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Archaeal proteasomes and other regulatory proteases
Archaeal proteasomes and other regulatory proteases Julie A Maupin-Furlow, Malgorzata A Gil, Matthew A Humbard, Phillip Aaron Kirkland, Wei Li, Christopher J Reuter and Amy J Wright Numerous proteases have been shown to catalyze the precisely-timed and rapid turnover of key cellular proteins. Often these regulatory proteases are either energy-dependent or intramembrane-cleaving. In archaea, two different types of energy-dependent proteases have been characterized: 20S proteasomes associated with proteasome-activating nucleotidases and membrane-associated Lon proteases. Interestingly, homologs of all three mechanistic classes of intramembrane-cleaving proteases are widely distributed in archaea. Similar to their eucaryal and bacterial counterparts, members of these uncharacterized proteases might promote the controlled release of membrane-anchored regulatory proteins or liberate small peptide reporters and/or effectors that function in cell signaling. Addresses Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA Corresponding author: Maupin-Furlow, Julie A ([email protected])
Current Opinion in Microbiology 2005, 8:720–728 This review comes from a themed issue on Growth and development Edited by John N Reeve and Ruth Schmitz Available online 26th October 2005 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2005.10.005
Introduction Insight into the world of proteolysis has expanded considerably over the past decade. Proteases are no longer viewed as non-specific degradative enzymes associated solely with protein catabolism, but are known to be intimately involved in controlling biological processes that span life to death. Proteases maintain this exquisite control by catalyzing the precisely-timed and rapid turnover of key regulatory proteins. Proteases also interplay with chaperones to ensure protein quality and to readjust the composition of the proteome following stress. Although much less is known about proteolysis in the context of an archaeal cell than that of bacteria and eukaryotes, recent advances in genomics and biochemistry provide unprecedented insight into the complete repertoire of archaeal proteases that might regulate cell function. In this review, we provide a brief overview of archaeal proteases that are likely to be involved in regulation. Emphasis Current Opinion in Microbiology 2005, 8:720–728
will be on our current understanding of energy-dependent proteases such as proteasomes and Lon as well as homologs of intramembrane-cleaving proteases. The reader is referred to additional reviews [1–4] for more detailed discussions of archaeal proteases and peptidases.
Proteasomes and proteasome-associated nucleotidases Proteasomes are proteolytic nanomachines that are distributed in all three domains of life: Bacteria, Archaea and Eukarya [5]. These enzymes maintain protein quality control by degrading misfolded and denatured proteins in response to cell stress [6]. They also mediate general protein turnover and play central roles in the regulation of cellular activities such as cell division, metabolism, DNA repair and transcription [7]. Proteasomes consist of a 20S proteolytic core particle (CP) that associates with AAA ‘unfoldases’ (a subfamily of the AAA+ superfamily) to catalyze the energy-dependent and processive degradation of proteins [1]. The CP is a large cylindrical complex of four stacked heptameric rings that are in an a7b7b7a7 stoichiometry. The outermost a-rings form a gate at each end of the cylinder, which restricts folded proteins from accessing the central channel that connects two antechambers to a central chamber (formed by the two b-rings). Proteolysis occurs within the central chamber at active centers. These are formed by autocatalytic processing of b-preproteins to expose the aminoterminal nucleophile (Ntn) threonine [8,9]. The high concentration of active sites (hundreds of mM) within the CP ensures multi-cleavage of most polypeptides into fragments of 3–30 amino acids in length [10]. The small cleavage products apparently diffuse out of the proteolytic chamber, whereas the export of longer peptides might require an ATPase partner [11]. AAA proteins associate with proteasomes and serve multiple roles in regulating the degradation of proteins in the cell [7,12]. Most archaea encode homologs of the regulatory particle type 1 AAA (Rpt) subunits of the 19S cap that associate with CPs to form 26S proteasomes in eukaryotes. The Rpt homolog of Methanocaldococcus jannaschii proteasome-activating nucleotidase (MjPAN, also known as MJ1176) forms an irregular ring-shaped ATPase dodecamer that associates with CPs [13,14]. In the presence of ATP or CTP, MjPAN stimulates CP-mediated degradation of proteins (e.g. casein and green fluorescent protein [GFP]-SsrA). Substrate binding to this AAA activates nucleotide hydrolysis, which successively promotes substrate unfolding, opening of the axial gate, and possibly www.sciencedirect.com
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substrate translocation into the CP [15,16]. Although proteasomal CPs are universally distributed, not all archaea encode Rpt homologs (i.e. Thermoplasma and Pyrobaculum sp.). Thus, it is tempting to speculate that homologs of the AAA Cdc48, which are widespread among archaea, might also facilitate proteasome-mediated degradation. This is based on the findings in eukaryotes that implicate Cdc48 in the ATP-dependent movement of substrates to proteasomes for degradation [17]. Complete genome sequences reveal that a wide variety of organisms, including archaea, carry duplicated genes predicted to encode the proteasomal CP and regulatory particle subtypes [1,18]. Differential expression of these genes might diversify the functional capacity of proteasomes in the cell. Examples that support this theory include vertebrates that encode an interferon-g-inducible immunoproteasome that facilitates immune responses [19] and insects that synthesize a spermatogenesis-specific proteasome [20]. CP and 19S cap isoforms have also been detected in plants [21,22] and in human erythrocytes [23]. In addition, a distant relative of the 19S cap, the constitutive photomorphogenic phenotype 9 (COP9) signalsome, has been shown to regulate proteasomemediated protein turnover in eukaryotes [24]. Like eukaryotes, several archaea encode paralogs of CP and AAA regulatory particle subunits, which suggests that they too synthesize proteasomal subtypes [1]. Consistent with this, the halophilic archaeon Haloferax volcanii produces three CP proteins (a1, a2 and b) and two PAN proteins (PanA and PanB) [25,26]. The levels of a1, b and PanA are relatively constant and abundant during ‘normal’ growth, which suggests that these are the housekeeping components [26]. By contrast, the levels of a2 and PanB are relatively low in log phase, but increase several-fold as cells enter stationary phase; this suggests these paralogs provide an ancillary role that diversifies proteasome function. The subunit topology of the Hf. volcanii CP subtypes has been determined, and it is now known to include two complexes — one of a1 and b composition and the other of a1, a2 and b composition [27]. The latter complex is asymmetric, with rings of a1 forming one end of the cylinder and those of a2 forming the other. Although the two a proteins are closely related (70% identical) they have structural differences, for example, variations in residues that precede the aminoterminal a-helix at each end of the cylinder and in the loop that restricts the channel opening. Thus, modulation of the ratio of these a subunits might control distinct structural domains at the ends of the CP. This in turn is expected to influence CP gating, the type of AAA regulatory proteins that associate with the CP, and/or the type of substrate recognized for destruction.
