Protein transport in the halophilic archaeon Halobacterium sp. NRC-1: a major role for the twin-arginine translocation pathway?

Albert Bolhuis1

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK1

Tel: +44 24 76528360. Fax: +44 24 76523701. e-mail: abolhuis{at}bio.warwick.ac.uk

Keywords: archaea, Sec pathway, Tat pathway, signal peptide, lipoprotein


   Background
TOP
Background
The Sec pathway
The Tat pathway
Signal peptides
Signal peptide predictions in...
Concluding remarks
REFERENCES
 
Trafficking of proteins, which is an essential process for all living organisms, has been extensively studied in bacteria and eukarya. Very little, however, is known about protein transport in the third domain of life, the Archaea. These organisms are prokaryotes, thus have the same cellular organization as bacteria, but phylogenetic analyses showed that archaea are evolutionarily distinct from both bacteria and eukarya (Woese et al., 1990 ). A unique aspect of archaea is the composition of their cytoplasmic membrane: whereas bacterial and eukaryal membranes are composed of fatty acids linked to glycerol by an ester bond, archaeal phospholipids are composed of branched isoprene units linked to glycerol by an ether group. These lipids play an important role in surviving the extreme environments in which archaea are frequently found, as they are less permeable to ions and protons and more resistant to extreme temperatures or high salt concentrations (van de Vossenberg et al., 1998 ). Because of the unique cytoplasmic membrane and the extreme environment in which many archaea thrive, it is likely that protein translocation machineries of archaea contain specific adaptations or even completely novel components that are important for protein transport.

Systems for the transport of proteins across membranes function by common principles in all domains of life (Schatz & Dobberstein, 1996 ). Proteins destined for export are usually synthesized as pre-proteins with an amino-terminal signal peptide that is required for targeting to the membrane and initiation of protein translocation (von Heijne, 1990 ). At the membrane, pre-proteins are transported through a proteinaceous translocation channel in a process that is driven by the binding and hydrolysis of nucleoside triphosphates and/or the proton motive force. During or shortly after translocation, the signal peptide is removed from the pre-protein by signal peptidases (SPases), and the mature protein is released at the trans side of the membrane. Systems of protein transport that function according to these basic principles include protein import into mitochondria, chloroplasts and the lumen of the endoplasmic reticulum (ER), and protein export to extracytoplasmic compartments of prokaryotes, such as the periplasm and outer membrane (Gram-negative bacteria), the cell wall (Gram-positive bacteria and archaea) and the growth medium.

Halobacterium species are excellent model organisms among the archaea. These organisms are true extremophiles that thrive in an environment that is nearly saturated with salt (4–5 M NaCl). In contrast to several other archaea, halobacteria are easy to culture as they usually grow aerobically between 30 and 40 °C. Furthermore, they are genetically amenable organisms for which several genetic tools are available, such as transformation, shuttle vectors and gene knockout strategies (Sowers & Schreier, 1999 ; Peck et al., 2000 ). As with other archaea, little is known about the transport of proteins in these organisms. Nevertheless, the availability of the genomic sequences, including that of Halobacterium sp. NRC-1 (Ng et al., 2000 ), provides a wealth of information that can be used to make predictions about various cellular processes. This review will focus primarily on the two major protein transport routes found in archaea, i.e. the Sec and the Tat pathways, and the identification of proteins in Halobacterium sp. NRC-1 that may use these pathways to reach their extracytoplasmic destination. The analyses described in this review show that, in contrast to most other organisms, the majority of secretory proteins of Halobacterium sp. NRC-1 use the Tat pathway for export. In addition, there is the unexpected finding of many putative lipoproteins in this archaeon, which, as discussed below, indicates the presence of a completely novel mechanism for lipo-modification and processing in archaea.


