoded by them? For one, the DNA sequence should give us the protein sequence. With proteins that are synthesized as larger precursors, the extra sequence in the precursor can be deduced. This will be much simpler than direct determination of the amino acid sequences of precursor polypeptides (if the N-terminal sequence of the mature protein is known). If the N-terminal regions of precursors operate as signals for import into mitochondria, it may be possible to discern recognizable features common to different precursors. However, this approach has limited use in defining the important information of the import signal; perhaps only part of the N-terminal region is responsible for providing the signal, and sequences present in the mature protein may be important, too. To define the nature of these signals, it would be useful to look at mutant forms which exhibit abnormal transport behavior. Such mutants can be generated in vitro by manipulation of the cloned precursor genes; deletions,

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insertions, and point mutations can be constructed. It will also be of interest to construct gene fusions. By fusing the signal region from a mitochondrial protein to an enzyme of nonmitochondrial origin, it may be possible to deliver the enzyme to the mitochondrion: if this does occur, one can determine how much of the precursor sequence is required to provide a functional signal. Analysis of cloned genes encoding mitochondria1 proteins will also be useful in studies of their regulation; this approach has already proved fruitful with a yeast cytochrome c gene (Guarente and Mason, 1983). 1 . MOLECULAR CLONING OF MITOCHONDRIAL PROTEINGENES

For several reasons the yeast Saccharomyces cerevisiae is generally the organism of choice for these studies. Apart from being well characterized genetically, yeast can be transformed with a variety of plasmid vectors, thus allowing the isolated genes to be returned into their homologous cell. Also, many of the biochemical studies of mitochondrial protein import have been performed with yeast. Most of the mitochondrial protein genes studied so far have indeed come from yeast. Some of the approaches used to isolate these genes are described below. A useful account of recombinant DNA technology in yeast may be found in Botstein and Davis (1982). a. Screening with u Synthetic Oligonucleotide Probe. The amino acid sequence of yeast iso- 1 -cytochrome c is known, but because of the redundancy in the genetic code the nucleotide sequence of the corresponding gene cannot be unambiguously predicted. Stewart and Sherman (1974) were able to infer the nucleotide sequence for part of this gene by analysis of a number of frameshift mutations. Montgomery et al. ( 1978) synthesized an oligonucleotide complementary to part of this region and identified a plasmid bearing the cytochrome c gene by its ability to hybridize this oligonucleotide. This approach can also be used where the nucleotide sequence in the region of interest is ambiguous; the “unknown” nucleotides can be guessed from known codon preferences in yeast (Bennetzen and Hall, 1982) or a mixed probe can be synthesized. b. Functional Complementation. Several yeast nuclear mutants have been described which are deficient in mitochondrial metabolism, particularly in oxidative phosphorylation (listed in Broach, 1981). If the chromosomal defect can be overcome by transformation of the mutant with a suitable plasmid containing a functional copy of the gene, the mutant phenotype will be suppressed. This approach has been used to isolate the yeast gene encoding the adenine nucleotide translocator (O’Malley et a!., 1982). The mutation pet9, or op, (Kovac et al., 1967; Beck et a f . , 1968), results in loss of oxidative phosphorylation and thus an inability of the mutant to grow on nonfermentable carbon sources. Several lines of evidence suggested that pet9 is the structural gene for the adenine nucleotide translocator, but the necessary confirmation of this came only with the charac-