CP proteolytic active site and GFP reporter proteins have been used in cell culture. An early study used tri-peptide carboxybenzyl–leucyl–leucyl–leucine vinyl sulfone (ZL3VS) to inhibit proteasomes in Thermoplasma acidophilum [28]. Z-L3VS is a potent inhibitor of CPs and was shown to modify 75–80% of the proteasomal b-subunits in cell culture. Inhibition of the CPs had only a marginal effect on growth of whole cells under normal conditions but arrested growth under heat shock conditions. By contrast, a more recent study used clasto-lactacystin blactone to inhibit the CPs of Hf. volcanii [29]. Addition of clasto-lactacystin b-lactone reduced the growth rate of these cells, suggesting that CP activity is required for their ‘normal’ growth. This study [29] also revealed that a soluble, modified, red-shifted derivative of GFP (smRSGFP) could be synthesized and readily detected in recombinant Hf. volcanii cells. The smRSGFP is a GFP variant with F99S, M153T, V163A and S65T modifications (where single letter code is used for amino acids). Addition of amino acid residues of various sequences and lengths to the carboxy-terminus of this GFP had a differential effect on the levels of this reporter protein in cell culture. Proteasomes were found to be responsible, at least in part, for modulating the levels of reporter proteins with carboxy-terminal extensions.
Ubiquitin enigma The way in which proteins are recognized for destruction by archaeal proteasomes remains to be elucidated. In eukaryotes, most proteins targeted for proteasomemediated degradation are covalently linked to chains of polyubiquitin (poly-Ub) [30]. The formation of these chains is often modulated by protein phosphorylation [24], glycosylation [31] and/or acetylation [32,33]. A combination of structural and sequence similarity approaches have predicted several proteins that have Ub-like folds distributed in all three domains [34–36]. In prokaryotes, these Ub-like homologs can be classified into two superfamilies: 2Fe-2S ferredoxins and MoaD/ ThiS proteins of the molybdenum and thiamin cofactor biosynthetic pathway [34]. The structural homology of these proteins to Ub combined with their similar sulfur chemistry suggests that they share a common ancestor with eukaryotic Ub. More recently, a crystal structure of the archaeal nascent polypeptide-associated complex (i.e. MTH177) was determined, and the carboxy-terminal domain was found to be structurally related to eukaryal Ub-associated proteins [37]. It is unknown if any of these proteins participate in proteasome-mediated proteolysis in archaea; however, it is quite probable that posttranslational mechanisms that alter protein conformation trigger regulated proteolysis.
Lon proteases To understand the role of archaeal proteasomes within the context of whole cells, irreversible inhibitors of the www.sciencedirect.com
Lon protease domains are highly conserved and widely distributed among Archaea, Bacteria and Eukarya [38]. Current Opinion in Microbiology 2005, 8:720–728
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Figure 1
Archaeal Lon homologs display a high degree of variability in domain organization. (a) Schematic representation of the domain organization of archaeal Lon homologs compared to bacterial, mitochondrial and birnaviridae Lon proteases. The arrow indicates the genetic cleavage used to
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Figure 2
Archaeal homologs of the intramembrane-cleaving proteases of the SPP family. These are aspartyl proteases. (a) Schematic of an archaeal homolog of the SPP family. Motifs that include the two catalytic aspartate acid residues are shown in relation to the cell membrane. F represents a conserved hydrophobic residue. (b) Multiple sequence alignment of the proteolytic active site motifs of archaeal SPP homologs. Residues that form the active site are highlighted in red in (a) and are also indicated with an arrowhead in (b). Identical and similar residues within each group highlighted in black and grey, respectively. GenBank accession and gene locus tag numbers are indicated on the left. Amino acid residue numbers are indicated on the right and left. VNG2285c* and rrnAC2525*, haloarchaeal SPP homologs, have been reannotated to include the upstream region that encodes the proteolytic active site Asp residues. Organism abbreviations are as in Figure 1, with the following additions: MK, Methanopyrus kandleri; Mvo, Methanococcus voltae; SSO, Sulfolobus solfataricus; ST, Sulfolobus tokodaii.
Of these, the LonA proteases of Escherichia coli (also known as La or EcLonA) and of the yeast mitochondria (also known as ScPIM1 or LonA) have been most thoroughly characterized. A single gene encodes the Lon amino-terminal domain (LAN), the central AAA+ domain, and the carboxy-terminal proteolytic domain
(P-domain) that contains a Ser–Lys catalytic dyad responsible for the hydrolysis of peptide bonds [39] (Figure 1). The Lon polypeptide associates as a hexameric (bacteria) or heptameric (yeast) ring-like particle that functions as an energy-dependent protease in the bacterial cytosol and in the mitochondrial matrix [40,41].
(Figure 1 Legend Continued) generate the separate ScLonA subdomains that only restore Lon function when expressed together in vivo [43]. GenBank accession numbers, gene locus tag numbers (e.g. MA1862) and cluster of orthologous group (COG) numbers are indicated on the right. Predicted signal sequences, transmembrane spanning domains (TMs), Lon amino-terminal domains (LAN), AAA+ domains and protease domains are included. (b) Multiple sequence alignment of Lon proteolytic active site motifs including the type I Ser–Lys dyad of E. coli LonA and type II Ser–Lys–Asp triad of MJ1417. Active site residues are highlighted in red and are indicated with an arrowhead. Proline residues that might promote formation of the Ser–Lys catalytic dyad and disrupt the a-helix of the catalytic triad are highlighted in green and are marked with a circle. Identical and similar residues highlighted in black and grey, respectively. Gene locus tag numbers or abbreviations used in the main body of text are indicated on left. Amino acid residue numbers are indicated on right and left. ‘_m’ indicates a polypeptide that has had its intein excised. Organism abbreviations are as follows: AF, Archaeoglobus fulgidus; APE, Aeropyrum pernix; Ec, Escherichia coli; FAC, Ferroplasma acidarmanus; MA, Methanosarcina acetivorans; MJ, Methanocaldococcus jannaschii; MM, Methanosarcina mazei; MMP, Methanococcus maripaludis; MTH, Methanothermobacter thermoautotrophicus; NEQ, Nanoarchaeum equitans; PAB, Pyrococcus abyssi; PAE, Pyrobaculum aerophilum; PF, Pyrococcus furiosus; PH, Pyrococcus horikoshii(shinkaj); PTO, Picrophilus torridus; rrnAC, Haloarcula marismortui; Sc, Saccharomyces cerevisiae; Ta, Thermoplasma acidophilum; TK, Thermococcus kodakaraensis; TVN, Thermoplasma volcanium; VNG, Halobacterium sp. NRC-1; VP4, virus protein 4 of infectious bursal disease virus strain P2. www.sciencedirect.com
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The Lon homologs of archaea are highly diverse in domain organization (Figure 1). With the rare exception of the LonA homologs of methanosarcina (MA1862 and MM3118), most lack the LAN domain. Interestingly, several of the Lon homologs of pyrococcus (PAB1313, PH0452 and PF0467) have retained inteins, which are protein splicing elements that catalyze their own excision and concomitant ligation of the flanking protein sequences [42]. Although the biological role of inteins is unclear, it has been proposed that genes encoding intein-containing proteins are vital for host survival [42]. One common theme among the Lon homologs of archaea is the presence of putative signal sequences and/or trans-
membrane-spanning helices, which suggests that most are membrane-associated. Many contain both AAA+ and P-domains, however, there are numerous members that have only a single ATPase or protease catalytic-site motif and, thus, they alone are not predicted to hydrolyze folded proteins. Instead, these latter homologs might associate with partners to facilitate this hydrolysis by way of an energy-dependent mechanism. This would be in analogy to the finding that the AAA+ and P-domains of ScLonA can be expressed as separate polypeptides yet still restore Lon function [43]. Archaeal Lon homologs might also function in the catalysis of protein folding [44] and in the binding of inorganic polyphosphate, DNA and RNA [45,46] in analogy to other Lon proteases.