   The Sec pathway
TOP
Background
The Sec pathway
The Tat pathway
Signal peptides
Signal peptide predictions in...
Concluding remarks
REFERENCES
 
One of the major pathways for protein translocation is the Sec pathway, which is conserved in all domains of life. In eukarya, most proteins destined for export to the lumen of the ER are translocated co-translationally via this pathway (Johnson & Waes, 1999 ). As soon as the signal peptide emerges from the ribosome, translation is arrested through binding of the signal recognition particle (SRP) to the nascent chain. In mammalian cells, SRP comprises six protein subunits (SRP9, SPR14, SRP19, SRP54, SRP68 and SRP72), which are bound to a 7S RNA scaffold (Keenan et al., 2001 ). The entire complex of the ribosome, the nascent chain and the SRP is targeted to the ER membrane with the aid of the SRP receptor subunits SR{alpha} and SRß. At the membrane, the SRP is released from the nascent chain in a process that depends on the binding of GTP, and translation is resumed. This occurs at the site of the translocase, and protein synthesis by the ribosome pushes the protein through the translocation channel. The channel is formed by the Sec61 complex, which comprises Sec61{alpha}, Sec61ß and Sec61{gamma} (Görlich & Rapoport, 1993 ). In yeast, in particular, several proteins are translocated post-translationally. In that case, transport is driven by a pulling action from Kar2p (called BiP in mammalian cells), which is an Hsp70 that resides in the lumen of the ER (Nguyen et al., 1991 ). Accessory components that are important for protein transport include the Sec62/63 and Sec71/72 complexes, both of which are involved in post-translational translocation (Johnson & Waes, 1999 ), and TRAM, which is important for the translocation and integration of most secretory proteins (Voigt et al., 1996 ). The signal peptidase complex (SPC; Evans et al., 1986 ) removes the signal peptide at the trans side of the membrane.

In bacteria, most extracytoplasmic proteins are targeted post-translationally, with the aid of a targeting factor such as SecB, to the translocase, which comprises SecY, SecE and SecG (for a review, see Manting & Driessen, 2000 ). SecY and SecE are related to the eukaryotic Sec61{alpha} and Sec61{gamma}, respectively. SecG and Sec61ß are not related, but may have the same role in protein translocation. A pivotal role in bacterial protein transport is played by the peripheral membrane protein SecA, which is an ATPase that pushes the pre-protein through the translocase channel (Economou, 1998 ). Protein translocation is assisted by accessory components such as SecD and SecF. During, or shortly after, translocation, the signal peptide is removed by a type I SPase (Dalbey et al., 1997 ) or, in case of lipoproteins (see below), by a type II SPase (Hayashi & Wu, 1990 ).

Several membrane proteins that use the Sec machinery for integration into the membrane are targeted by an SRP-like complex that comprises a homologue of SRP54 (Ffh) and a 4·5S RNA molecule (Seluanov & Bibi, 1997 ; Ulbrandt et al., 1997 ). Targeting is mediated by a homologue of the eukaryotic SR{alpha} called FtsY. For certain proteins SecA is still required for SRP-dependent translocation (Neumann-Haefelin et al., 2000 ), indicating that their translocation is not driven by the ribosome. The integral membrane protein YidC is also vital for the insertion of membrane proteins in Escherichia coli (Dalbey & Kuhn, 2000 ). This protein, which interacts with the SecYEG complex (Beck et al., 2001 ; Scotti et al., 2000 ), is also involved in the insertion of Sec-independent membrane proteins (Samuelson et al., 2000 ).