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terization of the isolated gene. It is not possible to select directly for growth of transformants on a nonfermentable carbon source since the Pet phenotype takes some time to be overcome. O’Malley et al. (1982) therefore used a two-step screening procedure. They constructed a mutant which contained not only the pet9 defect but also a mutant leu2 gene. This mutant was transformed with a plasmid capable of episomal replication in yeast which contained the selectable leu2 gene and random fragments of the yeast genome. First, transformants were selected by their ability to grow in the absence of leucine in order to select clones containing plasmid. Leu transformants were then selected for their ability to grow on a nonfermentable carbon source. In this way a plasmid containing the pet9 gene was found. The yeast DNA inserted in this plasmid was found to encode a 30-kDa protein. This protein was recognized as the adenine nucleotide translocator by its reaction with specific antiserum. Ebner et al. (1973a,b; Ebner and Schatz, 1973) and Tzagoloff et al. (1975) have described deficiencies in mitochondrial enzyme activities resulting from defined nuclear mutations. Complementation of these mutations could be used to isolate genes for subunits of the enzymes concerned. Not all of these mutations, however, are in the structural genes for components of the deficient enzymes. One group of per mutants, leading to mitochondrial cytochrome b deficiency, produces abnormal transcripts of the mitochondrial cytochrome b gene. Complementation of this mutation was used to select a nuclear gene (cbpl) responsible for correct expression of a mitochondrial gene (Dieckmann et al., 1982). Similarly, Faye and Simon (1983) isolated a gene (mss5l) involved in maturation of the mitochondrial RNA encoding subunit I of cytochrome c oxidase. If, as is very likely, cbpl and mss.51 are structural genes, their products are presumably imported into mitochondria. c. Immunological Screening. Some plasmid-borne eukaryotic genes (e.g., from yeast) can be expressed in E. coli without further genetic manipulation. It is thus possible to directly screen for E . coli colonies harbouring plasmids into which yeast gene fragments have been inserted by using antibodies to detect specific, expressed polypeptides. This method (Erlich et al., 1978; Henning et al., 1979) has been used to identify the yeast cytochrome c peroxidase gene (Goltz et al., 1982). Viebrock et al. (1982) constructed a Neurospora cDNA clone bank from which they wanted to select the gene encoding subunit 9 of the mitochondrial ATP synthase. A plasmid harboring this gene was identified after screening by in vitro translation of hybrid-selected mRNA: the plasmids were allowed to hybridize complementary RNA molecules from total Neurospora mRNA, and the hybridized RNA was translated in a wheat germ lysate. The translation products were immunoprecipitated with an antiserum against the purified subunit in order to recognize a plasmid containing the subunit 9 gene. +

7. TRANSPORT OF PROTEINS INTO MITOCHONDRIA

323

d. RNA Hybridization. A more general approach to the cloning of nuclear genes encoding mitochondrial polypeptides has been developed by van Loon et al. (1982). Many mitochondrial proteins in yeast are repressed by glucose. Their regulation appears to be transcriptional, so yeast cells grown with a nonfermentable carbon source contain much higher levels of the mRNAs encoding mitochondrial polypeptides than do yeast cells grown on a glucose-containing medium. mRNA from lactate-grown yeast was radioactively labeled and hybridized to a yeast clone bank ( E . coli colonies harboring plasmids into which random pieces of the yeast genome had been inserted). The hybridization was repeated in the presence of unlabeled mRNA from glucose-repressed yeast. The hybridization of labeled RNA to most colonies was reduced by competition, but those containing genes for mitochondrial polypeptides still gave strong signals. This procedure produced an enriched clone bank which could then be screened for the presence of specific genes by translation of hybrid-selected mRNA. This procedure has been used to identify the genes encoding three subunits of the cytochrome bc, complex (van Loon et a[., 1982) and several other mitochondrial polypeptides ( H . Riezman, A. P. G. M. van Loon, G. A . Reid, M. Suissa, and G . Schatz, unpublished results).

2. WHATTHE GENESHAVETOLDUS Since the work described in Section IV,B,I is rather recent, we have so far obtained only little information from the cloned genes for mitochondrial polypeptides. This will certainly change rapidly, but it is worth considering what we have learned so far, and where we might go from here. The nucleotide sequences of a few of the cloned genes have been determined, revealing the nature of the N-terminal extensions where present. The predicted amino acid sequences of the N-terminal regions of these precursors so far examined are shown in Table I. There are no obvious homologies, but this is not so surprising. It is known that the N-terminal extensions of different imported mitochondrial proteins vary widely in apparent size (Neupert and Schatz, 1981; Hay et al., 1983) and the proteins for which the sequence has so far been determined are transported to different submitochondrial locations, so presumably have recognizably different addressing signals. There are, however, some noteworthy features of these sequences. In all but one case, the N-terminal region is predominantly basic, as had been expected from comparison of precursor and mature proteins by isoelectric focusing (Anderson, 1981; Reid et al., 1982). The N-terminal extension of cytochrome c peroxidase precursor contains three lysine, four arginine, and three histidine residues and no acidic residues (Kaput et a l . , 1982). Similarly, the cleaved Nterminal peptide of Neurospora ATP synthase subunit 9 contains 12 basic and no

TABLE I THE N-TERMINAL AMINOACIDSEQUENCES OF IMPORTED MITOCHONDRIAL POLYPEPTIDES~ Polypeptide

Location of mature protein

N-Terminal amino acid sequence of precursor

+

+ ATP s y n k subunit 9

Inner membrane

+

+

Reference

++

+

++

MASTRVLASRLASQMAASAKVARPAVRVAQVSKRTIQTGSPLQTLKR

+

(Neurosporn)

++

Viebmck ef al

( 1982)

TQMTSIVNATTRQAFQKRAYSS.