Figure 3
Archaeal homologs of the intramembrane-cleaving proteases of the S2P family. These are metalloproteases. (a) Schematic of an archaeal homolog of the S2P family. The catalytic site motif is shown in relation to the cell membrane. The number of transmembrane domains between the two domains represented in the figure vary among the archaeal S2P homologs. (b) Multiple sequence alignment of the proteolytic active site motifs of archaeal S2P homologs. Residues that form the active site are highlighted in red in (a) and are also indicated with an arrowhead in (b). Identical and similar residues within each group are highlighted in black and grey, respectively. GenBank accession and gene locus tag numbers are indicated on the left. Amino acid residue numbers are indicated on the right and left. Organism abbreviations are as in Figure 1, with the following additions: MK, Methanopyrus kandleri; SSO, Sulfolobus solfataricus. The sequence alignment has been split into 4 groups (Grp 1–4) based on similarities and differences in the number and sequence of the amino acids that bridge the HExxH and LDG motifs or the absence of conserved amino acid residues. Current Opinion in Microbiology 2005, 8:720–728
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Many archaeal LonB homologs have been characterized including the Thermococcus kodakaraensis TK1264 (TkLonB) [47], Thermoplasma acidophilum Ta1081 (TaLonB) [48,49], Methanocaldococcus jannaschii MJ1417 (MjLonB) [50] and Archaeoglobus fulgidus AF0364 (AfLonB) [51]. All have similar domain organization, which includes an amino-terminal AAA+ domain that has two transmembrane-spanning helices and a carboxy-terminal P-domain (Figure 1). Biochemical studies of LonB have been limited to proteins purified from recombinant E. coli, such as full-length TkLonB [47], TaLonB [48,49] and AfLonB [51] as well as the P-domains of AfLonB [51] and MjLonB [50]. Of these proteins, only TaLonB was shown to form a membraneassociated, hexameric-ring-shaped particle [49]. Western blot analysis, however, suggested that TkLonB and TaLonB are membrane-associated in their native hosts [47,49]. All three of the full-length LonB proteins that have been purified catalyze ATP-dependent proteolysis [47,49,51]. To further understand this catalytic mechanism, site-directed mutagenesis has been per-
formed on TaLonB [48,49] and on AfLonB [51]. In addition, atomic resolution crystal structures of a dimeric form of the MjLonB P-domain [50] and of the hexameric AfLonB P-domain [51] have been determined and compared to that of EcLonA [40]. From these results, a Ser–Lys–Asp catalytic triad distinct from the canonical catalytic dyad of LonA proteases was proposed [50] (Figure 1b). However, in the future, studies that examine the structure of proteolytically active derivatives of LonB are required to fully confirm and understand this catalytic mechanism.
Intramembrane-cleaving protease homologs Intramembrane-cleaving proteases (I-CLiPs) catalyze the hydrolysis of peptide bonds within lipid bilyaers [52]. In eukaryotes and bacteria, many of these integral membrane proteases have been shown to promote the controlled release of membrane-anchored regulatory proteins (e.g. transcription factors) or to liberate small peptides that can function as reporters and/or effectors in cell signaling (e.g. quorum sensing).
Figure 4
Archaeal homologs of the intramembrane-cleaving proteases of the RHO family. These are serine proteases. (a) Schematic of an archaeal homolog of the RHO family. The catalytic site motif is shown in relation to the cell membrane. (b) Multiple sequence alignment of the proteolytic active site motifs of archaeal RHO homologs. Residues that form the active site are highlighted in red in (a) and are also indicated with an arrowhead in (b). The RHO Asn residue that might provide a water molecule for the deacylation step of proteolysis is highlighted in green in (a) and is also marked with a circle in (b). Identical and similar residues within each group are highlighted in black and grey, respectively. GenBank accession and gene locus tag numbers are indicated on the left. Amino acid residue numbers are indicated on the right and left. Organism abbreviations are as in Figure 1, with the following additions: Dm, Drosophila melanogaster; MK, Methanopyrus kandleri; SSO, Sulfolobus solfataricus. www.sciencedirect.com
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Homologs of all three mechanistic classes of I-CLiPs are widely distributed in archaea, and all contain putative multi-spanning transmembrane regions. These include members of the g-secretase or type IV signal peptide peptidase (SPP), site-2 protease (S2P) and rhomboid (RHO) families (Figures 2, 3 and 4). Of these, only the SPPs of Methanococcus voltae (FlaK) [53] and Sulfolobus solfataricus (PibD, SSO0131) [54] have been characterized. When heterologously expressed in E. coli, the genes encoding FlaK and PibD promote the processing of type IV prepilin-like signal sequences of flagellins and of other secretory proteins. Site-directed mutagenesis of the M. voltae FlaK revealed that two highly conserved aspartic acid residues (Asp17 and Asp79) form a proteolytic active-site that is likely to reside close to the membrane [53].
The study of archaeal proteases and their mode of regulation has contributed to our overall understanding of the physiology of these unusual organisms, many of which are extremophiles, and will continue to do so in the future. It will be interesting to see what proteins and pathways are regulated by proteases and how these are recognized for destruction in the archaeal cell.
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The S2P and RHO homologs of archaea are likely to be regulatory proteases, based on analogy to their counterparts in eukaryotes and bacteria [52]. However, none of the archaeal homologs have been characterized to date. Most archaeal S2P homologs have HExxH and LDG motifs, in which the conserved Asp residue is predicted to act together with the two His residues to coordinate the proteolytic active-site zinc (Figure 2). Like the archaeal SPPs, the catalytic-site motifs of S2P homologs are often close to, but not entirely within, the membrane. Thus, these homologs are not likely to be true I-CLiPs and might represent ancestors of the eukaryal S2Ps that exploit the interior of lipid bilayers for proteolysis. In contrast to SPP and S2P, archaeal homologs of RHO have retained catalytic-site motifs that reside within putative transmembrane-spanning helices [55]. These include the GxS(G/A) and Hxx(G/A) motifs proposed to form a Ser– His catalytic dyad [56]. The Asn residue, recently postulated to provide a water molecule for the deacylation step of proteolysis, is also conserved [56,57]. It remains to be determined if these putative archaeal proteases are involved in the regulation of quorum sensing, signal transduction or other cellular processes.