Archaea lack the chaperone SecB, but do contain an SRP pathway (Eichler & Moll, 2001 ). The archaeal components of this pathway are SRP54, SRP19 (which is absent in bacteria), 7S RNA, and a homologue of SR{alpha}/FtsY (denoted Dpa in Halobacterium sp. NRC-1). Analysis of the SRP pathway in the halophilic archaeon Haloferax volcanii showed that SRP54 is essential for viability and that it interacts with SRP19 and 7S RNA (Rose & Pohlschröder, 2002 ). All archaea contain homologues of SecY/Sec61{alpha} and SecE/Sec61{gamma} but, interestingly, these are more similar to their eukaryotic counterparts (Pohlschröder et al., 1997 ; Eichler, 2000 ). A SecG homologue is not present in archaea, but a recent study suggested the presence of a homologue of the eukaryotic protein Sec61ß (Kinch et al., 2002 ). Strikingly, archaea lack the bacterial translocation motor SecA. Thus archaea may use an alternative unique form of motor protein, or use an entirely different translocation mechanism that is possibly similar to the eukaryotic co-translational translocation mechanism. Since the archaeal translocase and SRP show more similarity to those found in eukaryotes, the latter possibility seems to be the most likely one. It is interesting to note, however, that of the accessory components only homologues of the bacterial SecD and SecF proteins are present. The role of these proteins is not clear, but they have been proposed to be involved in the membrane cycling of SecA (Duong & Wickner, 1997 ). Thus it is conceivable that SecD and SecF are involved in the cycling of a protein with a function similar to SecA. It is also possible that SecD and SecF serve a completely different role in archaea, or that the effect of SecD and SecF on SecA cycling is only indirect and that they have a different function in bacterial protein translocation as well. Strikingly, other roles have also been proposed for SecD/F in bacteria, such as involvement in the release of the mature secretory protein from the translocase (Matsuyama et al., 1993 ), gating or assembly of the translocase channel (Pohlschröder et al., 1997 ), or removal of signal peptides from the translocase (Bolhuis et al., 1998 ). The latter function is compatible with the observation that SecD and SecF show similarity to secondary solute transporters (Bolhuis et al., 1998 ; Tseng et al., 1999 ). Notably, the previously suggested function of SecD and SecF in maintenance of the proton motive force (Arkowitz & Wickner, 1994 ) was recently refuted (Nouwen et al., 2001 ). Archaea lack a number of components that are pivotal for protein transport in bacteria or eukarya, including homologues of the bacterial YidC protein and the eukaryotic TRAM and Sec63 proteins. Therefore, it is very likely that novel components will be involved in the archaeal translocation process.


   The Tat pathway
TOP
Background
The Sec pathway
The Tat pathway
Signal peptides
Signal peptide predictions in...
Concluding remarks
REFERENCES
 
Recently, a novel Sec-independent system for protein export was discovered that is present in most prokaryotes and thylakoid membranes of chloroplasts. Proteins using this pathway contain a typical twin-arginine motif in their signal peptide (Chaddock et al., 1995 ; Berks, 1996 ) and, therefore, it was denoted the twin-arginine translocation (Tat) pathway. In contrast to the Sec pathway, the Tat system has the unique ability to transport fully folded proteins and does not seem to depend on the presence of nucleoside triphosphates (for a review, see Robinson & Bolhuis, 2001 ). This pathway is involved in the export of proteins that either have to fold before translocation, such as certain co-factor containing proteins, or just fold too quickly. These folded proteins are incompatible with the Sec machinery and can only be exported via the Tat system.

Bacteria and thylakoid membranes of chloroplasts usually contain three major membrane-bound components that are required for Tat-dependent export (for reviews, see Berks et al., 2000 ; Robinson & Bolhuis, 2001 ). The core of the E. coli Tat-translocase is probably formed by TatB and TatC, both of which are present in a strict 1:1 ratio (Bolhuis et al., 2001 ). These proteins function together with a third component, TatA. Similarly, chloroplast Hcf106 (a TatB homologue) and TatC are tightly associated with each other and interact only with Tha4 (a TatA homologue) in the presence of precursor protein (Mori & Cline, 2002 ). TatA and TatB are similar in structure and contain one transmembrane helix at the N-terminus followed by a cytoplasmic domain. TatC is an integral membrane protein with six transmembrane helices (Fig. 1a).



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Fig. 1. Predicted membrane topologies of E. coli TatC, Halobacterium sp. NRC-1 TatC1 and Halobacterium sp. NRC-1 TatC2. The topologies were determined using the TMHMM2.0 server at www.cbs.dtu.dk/services. Transmembrane helices of the TatC proteins or domains are filled, and transmembrane helices of the linker domain in TatC2 are open.