+

-

t

+

+

++

.

Cyt c reductase ICkDa subunit

Inner membrane

MPQSFTSIARIGDYILKSPVLSKLCVPVANQFINLAGYKKLCL

Cyt c reductase 17-kDasubunit

Inner membrane

MDMLELVGEYWEQLKITVVPVVAAAEDDDDNEQHEEKAA

Citrate synthase

Matrix

MSAILSTTSKSFLSRGSTRQCQNMQKALFALLNARHYSS

EF-Tu

Mamx

MSALLPRLLTRTAFKASGKLLRLSSVISRTFSQTTTSYAAA

MSS 51

Unknown (probably mamx)

MT V L Y A P S GA TQ L Y F H L LR K S P HN R L V V S HQTR R H LMG F V R N A

Cytochrome c pemxidase

Intermembrane space

MTTAVRLLPSLGRTAHKRSLYLFSAAAAAAAAAATFAYSQSHKRSSS

-

-

-

-

-_-__

+

+

+

+

+

+

+

+

+

++

Suissa er a/. ( 1984)

+

+

+

+ +

Nagata ef

+

+++

a/ (1983)

Faye and Simon (1983)

+++

+++

+

+

+--+

-

+

++

de Haan el a / . (1983)

+

Kaput ef a/. ( 1982)

SPGGGSNHGWNNWGKAAALASTTPLV

++

-

7 t

+

+

-+

++

+

++

Cytochrome c

Intermembrane space

MTEFKAGSAKKGATIFKTRCLQCHTVEKGGPHKVGPNLHGIFGRH..

7&kDa polypeptide

Outer membrane

MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKKNT

+

+ +

+ ++

Narita and Titan,(1969); Lederer el a / . (1972)

Hase ef

(21.

(1983)

0 The single-letter code for amino acids is used. The basic amino acids arginine, lysine, and histidine are marked +; the acidic aspartate and glutamate are marked - . The proteolytic cleavage sites generating the mature forms of ATP synthase subunit 9 and cytochrome c peroxidase are marked by arrows. Cytochrome c and the 70-kDa outer membrane protein are not cleaved except that the N-terminal methionine is removed from the former. The possible cleavage sites of the other precursors are not known. All sequences other than that of ATP synthase subunit 9 (from Neurospora) are derived from yeast.

7. TRANSPORT OF PROTEINS INTO MITOCHONDRIA

325

acidic amino acids (Viebrock et uf.. 1982). The signal sequences of proteins secreted across the eukaryotic endoplasmic reticulum or across prokaryotic membranes (Austen, 1979: lnouye and Halegoua, 1980) are predominantly basic and generally also contain a stretch of uncharged amino acids which may form a transmembrane segment during translocation. The precursors of the ATP synthase subunit 9, and of several other proteins contain no such hydrophobic segments near the N-terminus; indeed the ATP synthase subunit 9 precursor has a remarkably polar N-terminal region, whereas the mature protein is generally apolar (Viebrock et al., 1982). On the other hand, the 70-kDa mitochondrial outer membrane protein has an uncharged stretch of 28 amino acids near the N-terminus which may act as a membrane anchor (Hase et al., 1983). This precursor is not proteolytically processed during import into mitochondria (Gasser and Schatz, 1983). Cytochrome c peroxidase precursor also has an unusual stretch of nonpolar amino acids, which is proposed to specify membrane binding (Kaput et ul., 1982). Thus, at the moment it is difficult to propose unifying concepts describing the signals involved in directing different precursors to the mitochondria, but specific roles have been proposed for the Nterminal extension of Neurospora ATP synthase subunit 9 and of yeast cytochrome c peroxidase. Subunit 9 of ATP synthase is an extremely hydrophobic polypeptide of 81 amino acid residues (Sebald et al., 1980). Its polarity (Capaldi and Vanderkooi, 1972) is only 25.9%, making it one of the most hydrophobic proteins known. In Neurospora this polypeptide is synthesized as a precursor in the cytoplasm (Michel ef ul., 1979; Schmidt et ul., 1983a). How is it maintained in solution despite such a hydrophobic nature'?The precursor is much larger than the mature polypeptide (Michel et al., 1979); indeed, it consists of 147 amino acid residues, of which 66 are removed during maturation (Viebrock et al., 1982). This 66amino acid extension is extremely hydrophilic, with a polarity of 53%, so that the overall polarity of the precursor is similar to that of a typical water-soluble protein. It is suggested (Viebrock er al., 1982) that an important function of the N-terminal extension of this protein is to render the precursor soluble, thus allowing its posttranslational import into mitochondria. The precursor must also contain specific information directing subunit 9 to the mitochondrial inner membrane. Interestingly, the yeast ATP syntase subunit 9 is encoded on mitochondrial DNA and translated without a cleaved N-terminal extension (Macino and Tzagoloff, 1979; Hensgens et al., 1979). Cytochrome c peroxidase is a soluble protein of the mitochondrial intermembrane space which appears to be imported into mitochondria by a two-step processing mechanism involving initial translocation of the precursor such that its N-terminus protrudes across the inner membrane into the matrix space (Gasser et al., 1982b; Reid et al., 1982; see Section 11,C,3). The precursor is larger than the mature protein (Maccecchini et al., 1979b) by 68 amino acid residues (Kaput