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Conclusions Regulatory proteases, which catalyze the precisely-timed and rapid turnover of key cellular proteins, are widely distributed in all three domains of life. In archaea, regulatory proteases are likely to include not only energydependent proteasomes and Lon proteases but also I-CLiPs of the S2P and RHO classes. Archaeal 20S proteasomes are anticipated to combine with a variety of AAA+ proteins (e.g. PAN and Cdc48 homologs) to mediate the quality control and regulated turnover of most cytosolic proteins. Archaeal proteasomes might also participate with membrane-associated Lon proteases in the retrograde translocation and degradation of proteins in the cell membrane. Although the archaeal I-CliP homologs are not likely to mediate protein quality control, these putative proteases might function in cell signaling. Current Opinion in Microbiology 2005, 8:720–728
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
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17. Richly H, Rape M, Braun S, Rumpf S, Hoege C, Jentsch S: A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting. Cell 2005, 120:73-84. The authors present a model to describe the delivery of a subset of substrates to eukaryal proteasomes. This includes a sequential hand-off of substrate proteins from a recruiting complex (Cdc48–Ufd1–Npl4) to a multi-Ub chain assembly complex (Ufd2) and then to either Rad23 or Dsk2, which ultimately deliver substrates to the proteasome. Cdc48 homologs are highly conserved in archaea and might provide a mechanism for the recruitment of substrates to proteasomes. 18. Smalle J, Vierstra RD: The ubiquitin 26S proteasome proteolytic pathway. Annu Rev Plant Biol 2004, 55:555-590. 19. Van den Eynde BJ, Morel S: Differential processing of class-Irestricted epitopes by the standard proteasome and the immunoproteasome. Curr Opin Immunol 2001, 13:147-153. 20. Ma J, Katz E, Belote JM: Expression of proteasome subunit isoforms during spermatogenesis in Drosophila melanogaster. Insect Mol Biol 2002, 11:627-639. 21. Shibahara T, Kawasaki H, Hirano H: Mass spectrometric analysis of expression of ATPase subunits encoded by duplicated genes in the 19S regulatory particle of rice 26S proteasome. Arch Biochem Biophys 2004, 421:34-41. 22. Yang P, Fu H, Walker J, Papa CM, Smalle J, Ju YM, Vierstra RD: Purification of the Arabidopsis 26 S proteasome: biochemical and molecular analyses revealed the presence of multiple isoforms. J Biol Chem 2004, 279:6401-6413. 23. Claverol S, Burlet-Schiltz O, Girbal-Neuhauser E, Gairin JE, Monsarrat B: Mapping and structural dissection of human 20 S proteasome using proteomic approaches. Mol Cell Proteomics 2002, 1:567-578. 24. Harari-Steinberg O, Chamovitz DA: The COP9 signalosome: mediating between kinase signaling and protein degradation. Curr Protein Pept Sci 2004, 5:185-189. 25. Wilson HL, Aldrich HC, Maupin-Furlow JA: Halophilic 20S proteasomes of the archaeon Haloferax volcanii: purification, characterization, and gene sequence analysis. J Bacteriol 1999, 181:5814-5824. 26. Reuter CJ, Kaczowka SJ, Maupin-Furlow JA: Differential regulation of the PanA and PanB proteasome-activating nucleotidase and 20S proteasomal proteins of the haloarchaeon Haloferax volcanii. J Bacteriol 2004, 186:7763-7772. A model is presented in which the haloarchaeon Hf. volcanii regulates the ratio of the different 20S proteasome proteins (a1, a2 and b) and PAN proteins (PanA and PanB) to modulate the structure and ultimately the function of proteasomes in the cell. 27. Kaczowka SJ, Maupin-Furlow JA: Subunit topology of two 20S proteasomes from Haloferax volcanii. J Bacteriol 2003, 185:165-174. 28. Ruepp A, Eckerskorn C, Bogyo M, Baumeister W: Proteasome function is dispensable under normal but not under heat shock conditions in Thermoplasma acidophilum. FEBS Lett 1998, 425:87-90. 29. Reuter CJ, Maupin-Furlow JA: Analysis of proteasome dependent proteolysis in Haloferax volcanii cells using shortlived green fluorescent proteins. Appl Environ Microbiol 2004, 70:7530-7538. A convenient reporter system was developed by functional expression of GFP variants that had carboxy-terminal fusions in Hf. volcanii. Accumulation of reporter protein was dependent on the sequence of the carboxyterminal fusion, as well as on the presence of an irreversible, proteasomespecific inhibitor in cell culture. These findings provide a powerful tool for the elucidation of protein substrate recognition motifs and for the identification of new genes that might be involved in the proteasome pathway of archaea. 30. Ciechanover A: The ubiquitin-proteasome proteolytic pathway. Cell 1994, 79:13-21. 31. Yoshida Y, Chiba T, Tokunaga F, Kawasaki H, Iwai K, Suzuki T, Ito Y, Matsuoka K, Yoshida M, Tanaka K, Tai T: E3 ubiquitin ligase that recognizes sugar chains. Nature 2002, 418:438-442. www.sciencedirect.com
32. Li M, Luo J, Brooks CL, Gu W: Acetylation of p53 inhibits its ubiquitination by Mdm2. J Biol Chem 2002, 277:50607-50611. 33. Giandomenico V, Simonsson M, Gronroos E, Ericsson J: Coactivator-dependent acetylation stabilizes members of the SREBP family of transcription factors. Mol Cell Biol 2003, 23:2587-2599. 34. Bienkowska JR, Hartman H, Smith TF: A search method for homologs of small proteins. Ubiquitin-like proteins in prokaryotic cells? Protein Eng 2003, 16:897-904. 35. Rudolph MJ, Wuebbens MM, Rajagopalan KV, Schindelin H: Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation. Nat Struct Biol 2001, 8:42-46. 36. Wang C, Xi J, Begley TP, Nicholson LK: Solution structure of ThiS and implications for the evolutionary roots of ubiquitin. Nat Struct Biol 2001, 8:47-51. 37. Spreter T, Pech M, Beatrix B: The crystal structure of archaeal nascent polypeptide-associated complex (NAC) reveals a unique fold and the presence of a ubiquitin-associated domain. J Biol Chem 2005, 280:15849-15854. This study presents the first crystal structure of a nascent polypeptideassociated complex protein (i.e. MTH177). A novel protein fold that mediates complex dimerization was identified and a Ub-associated domain was discovered at the carboxy terminus of the protein. The role that this Ub-association domain plays in archaea is not known. 38. Iyer LM, Leipe DD, Koonin EV, Aravind L: Evolutionary history and higher order classification of AAA+ ATPases. J Struct Biol 2004, 146:11-31. All available sequences and structures of AAA+ protein domains were compared in silico. Based on these results, the authors suggest that AAA+ proteins cluster into 26 major families, which include previously undetected protein domains and new relationships. The results are likely to provide leads for investigating the biology of AAA+ ATPases. 39. Rotanova TV, Melnikov EE, Khalatova AG, Makhovskaya OV, Botos I, Wlodawer A, Gustchina A: Classification of ATPdependent proteases Lon and comparison of the active sites of their proteolytic domains. Eur J Biochem 2004, 271:4865-4871. 40. Botos I, Melnikov EE, Cherry S, Tropea JE, Khalatova AG, Rasulova F, Dauter Z, Maurizi MR, Rotanova TV, Wlodawer A, Gustchina A: The catalytic domain of E.coli Lon protease has a unique fold and a Ser–Lys dyad in the active site. J Biol Chem 2004, 279:8140-8148. A crystal structure of a hexameric ring of the EcLonA P-domain was solved to 1.75 A˚ resolution. Based on alignment of the active site pocket with those of several Ser–Lys dyad peptidases, a model of substrate binding and polypeptide bond hydrolysis by way of a Ser–Lys catalytic dyad was proposed. 