 
Most archaea contain components for the Tat machinery, as a BLAST search against available genomic sequences (http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi) showed that 9 out of 15 archaea of which the genome has been fully sequenced contain at least one or two TatC-like proteins (also see Yen et al., 2002 ). Several of these also seem to contain one or two TatA and/or TatB-like proteins but, as these are not very similar to each other in general (Robinson & Bolhuis, 2001 ), the role of these proteins in translocation needs to be determined experimentally. Halobacterium sp. NRC-1 contains two TatC proteins, denoted TatC1 and TatC2 (Ng et al., 2000 ). TatC1, which has six predicted membrane-spanning domains, resembles other prokaryotic TatC-like proteins, except for its large N-terminal cytoplasmic domain (approx. 100 residues), which is absent in other TatC proteins (Fig. 1b). The TatC2 protein has a very unusual topology as it comprises two domains, each of which is similar to TatC proteins. These two domains, which share 45% identical and conserved residues with each other, are joined together by a linker region with two membrane-spanning domains (Fig. 1c). Thus the TatC2 protein contains 14 membrane-spanning domains in total. Interestingly, the cytoplasmic domain of the linker region is homologous to the N-terminal cytoplasmic domain of TatC1 (40% identical and conserved residues). The only other organism containing a TatC-like protein with a similar topology is the archaeon Hf. volcanii (http://wit-scranton.mbi.scranton.edu/Haloferax/), which is a halophile that is closely related to Halobacterium species.

Simple BLAST searches did not immediately show the presence of TatA- or TatB-like proteins in Halobacterium sp. NRC-1, but with PSI-BLAST (Altschul et al., 1997 ) a protein was identified showing similarity to TatA of E. coli (45% identical and conservative replacements; Fig. 2). This protein, denoted Vng0801, is predicted to contain an N-terminal membrane-spanning domain that ends on the FG motif that is diagnostic for proteins belonging to the TatA family (Robinson & Bolhuis, 2001 ). Like other TatA- and TatB-like proteins (Settles et al., 1997 ), the membrane-spanning domain of Vng0801 is followed by a stretch of approximately 30 residues that may form an amphipathic helix. A protein belonging to the TatB family was not identified in Halobacterium sp. NRC-1.



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Fig. 2. Comparison of Vng0801 of Halobacterium sp. NRC-1 with TatA of E. coli. Identical (*) and conservative (:) replacements are marked. The transmembrane helix is indicated in grey shading, and the FG motif at the end of the transmembrane helix, which is diagnostic for proteins belonging to the TatA family, is indicated in bold letters.

 

   Signal peptides
TOP
Background
The Sec pathway
The Tat pathway
Signal peptides
Signal peptide predictions in...
Concluding remarks
REFERENCES
 
Based upon cleavage by SPases, signal peptides can be classified in four distinct groups (Tjalsma et al., 2000 ). Class I signal peptides are typical Sec-type signal peptides that are cleaved by type I SPases. These signal peptides are usually 18–35 amino acids long and do not contain a strict consensus sequence. They share, however, a tripartite structure. The amino-terminal N-domain, which is usually two to eight residues, contains one or more positively charged residues. The N-domain is important for interaction with the protein translocation machinery (Akita et al., 1990 ) and negatively charged lipid head groups on the cytoplasmic face of the lipid bilayer (de Vrije et al., 1990 ; Phoenix et al., 1993 ). The hydrophobic H-domain that follows the N-domain varies in length from 8 to 15 residues. This region has been proposed to form an {alpha}-helical conformation in the membrane (Briggs et al., 1986 ). The third domain (C-domain) of the signal peptide contains the cleavage site for SPase. The residues at positions -3 and -1 (relative to the start of the mature protein) are usually those with small neutral side chains, such as alanine, glycine, serine and threonine (von Heijne, 1984 ). A subgroup of Class I signal peptides directs proteins to the Tat pathway. These signal peptides are very similar to Sec-type signal peptides but contain, in addition, a typical twin-arginine motif just before the H-domain. The consensus sequence of this motif is (S/T)RRx{phi}{phi}, where the arginines are invariant, x is any residue, and {phi} is a hydrophobic residue (Berks, 1996 ; Cristóbal et al., 1999 ). In Gram-negative bacteria, in particular, the hydrophobic residues (often FL) are frequently followed by a lysine residue (Berks, 1996 ). Surprisingly, the E. coli Tat substrate pre-SufI is still exported (albeit inefficiently) by the Tat pathway when one of the two arginine residues is replaced by a lysine (Stanley et al., 2000 ). There even exists a natural Tat-dependent substrate in Salmonella enterica that contains a ‘KR’ motif instead of ‘RR’ (Hinsley et al., 2001 ). Such examples are, however, still very rare and may be more the exception than the rule.