326

GRAEME A. REID

et al., 1982). Apart from the basic nature of this N-terminal extension, there are several striking features of the sequence, e.g., a stretch of 23 uncharged amino acids including 10 consecutive alanine residues. This hydrophobic region is expected to form an a-helix and is suggested to span the bilayer of the inner membrane when the precursor is imported into the mitochondrion (Kaput et al., 1982), acting as a stop-transfer sequence (Blobel, 1980). This hydrophobic stretch is flanked on either side by basic residues, perhaps to anchor it firmly in the membrane. The predicted sequence of the precursor of the 17-kDa subunit of ubiquino1:cytochrome c reductase is striking in its content of acidic amino acid residues (van Loon et al., 1984), not only in the mature protein, but also in the N-terminal region of the precursor. It will be of interest to compare the transport of this precursor into mitochondria with the transport of other polypeptides. To define the signals directing mitochondrial protein import, considerably more data are required. We have seen so far that basic amino acid residues near the N-terminus provide a common feature of most imported polypeptides. That these may be important in protein transport has been suggested by the finding that some basic compounds can inhibit mitochondrial protein import (Miura et al., 1982b). However, the basic nature of these sequences is clearly not sufficient for either intracellular or intramitochondrial sorting. Hydrophobic stretches may determine that a protein becomes membrane bound, but what determines whether it becomes a component of the inner or the outer membrane? In addition, the information in larger precursors must specify cleavage sites for processing enzymes. The most useful approach to the study of import signals will be to investigate the import of various mutated precursor polypeptides in order to delimit those regions of the precursor sequence which are essential for correct localization and processing. It has been shown that a truncated form of the 70-kDa outer membrane protein, lacking 203 amino acids from the C-terminus, is still transported to the mitochondrion (Riezman et al., 1983c), a finding suggesting that the C terminal region is not required for correct localization of this protein. Mutants in the signal sequence of the E . coli lamB protein that are deficient in the transport of this polypeptide have been used to select second-site revertants which restore secretion (Emr et al., 1981). The compensating mutations are likely to occur in components of the secretory machinery. A similar approach could be useful in recognizing the components of the mitochondrial import machinery: receptors, translocating components, proteases, and perhaps other, as yet undefined, molecules. It may also be possible to isolate such mutants by a conventional genetic approach. Matner and Sherman (1982) have described two groups of yeast mutants deficient in cytochrome c which may be lacking in heme-attaching activity or cytochrome c transport into mitochondria. Further biochemical studies are required to confirm this suggestion. Schekman (1982) has isolated temperature-

7. TRANSPORT OF PROTEINS INTO MITOCHONDRIA

327

sensitive yeast mutants deficient in protein secretion, thereby defining many genes required for function of the secretory pathway. It should be possible to isolate conditional mutants in mitochondrial protein import. This would be useful in defining how many gene products are required to direct functional import and in determining the molecular nature of these components. While the ability to analyze isolated genes will be of great importance in the study of mitochondrial protein import, such investigations cannot replace the biochemical approach. Indeed, the two lines of research should be complementary in elucidating molecular mechanisms in the import pathway.