41. Stahlberg H, Kutejova E, Suda K, Wolpensinger B, Lustig A, Schatz G, Engel A, Suzuki CK: Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits. Proc Natl Acad Sci USA 1999, 96:6787-6790. 42. Pietrokovski S: Intein spread and extinction in evolution. Trends Genet 2001, 17:465-472. 43. van Dijl JM, Kutejova E, Suda K, Perecko D, Schatz G, Suzuki CK: The ATPase and protease domains of yeast mitochondrial Lon: roles in proteolysis and respiration-dependent growth. Proc Natl Acad Sci USA 1998, 95:10584-10589. 44. Rep M, van Dijl JM, Suda K, Schatz G, Grivell LA, Suzuki CK: Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon. Science 1996, 274:103-106. 45. Liu T, Lu B, Lee I, Ondrovicova G, Kutejova E, Suzuki CK: DNA and RNA binding by the mitochondrial lon protease is regulated by nucleotide and protein substrate. J Biol Chem 2004, 279:13902-13910. 46. Nomura K, Kato J, Takiguchi N, Ohtake H, Kuroda A: Effects of inorganic polyphosphate on the proteolytic and DNA-binding activities of Lon in Escherichia coli. J Biol Chem 2004, 279:34406-34410. Current Opinion in Microbiology 2005, 8:720–728
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47. Fukui T, Eguchi T, Atomi H, Imanaka T: A membrane-bound archaeal Lon protease displays ATP-independent proteolytic activity towards unfolded proteins and ATP-dependent activity for folded proteins. J Bacteriol 2002, 184:3689-3698. 48. Besche H, Zwickl P: The Thermoplasma acidophilum Lon protease has a Ser–Lys dyad active site. Eur J Biochem 2004, 271:4361-4365. 49. Besche H, Tamura N, Tamura T, Zwickl P: Mutational analysis of conserved AAA+ residues in the archaeal Lon protease from Thermoplasma acidophilum. FEBS Lett 2004, 574:161-166. This study provides the only example to date of an archaeal LonB protease that forms a membrane-associated, hexameric ring-shaped particle when produced in recombinant E. coli. 50. Im YJ, Na Y, Kang GB, Rho SH, Kim MK, Lee JH, Chung CH, Eom SH: The active site of a Lon protease from Methanococcus jannaschii distinctly differs from the canonical catalytic dyad of Lon proteases. J Biol Chem 2004, 279:53451-53457. A crystal structure of the P-domain of MjLonB was solved to 1.9 A˚ resolution. Although the structure displays an overall fold comparable to the P-domain of EcLonA, uniquely configured Ser–Lys–Asp residues are proposed to form a catalytic triad. 51. Botos I, Melnikov EE, Cherry S, Kozlov S, Makhovskaya OV, Tropea JE, Gustchina A, Rotanova TV, Wlodawer A: Atomic-resolution crystal structure of the proteolytic domain of Archaeoglobus fulgidus Lon reveals the conformational variability in the active sites of Lon proteases. J Mol Biol 2005, 351:144-157. A 1.2 A˚ crystal structure of the P-domain of AfLonB was solved and compared to that of MjLonB and EcLonA. Despite their similarity in overall fold, the conformation of the active-site residues differs in all three structures. Site-directed mutagenesis of full-length AfLonB revealed that Ser509 was essential for proteolytic activity, whereas Glu506 and Asp508 were not. The Glu506 corresponds in primary sequence alignment to MjLonB Asp547 residue implicated by Im et al. [50] in the Ser–His–Asp
catalytic triad. Thus, the authors caution that the triad might be a premature proposal and suggest that the different arrangements of the active site residues observed in all three crystal structures might have been seen because analysis was performed on only the P-domains and not on the native enzyme. 52. Weihofen A, Martoglio B: Intramembrane-cleaving proteases: controlled liberation of proteins and bioactive peptides. Trends Cell Biol 2003, 13:71-78. 53. Bardy SL, Jarrell KF: Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae. Mol Microbiol 2003, 50:1339-1347. 54. Albers S-V, Szabo Z, Driessen AJM: Archaeal homolog of bacterial Type IV prepilin signal peptidases with broad substrate specificity. J Bacteriol 2003, 185:3918-3925. 55. Koonin EV, Makarova KS, Rogozin IB, Davidovic L, Letellier MC, Pellegrini L: The rhomboids: a nearly ubiquitous family of intramembrane serine proteases that probably evolved by multiple ancient horizontal gene transfers. Genome Biol 2003, 4:R19. 56. Lemberg MK, Menendez J, Misik A, Garcia M, Koth CM, Freeman M: Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases. EMBO J 2005, 24:464-472. Owing to the extreme hydrophobicity and the presence of multiple transmembrane-spanning helices, intramembrane proteases have been difficult to purify. The authors developed an in vitro assay to monitor RHO activity, which allowed purification of proteolytically active forms of the Bacillus subtilis YqgP (BsYqgP) and E. coli GlpG RHOs from recombinant E. coli. Site-directed mutagenesis of BsYqgP suggests that RHOs use a Ser–His catalytic dyad instead of a Ser–His–Asn triad. 57. Urban S, Lee JR, Freeman M: Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases. Cell 2001, 107:173-182.
Free journals for developing countries The WHO and six medical journal publishers have launched the Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the Internet. The science publishers, Blackwell, Elsevier, the Harcourt Worldwide STM group, Wolters Kluwer International Health and Science, Springer-Verlag and John Wiley, were approached by the WHO and the British Medical Journal in 2001. Initially, more than 1000 journals will be available for free or at significantly reduced prices to universities, medical schools, research and public institutions in developing countries. The second stage involves extending this initiative to institutions in other countries. Gro Harlem Brundtland, director-general for the WHO, said that this initiative was ‘perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’. For more information, visit http://www.healthinternetwork.net
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Current Opinion in Microbiology 2005, 8:720–728 This review comes from a themed issue on Growth and development Edited by John N Reeve and Ruth Schmitz Available online 26th October 2005 1369-5274/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2005.10.005
Introduction Insight into the world of proteolysis has expanded considerably over the past decade. Proteases are no longer viewed as non-specific degradative enzymes associated solely with protein catabolism, but are known to be intimately involved in controlling biological processes that span life to death. Proteases maintain this exquisite control by catalyzing the precisely-timed and rapid turnover of key regulatory proteins. Proteases also interplay with chaperones to ensure protein quality and to readjust the composition of the proteome following stress. Although much less is known about proteolysis in the context of an archaeal cell than that of bacteria and eukaryotes, recent advances in genomics and biochemistry provide unprecedented insight into the complete repertoire of archaeal proteases that might regulate cell function. In this review, we provide a brief overview of archaeal proteases that are likely to be involved in regulation. Emphasis Current Opinion in Microbiology 2005, 8:720–728
will be on our current understanding of energy-dependent proteases such as proteasomes and Lon as well as homologs of intramembrane-cleaving proteases. The reader is referred to additional reviews [1–4] for more detailed discussions of archaeal proteases and peptidases.
Proteasomes and proteasome-associated nucleotidases Proteasomes are proteolytic nanomachines that are distributed in all three domains of life: Bacteria, Archaea and Eukarya [5]. These enzymes maintain protein quality control by degrading misfolded and denatured proteins in response to cell stress [6]. They also mediate general protein turnover and play central roles in the regulation of cellular activities such as cell division, metabolism, DNA repair and transcription [7]. Proteasomes consist of a 20S proteolytic core particle (CP) that associates with AAA ‘unfoldases’ (a subfamily of the AAA+ superfamily) to catalyze the energy-dependent and processive degradation of proteins [1]. The CP is a large cylindrical complex of four stacked heptameric rings that are in an a7b7b7a7 stoichiometry. The outermost a-rings form a gate at each end of the cylinder, which restricts folded proteins from accessing the central channel that connects two antechambers to a central chamber (formed by the two b-rings). Proteolysis occurs within the central chamber at active centers. These are formed by autocatalytic processing of b-preproteins to expose the aminoterminal nucleophile (Ntn) threonine [8,9]. The high concentration of active sites (hundreds of mM) within the CP ensures multi-cleavage of most polypeptides into fragments of 3–30 amino acids in length [10]. The small cleavage products apparently diffuse out of the proteolytic chamber, whereas the export of longer peptides might require an ATPase partner [11]. AAA proteins associate with proteasomes and serve multiple roles in regulating the degradation of proteins in the cell [7,12]. Most archaea encode homologs of the regulatory particle type 1 AAA (Rpt) subunits of the 19S cap that associate with CPs to form 26S proteasomes in eukaryotes. The Rpt homolog of Methanocaldococcus jannaschii proteasome-activating nucleotidase (MjPAN, also known as MJ1176) forms an irregular ring-shaped ATPase dodecamer that associates with CPs [13,14]. In the presence of ATP or CTP, MjPAN stimulates CP-mediated degradation of proteins (e.g. casein and green fluorescent protein [GFP]-SsrA). Substrate binding to this AAA activates nucleotide hydrolysis, which successively promotes substrate unfolding, opening of the axial gate, and possibly www.sciencedirect.com
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substrate translocation into the CP [15,16]. Although proteasomal CPs are universally distributed, not all archaea encode Rpt homologs (i.e. Thermoplasma and Pyrobaculum sp.). Thus, it is tempting to speculate that homologs of the AAA Cdc48, which are widespread among archaea, might also facilitate proteasome-mediated degradation. This is based on the findings in eukaryotes that implicate Cdc48 in the ATP-dependent movement of substrates to proteasomes for degradation [17]. Complete genome sequences reveal that a wide variety of organisms, including archaea, carry duplicated genes predicted to encode the proteasomal CP and regulatory particle subtypes [1,18]. Differential expression of these genes might diversify the functional capacity of proteasomes in the cell. Examples that support this theory include vertebrates that encode an interferon-g-inducible immunoproteasome that facilitates immune responses [19] and insects that synthesize a spermatogenesis-specific proteasome [20]. CP and 19S cap isoforms have also been detected in plants [21,22] and in human erythrocytes [23]. In addition, a distant relative of the 19S cap, the constitutive photomorphogenic phenotype 9 (COP9) signalsome, has been shown to regulate proteasomemediated protein turnover in eukaryotes [24]. Like eukaryotes, several archaea encode paralogs of CP and AAA regulatory particle subunits, which suggests that they too synthesize proteasomal subtypes [1]. Consistent with this, the halophilic archaeon Haloferax volcanii produces three CP proteins (a1, a2 and b) and two PAN proteins (PanA and PanB) [25,26]. The levels of a1, b and PanA are relatively constant and abundant during ‘normal’ growth, which suggests that these are the housekeeping components [26]. By contrast, the levels of a2 and PanB are relatively low in log phase, but increase several-fold as cells enter stationary phase; this suggests these paralogs provide an ancillary role that diversifies proteasome function. The subunit topology of the Hf. volcanii CP subtypes has been determined, and it is now known to include two complexes — one of a1 and b composition and the other of a1, a2 and b composition [27]. The latter complex is asymmetric, with rings of a1 forming one end of the cylinder and those of a2 forming the other. Although the two a proteins are closely related (70% identical) they have structural differences, for example, variations in residues that precede the aminoterminal a-helix at each end of the cylinder and in the loop that restricts the channel opening. Thus, modulation of the ratio of these a subunits might control distinct structural domains at the ends of the CP. This in turn is expected to influence CP gating, the type of AAA regulatory proteins that associate with the CP, and/or the type of substrate recognized for destruction.
CP proteolytic active site and GFP reporter proteins have been used in cell culture. An early study used tri-peptide carboxybenzyl–leucyl–leucyl–leucine vinyl sulfone (ZL3VS) to inhibit proteasomes in Thermoplasma acidophilum [28]. Z-L3VS is a potent inhibitor of CPs and was shown to modify 75–80% of the proteasomal b-subunits in cell culture. Inhibition of the CPs had only a marginal effect on growth of whole cells under normal conditions but arrested growth under heat shock conditions. By contrast, a more recent study used clasto-lactacystin blactone to inhibit the CPs of Hf. volcanii [29]. Addition of clasto-lactacystin b-lactone reduced the growth rate of these cells, suggesting that CP activity is required for their ‘normal’ growth. This study [29] also revealed that a soluble, modified, red-shifted derivative of GFP (smRSGFP) could be synthesized and readily detected in recombinant Hf. volcanii cells. The smRSGFP is a GFP variant with F99S, M153T, V163A and S65T modifications (where single letter code is used for amino acids). Addition of amino acid residues of various sequences and lengths to the carboxy-terminus of this GFP had a differential effect on the levels of this reporter protein in cell culture. Proteasomes were found to be responsible, at least in part, for modulating the levels of reporter proteins with carboxy-terminal extensions.