V.

SUMMARY

Because we now have considerable information describing several important biochemical features of mitochondrial protein transport, many exciting questions can now be asked, particularly regarding the molecular mechanisms involved. We do not yet know how many different pathways are used to import proteins into mitochondria, how many molecules are required to catalyze the process, or how these molecules work to convert extramitochondrial precursor polypeptides into active intramitochondrial enzymes. At present it seems that there are at least three pathways leading to mitochondrial protein import. Cytochrome c is transported by a route different from that required for import of all other proteins tested (Zimmermann et ul., 1981; Teintze et al., 1982), including an outer membrane protein (W. Neupert, personal communication). The transport of proteins to the mitochondrial outer membrane is very different from transport to the inner membrane and matrix; the two are distinguished by protease sensitivity of mitochondrial surface components, by energy dependence, and generally by the involvement of proteolytic processing. The initial steps of the two-step pathway by which some intermembrane space polypeptides are imported are very similar to the initial steps of import to the inner membrane and matrix. Each requires a protease-sensitive receptor, an electrochemical potential difference across the inner membrane, and usually a matrix-located protease. It will be interesting to test whether the same molecules are involved in the import of these proteins to different final locations, as seems likely. This pathway then could be largely responsible for the biogenesis of the mitochondrial matrix, inner membrane, and intermembrane space. The essential feature of polypeptides imported by this route must be a signal directing the initiation of translocation across the inner membrane. Some polypeptides will be completely translocated to become components of the mitochondrial matrix, others having stop-transfer signals (Blobel, 1980) will become components of the inner membrane. When such membrane-bound proteins are cleaved by a protease at the outer face of the inner membrane (Gasser et a/., I982b), they may

328

GRAEME A. REID

become components of the intermembrane space. Other factors may also be significant in defining the ultimate topology of a polypeptide: cytochrome c , apparently binds to the inner membrane by its rather hydrophobic C-terminus; interactions with other subunits of an enzyme may also be important. The above model poses an interesting question: Why would a stop-transfer signal determine that an imported polypeptide become attached to the inner membrane and yet allow its passage through the outer membrane? The relationship between the two membranes in protein import is not well understood. It has, however, been suggested that junctions between the two membranes may be sites of protein import (Schatz and Butow, 1983). Proteins can apparently be imported to a single compartment by more than one route. Cytochrome b, is transported to the intermembrane space by an energy-dependent, two-step processing pathway, whereas cytochrome c and apparently also adenylate kinase (Watanabe and Kubo, 1982) are imported without cleavage. By isolating the components of the import pathway, it may be possible to reconstitute the process in vitro and examine the molecular details. Such a system may be useful in determining how the electrochemical gradients across the mitochondrial inner membrane are involved in protein movement. We can begin to ask how mitochondrial biogenesis is regulated and what role protein import plays in this process. Some polypeptides are synthesized in the mitochondrion and become components of multisubunit enzymes. How is the synthesis of the various subunits coordinated, particularly when some subunits of an enzyme, such as cytochrome c oxidase, are synthesized intramitochondrially and others have to be imported from the cytoplasm? The 45-kDa subunit of mitochondrial RNA polymerase is encoded on nuclear DNA and is subject to glucose repression (Lustig et al., 1982). This control at the nuclear level may then be involved in the regulation of transcription of mitochondrial genes. Genetic evidence (Dieckmann et al., 1982; Faye and Simon, 1983) indicates the importance of nuclear genes in the maturation of mitochondrial transcripts. Thus, the nucleus communicates with the mitochondrial genetic system at least in part by means of polypeptides which are imported into the mitochondrion. In this article we have discussed the import of proteins into mitochondria from very diverse organisms. There is good reason to suspect that the processes involved are well conserved among different species. The yeast F,-ATPase psubunit precursor is matured by the matrix protease from rat and from maize. The precursor of subunit 9 of Neurospora ATP synthase is imported into yeast mitochondria and correctly processed (Schmidt et al., 1983b) even though the equivalent yeast polypeptide is synthesized intramitochondrially without an N-terminal extension. Presumably the Neurospora precursor has structural features similar to those of yeast precursor polypeptides and can be imported by a pathway normally used for import of these other yeast proteins. The import of proteins into chloroplasts shares many features with mitochondrial protein import