Ubiquitin enigma The way in which proteins are recognized for destruction by archaeal proteasomes remains to be elucidated. In eukaryotes, most proteins targeted for proteasomemediated degradation are covalently linked to chains of polyubiquitin (poly-Ub) [30]. The formation of these chains is often modulated by protein phosphorylation [24], glycosylation [31] and/or acetylation [32,33]. A combination of structural and sequence similarity approaches have predicted several proteins that have Ub-like folds distributed in all three domains [34–36]. In prokaryotes, these Ub-like homologs can be classified into two superfamilies: 2Fe-2S ferredoxins and MoaD/ ThiS proteins of the molybdenum and thiamin cofactor biosynthetic pathway [34]. The structural homology of these proteins to Ub combined with their similar sulfur chemistry suggests that they share a common ancestor with eukaryotic Ub. More recently, a crystal structure of the archaeal nascent polypeptide-associated complex (i.e. MTH177) was determined, and the carboxy-terminal domain was found to be structurally related to eukaryal Ub-associated proteins [37]. It is unknown if any of these proteins participate in proteasome-mediated proteolysis in archaea; however, it is quite probable that posttranslational mechanisms that alter protein conformation trigger regulated proteolysis.
Lon proteases To understand the role of archaeal proteasomes within the context of whole cells, irreversible inhibitors of the www.sciencedirect.com
Lon protease domains are highly conserved and widely distributed among Archaea, Bacteria and Eukarya [38]. Current Opinion in Microbiology 2005, 8:720–728
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Figure 1
Archaeal Lon homologs display a high degree of variability in domain organization. (a) Schematic representation of the domain organization of archaeal Lon homologs compared to bacterial, mitochondrial and birnaviridae Lon proteases. The arrow indicates the genetic cleavage used to
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Figure 2
Archaeal homologs of the intramembrane-cleaving proteases of the SPP family. These are aspartyl proteases. (a) Schematic of an archaeal homolog of the SPP family. Motifs that include the two catalytic aspartate acid residues are shown in relation to the cell membrane. F represents a conserved hydrophobic residue. (b) Multiple sequence alignment of the proteolytic active site motifs of archaeal SPP homologs. Residues that form the active site are highlighted in red in (a) and are also indicated with an arrowhead in (b). Identical and similar residues within each group highlighted in black and grey, respectively. GenBank accession and gene locus tag numbers are indicated on the left. Amino acid residue numbers are indicated on the right and left. VNG2285c* and rrnAC2525*, haloarchaeal SPP homologs, have been reannotated to include the upstream region that encodes the proteolytic active site Asp residues. Organism abbreviations are as in Figure 1, with the following additions: MK, Methanopyrus kandleri; Mvo, Methanococcus voltae; SSO, Sulfolobus solfataricus; ST, Sulfolobus tokodaii.
Of these, the LonA proteases of Escherichia coli (also known as La or EcLonA) and of the yeast mitochondria (also known as ScPIM1 or LonA) have been most thoroughly characterized. A single gene encodes the Lon amino-terminal domain (LAN), the central AAA+ domain, and the carboxy-terminal proteolytic domain
(P-domain) that contains a Ser–Lys catalytic dyad responsible for the hydrolysis of peptide bonds [39] (Figure 1). The Lon polypeptide associates as a hexameric (bacteria) or heptameric (yeast) ring-like particle that functions as an energy-dependent protease in the bacterial cytosol and in the mitochondrial matrix [40,41].
(Figure 1 Legend Continued) generate the separate ScLonA subdomains that only restore Lon function when expressed together in vivo [43]. GenBank accession numbers, gene locus tag numbers (e.g. MA1862) and cluster of orthologous group (COG) numbers are indicated on the right. Predicted signal sequences, transmembrane spanning domains (TMs), Lon amino-terminal domains (LAN), AAA+ domains and protease domains are included. (b) Multiple sequence alignment of Lon proteolytic active site motifs including the type I Ser–Lys dyad of E. coli LonA and type II Ser–Lys–Asp triad of MJ1417. Active site residues are highlighted in red and are indicated with an arrowhead. Proline residues that might promote formation of the Ser–Lys catalytic dyad and disrupt the a-helix of the catalytic triad are highlighted in green and are marked with a circle. Identical and similar residues highlighted in black and grey, respectively. Gene locus tag numbers or abbreviations used in the main body of text are indicated on left. Amino acid residue numbers are indicated on right and left. ‘_m’ indicates a polypeptide that has had its intein excised. Organism abbreviations are as follows: AF, Archaeoglobus fulgidus; APE, Aeropyrum pernix; Ec, Escherichia coli; FAC, Ferroplasma acidarmanus; MA, Methanosarcina acetivorans; MJ, Methanocaldococcus jannaschii; MM, Methanosarcina mazei; MMP, Methanococcus maripaludis; MTH, Methanothermobacter thermoautotrophicus; NEQ, Nanoarchaeum equitans; PAB, Pyrococcus abyssi; PAE, Pyrobaculum aerophilum; PF, Pyrococcus furiosus; PH, Pyrococcus horikoshii(shinkaj); PTO, Picrophilus torridus; rrnAC, Haloarcula marismortui; Sc, Saccharomyces cerevisiae; Ta, Thermoplasma acidophilum; TK, Thermococcus kodakaraensis; TVN, Thermoplasma volcanium; VNG, Halobacterium sp. NRC-1; VP4, virus protein 4 of infectious bursal disease virus strain P2. www.sciencedirect.com
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The Lon homologs of archaea are highly diverse in domain organization (Figure 1). With the rare exception of the LonA homologs of methanosarcina (MA1862 and MM3118), most lack the LAN domain. Interestingly, several of the Lon homologs of pyrococcus (PAB1313, PH0452 and PF0467) have retained inteins, which are protein splicing elements that catalyze their own excision and concomitant ligation of the flanking protein sequences [42]. Although the biological role of inteins is unclear, it has been proposed that genes encoding intein-containing proteins are vital for host survival [42]. One common theme among the Lon homologs of archaea is the presence of putative signal sequences and/or trans-
membrane-spanning helices, which suggests that most are membrane-associated. Many contain both AAA+ and P-domains, however, there are numerous members that have only a single ATPase or protease catalytic-site motif and, thus, they alone are not predicted to hydrolyze folded proteins. Instead, these latter homologs might associate with partners to facilitate this hydrolysis by way of an energy-dependent mechanism. This would be in analogy to the finding that the AAA+ and P-domains of ScLonA can be expressed as separate polypeptides yet still restore Lon function [43]. Archaeal Lon homologs might also function in the catalysis of protein folding [44] and in the binding of inorganic polyphosphate, DNA and RNA [45,46] in analogy to other Lon proteases.