7. TRANSPORT OF PROTEINS INTO MITOCHONDRIA

329

(Chua and Schmidt, 1979): larger precursors, posttranslational and energy-dependent import, and chelator-sensitive processing by a soluble protease (Highfield and Ellis, 1978; Grossman e t d . , 1980; Ellis, 1981). There must, however, be clearly recognizable differences between the cytoplasmically synthesized precursors destined for mitochondria and those en route to chloroplasts since the two import processes must be able to coexist in the plant cell cytoplasm. ACKNOWLEDGMENTS I sincerely thank Jeff Schatz and Rick Hay for critical evaluation of the manuscript. I am particularly grateful to Jeff for his immense support and encouragement during my stay in his lab. I also thank Ilona Durring for typing this article. REFERENCES Ades, I. Z . . and Butow, R. A. (198Oa). The products of mitochondria-bound cytoplasmic polysomes in yeast. J . Biol. Chem. 255, 9918-9924. Ades, 1. 2.. and Butow, R. A. (1980b). The transport of proteins into yeast mitochondria. Kinetics and pools. J . B i d . Chetn. 255, 9925-9935. Anderson, L. (1981). Identification of mitochondrial proteins and some of their precursors in twodimensional electrophoretic maps of human cells. Proc. Nafl. Acud. Sci. U.S.A. 78, 24072411. Anderson, S . , Bankier, A. T., Barrell, B. G., de Bruijn, M. H. L., Coulson. A. R., Drouin, J.. Eperon, I. C., Nierlich, D. P., Roe, B. A,, Sanger, F.. Schreier. P. H., Smith, A. J. H . , Staden, R.. and Young, I. G. (1981). Sequence and organization of the human mitochondrial genome. Nafrire (London) 290, 457-465. Anderson, S . , de Bruijn, M. H. L., Coulson, A. R.. Eperon, I . C., Sanger, F., and Young, I. G. (1982). Complete sequence of bovine mitochondrial DNA. Conserved features of the mammalian mitochondrial genome. J . Mol. Biol. 156, 683-7 17. Argan, C., Lusty, C. J . , and Shore, G . C. (1983). Membrane and cytosolic components affecting transport of the precursor for omithine carbamyltransferase into mitochondria. J . Biof. Chem. 258, 6667-6610. Austen. B. M. (1979). Predicted secondary structures of aminoterminal extension sequences of secreted proteins. FEES Left. 103, 308-309. Beck, J . C., Mattoon, J . R.. Hawthome. D. C., and Sherman, F. (1968). Genetic modification of energy-conserving systems in yeast mitochondria. Proc. Nut/. Acad. Sci. U.S.A. 60, 186- 193. Bennetzen. J. L., and Hall, B. D. (1982). Codon selection in yeast. J . Biol. Chem. 257, 3026-3031. Bibb, M. J . , van Etten, R. A., Wright, C. T . , Walberg, M. W., and Clayton, D. A. (1981). Sequence and gene organization of mouse mitochondrial DNA. Cell 26, 167-180. Blobel, G. (1980). lntracellular protein topogenesis. Proc. Nut/. Acad. Sci. U.S.A. 77, 1496-1500. Bohni. P. C.. Gasser. S . , Leaver, C., and Schatz, G . (1980). A matrix-localized mitochondrial protease processing cytoplasmically-made precursors to mitochondria1 proteins. In “The Organization and Expression of the Mitochondria1 Genome’’ (A. M. Kroon and C. Saccone, eds.), pp. 423-433. North-Holland Publ., Amsterdam. Bohni, P. C., Daum. G . , and Schatz, G . (1983). Import of proteins into mitochondria. Partial purification of a matrix-located protease involved in cleavage of mitochondrial precursor polypeptides. J . B i d . Chem. 258, 4937- 4943. Borst, P., and Grivell, L. A. (1978). The mitochondrial genome of yeast. Cell 15, 705-723.

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mutants of Sacchuromyces cerevisiue. 1. Effect of nuclear mutations on mitochondria1 protein synthesis. J . Eiol. ('hem. 248, 5360-5368. Ebner, E., Mason, T. L., and Schatz, G. (1973b). Mitochondria1 assembly in respiration-deficient mutants of Sacchurtrmyces cerevisiue. 11. Effect of nuclear and extrachromosomal mutations on the formation of cytochrome c oxidase. J . Eiul. Chem. 248, 5369-5378. Ellis, R. J. ( 1981). Chloroplast proteins: Synthesis, transport and assembly. Annu. Rev. P l m t Physiol. 32, 111-137. Emr, S. D.. Hanley-Way, S., and Silhavy, T. J. (1981). Suppressor mutations that restore export o f a protein with a defective signal sequence. Cell 23, 79-88. Erlich, H. A , , Cohen, S. N . , and McDevitt, H. 0. (1978). A sensitive radioimmunoassay for detecting products translated from cloned DNA fragments. Cell 13, 681-689. Faye, G . , and Simon, M. (1983). Analysis of a yeast nuclear gene involved in the maturation of mitochondrial pre-messenger RNA of the cytochrome oxidase subunit I . Cell 32, 77-87. Freitag, H., Neupert. W., and Benz, R. (1982a). Purification and characterisation of a pore protein of the outer mitochondrial membrane from Neurosporu erassu. Eur. J . Eiochem. 123,629-639. Freitag, H., Janes, M., and Neupert, W. (l982b). Biosynthesis of mitochondrial porin and insertion into the outer mitochondrial menihrane of Neurospora crassa. Eur. J . Eiochem. 126, 197-202. Gasser, S. M., and Schatz, G . (1983). Import of proteins into mitochondria. In vitro studies on the biogenesis of the outer membrane. J . Biol. Chem. 2.58, 3427-3430. Gasser, S . M., Daum, G . , and Schatz, G. (1982a). Import of proteins into mitochondria. Energydependent uptake of precursors by isolated mitochondria. J . B i d . Chem. 257, 13034- 13041. Gasser, S . M., Ohashi, A , , Daum, G., Bohni, P. C., Gibson, J., Reid, G. A,, Yonetani, T., and Schatz, G. (l982b). Imported mitochondrial proteins cytochrome bz and cytochrome c, are processed in two steps. Proc. Natl. Acad. Sci. U.S.A. 79, 267-271. Goltz, S . , Kaput, J., and Blobel, G . (1982). Isolation of the yeast nuclear gene encoding the mitochondrial protein, cytochrome c peroxidase. J . Eiol. Chem. 2.57, 1 1186-1 1190. Grossmann, A,, Bartlett, S . , and Chua, N.-H. (1980). Energy-dependent uptake of cytoplasmically synthesized polypeptides by chlomplasts. Nature (London) 28.5, 625-628. Guarente, L., and Mason, T. (1983). Heme regulates transcription of the CYC 1 gene of S. cerevisiue via an upstream activation site. Cell 32, 1279-1286. Hallermayer, G . , Zimmermann, R.. and Neupert. W. (1977). Kinetic studies on the transport of cytoplasmically synthesized proteins into the mitochondria in intact cells of Neurosporu c'rassa. Eur. J . Eiochem. 81, 523-532. Hampsey, D. M., Lewin, A. S . , and Kohlhaw, G. B. (1983). Subniitochondrial localization, cellfree synthesis, and mitochondria1 import of 2-isopropylmalate synthase of yeast. Proc. Nutl. Acad. Sci. U . S . A . 80, 1270-1274. Harmey, M. A,, Hallermayer, G., Korb, H.. and Neupert, W. (1977). Transport of cytoplasmically synthesized proteins into the mitochondria in a cell free system from Neurospora crassa. Eur. J . Eiochem. 81, 533-544. Hase, T.. Riezman, H., Suda, K., and Schatz, G. (1983). Import of proteins into mitochondria. Nucleotide sequence of the gene for a 70kd protein of the yeast mitochondrial outer membrane. EMBO J . 2, 2169-1172. Hay, R.. Bohni. P., and Gasser, S. (1983). How mitochondria import proteins. Eiochim. Eiuphys. Actu 779, 65-87. Hennig. B.. and Neupert. W. (1981). Assembly of cytochrome c. Apocytochrome c is bound to specific sites on mitochondria before its conversion to holocytochrome c. Eur. J . Eiorhem. 121, 203-2 12. Hennig, B., Koehler, H., and Neupert, W. (1983). Receptor sites involved in post-translational transport of apocytochrome c into mitochondria: Specificity, affinity and number of sites. Proc. Narl. Acad. Sci. U . S . A . 80, 4963-4967.

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