Figure 3
Archaeal homologs of the intramembrane-cleaving proteases of the S2P family. These are metalloproteases. (a) Schematic of an archaeal homolog of the S2P family. The catalytic site motif is shown in relation to the cell membrane. The number of transmembrane domains between the two domains represented in the figure vary among the archaeal S2P homologs. (b) Multiple sequence alignment of the proteolytic active site motifs of archaeal S2P homologs. Residues that form the active site are highlighted in red in (a) and are also indicated with an arrowhead in (b). Identical and similar residues within each group are highlighted in black and grey, respectively. GenBank accession and gene locus tag numbers are indicated on the left. Amino acid residue numbers are indicated on the right and left. Organism abbreviations are as in Figure 1, with the following additions: MK, Methanopyrus kandleri; SSO, Sulfolobus solfataricus. The sequence alignment has been split into 4 groups (Grp 1–4) based on similarities and differences in the number and sequence of the amino acids that bridge the HExxH and LDG motifs or the absence of conserved amino acid residues. Current Opinion in Microbiology 2005, 8:720–728
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Many archaeal LonB homologs have been characterized including the Thermococcus kodakaraensis TK1264 (TkLonB) [47], Thermoplasma acidophilum Ta1081 (TaLonB) [48,49], Methanocaldococcus jannaschii MJ1417 (MjLonB) [50] and Archaeoglobus fulgidus AF0364 (AfLonB) [51]. All have similar domain organization, which includes an amino-terminal AAA+ domain that has two transmembrane-spanning helices and a carboxy-terminal P-domain (Figure 1). Biochemical studies of LonB have been limited to proteins purified from recombinant E. coli, such as full-length TkLonB [47], TaLonB [48,49] and AfLonB [51] as well as the P-domains of AfLonB [51] and MjLonB [50]. Of these proteins, only TaLonB was shown to form a membraneassociated, hexameric-ring-shaped particle [49]. Western blot analysis, however, suggested that TkLonB and TaLonB are membrane-associated in their native hosts [47,49]. All three of the full-length LonB proteins that have been purified catalyze ATP-dependent proteolysis [47,49,51]. To further understand this catalytic mechanism, site-directed mutagenesis has been per-
formed on TaLonB [48,49] and on AfLonB [51]. In addition, atomic resolution crystal structures of a dimeric form of the MjLonB P-domain [50] and of the hexameric AfLonB P-domain [51] have been determined and compared to that of EcLonA [40]. From these results, a Ser–Lys–Asp catalytic triad distinct from the canonical catalytic dyad of LonA proteases was proposed [50] (Figure 1b). However, in the future, studies that examine the structure of proteolytically active derivatives of LonB are required to fully confirm and understand this catalytic mechanism.
Intramembrane-cleaving protease homologs Intramembrane-cleaving proteases (I-CLiPs) catalyze the hydrolysis of peptide bonds within lipid bilyaers [52]. In eukaryotes and bacteria, many of these integral membrane proteases have been shown to promote the controlled release of membrane-anchored regulatory proteins (e.g. transcription factors) or to liberate small peptides that can function as reporters and/or effectors in cell signaling (e.g. quorum sensing).
Figure 4
Archaeal homologs of the intramembrane-cleaving proteases of the RHO family. These are serine proteases. (a) Schematic of an archaeal homolog of the RHO family. The catalytic site motif is shown in relation to the cell membrane. (b) Multiple sequence alignment of the proteolytic active site motifs of archaeal RHO homologs. Residues that form the active site are highlighted in red in (a) and are also indicated with an arrowhead in (b). The RHO Asn residue that might provide a water molecule for the deacylation step of proteolysis is highlighted in green in (a) and is also marked with a circle in (b). Identical and similar residues within each group are highlighted in black and grey, respectively. GenBank accession and gene locus tag numbers are indicated on the left. Amino acid residue numbers are indicated on the right and left. Organism abbreviations are as in Figure 1, with the following additions: Dm, Drosophila melanogaster; MK, Methanopyrus kandleri; SSO, Sulfolobus solfataricus. www.sciencedirect.com
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726 Growth and development
Homologs of all three mechanistic classes of I-CLiPs are widely distributed in archaea, and all contain putative multi-spanning transmembrane regions. These include members of the g-secretase or type IV signal peptide peptidase (SPP), site-2 protease (S2P) and rhomboid (RHO) families (Figures 2, 3 and 4). Of these, only the SPPs of Methanococcus voltae (FlaK) [53] and Sulfolobus solfataricus (PibD, SSO0131) [54] have been characterized. When heterologously expressed in E. coli, the genes encoding FlaK and PibD promote the processing of type IV prepilin-like signal sequences of flagellins and of other secretory proteins. Site-directed mutagenesis of the M. voltae FlaK revealed that two highly conserved aspartic acid residues (Asp17 and Asp79) form a proteolytic active-site that is likely to reside close to the membrane [53].
The study of archaeal proteases and their mode of regulation has contributed to our overall understanding of the physiology of these unusual organisms, many of which are extremophiles, and will continue to do so in the future. It will be interesting to see what proteins and pathways are regulated by proteases and how these are recognized for destruction in the archaeal cell.
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The S2P and RHO homologs of archaea are likely to be regulatory proteases, based on analogy to their counterparts in eukaryotes and bacteria [52]. However, none of the archaeal homologs have been characterized to date. Most archaeal S2P homologs have HExxH and LDG motifs, in which the conserved Asp residue is predicted to act together with the two His residues to coordinate the proteolytic active-site zinc (Figure 2). Like the archaeal SPPs, the catalytic-site motifs of S2P homologs are often close to, but not entirely within, the membrane. Thus, these homologs are not likely to be true I-CLiPs and might represent ancestors of the eukaryal S2Ps that exploit the interior of lipid bilayers for proteolysis. In contrast to SPP and S2P, archaeal homologs of RHO have retained catalytic-site motifs that reside within putative transmembrane-spanning helices [55]. These include the GxS(G/A) and Hxx(G/A) motifs proposed to form a Ser– His catalytic dyad [56]. The Asn residue, recently postulated to provide a water molecule for the deacylation step of proteolysis, is also conserved [56,57]. It remains to be determined if these putative archaeal proteases are involved in the regulation of quorum sensing, signal transduction or other cellular processes.
2.
Ward DE, Shockley KR, Chang LS, Levy RD, Michel JK, Conners SB, Kelly RM: Proteolysis in hyperthermophilic microorganisms. Archaea 2002, 1:63-74.
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De Castro RE, Maupin-Furlow JA, Gime´nez MI, Herrera Seitz MK, Sa´nchez JJ: Haloarchaeal proteases and proteolytic systems. FEMS Microbiol Rev 2006, in press.
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Volker C, Lupas AN: Molecular evolution of proteasomes. Curr Top Microbiol Immunol 2002, 268:1-22.
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Wolf DH, Hilt W: The proteasome: a proteolytic nanomachine of cell regulation and waste disposal. Biochim Biophys Acta 2004, 1695:19-31.
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Conclusions Regulatory proteases, which catalyze the precisely-timed and rapid turnover of key cellular proteins, are widely distributed in all three domains of life. In archaea, regulatory proteases are likely to include not only energydependent proteasomes and Lon proteases but also I-CLiPs of the S2P and RHO classes. Archaeal 20S proteasomes are anticipated to combine with a variety of AAA+ proteins (e.g. PAN and Cdc48 homologs) to mediate the quality control and regulated turnover of most cytosolic proteins. Archaeal proteasomes might also participate with membrane-associated Lon proteases in the retrograde translocation and degradation of proteins in the cell membrane. Although the archaeal I-CliP homologs are not likely to mediate protein quality control, these putative proteases might function in cell signaling. Current Opinion in Microbiology 2005, 8:720–728
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
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Current Opinion in Microbiology 2005, 8:720–728
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