Class II signal peptides are found in bacterial lipoproteins. These signal peptides, which contain similar N- and H-domains to Class I signal peptides, are characterized by a conserved lipobox in the C-domain with the consensus sequence L(A/S)(G/A)C (von Heijne, 1989 ; Hayashi & Wu, 1990 ). The invariable cysteine in this lipobox is lipid-modified by a diacylglyceryl transferase and becomes the first residue of the mature protein after cleavage by a lipoprotein-specific type II SPase. Due to the lipid-modified cysteine, the protein remains anchored to the cytoplasmic membrane. Most Class II signal peptides are predicted to be Sec-dependent, since twin-arginine motifs are only rarely found in these signal peptides (Tjalsma et al., 2000 ).

The third class of signal peptides are present in prepilin-like proteins. These signal peptides also have a positively charged N-domain and a hydrophobic H-domain. In contrast to other signal peptides, prepilin signal peptides are cleaved just before the H-domain by a specific SPase that has its active site on the cytoplasmic face of the membrane (Lory, 1994 ). The H-domain, which remains attached to the mature protein, plays an important role in the assembly of the prepilin-like structures (Forest & Tainer, 1997 ).

The fourth class of signal peptides are found in certain bacteriocins and pheromones that are exported by ABC transporters. These lack a hydrophobic domain, and only some of them contain a double glycine motif at the -1 and -2 positions relative to the cleavage site (Michiels et al., 2001 ).

Only few extracellular or wall-bound proteins from archaea have been described. Through genomic analysis, the eurarchaeon Methanococcus jannaschii was predicted to contain at least 34 secretory proteins (Nielsen et al., 1999 ), but the criteria used for identification of proteins containing signal peptides were rather conservative and it is likely that several were not identified. Recently, a more extensive survey was performed on the genome of Sulfolobus solfataricus, a member of the crenarchaeotes (Albers & Driessen, 2002 ). In that case, 131 putative secretory proteins were identified. The main conclusions from these studies were that archaeal class I signal peptides have a bacterial-like charge distribution and a eukaryotic-like cleavage site. Very recently, Rose et al. (2002) developed a software program called ‘TATFIND that is able to identify Tat-dependent substrates in any sequenced genome. They identified several Tat-dependent substrates in Halobacterium sp. NRC-1 and other archaea, and concluded that haloarchaea use the Tat pathway extensively.


   Signal peptide predictions in Halobacterium sp. NRC-1
TOP
Background
The Sec pathway
The Tat pathway
Signal peptides
Signal peptide predictions in...
Concluding remarks
REFERENCES
 
The availability of the genome sequence of Halobacterium sp. NRC-1 facilitates the identification of secretory proteins using SignalP2.0 (Nielsen et al., 1999 ; www.cbs.dtu.dk/services/SignalP-2.0/). This is a neural network that, due to the lack of data, has not been trained on archaeal proteins. The complete proteome of Halobacterium sp. NRC-1 (www.ebi.ac.uk/proteome/index.html) was, therefore, analysed with the available datasets (eukaryotic, Gram-positive and Gram-negative). This resulted in a dataset containing proteins with putative signal peptides. These proteins were further analysed for the presence of additional membrane-spanning domains, and those predicted to be polytopic membrane proteins were excluded. The signal peptides in the final dataset were screened for the presence of an RR motif, a lipobox, or a cleavage site for prepilin SPase. Class IV signal peptides were not identified using this method.

Sec-type signal peptides
The analysis resulted in the identification of 28 proteins that are putative substrates for the Sec pathway and cleaved by type I SPase (Table 1). This represents 1% of the proteome, which is relatively low as compared with for instance Bacillus subtilis (4%; Tjalsma et al., 2000 ), E. coli (6%; unpublished results) or S. solfataricus (4%; Albers & Driessen, 2002 ). The N-regions of these signal peptides usually contain one or two positively charged residues, with a strong bias to arginines (18% Lys, 82% Arg). This bias, however, does not seem to be limited to N-regions of signal peptides, as a similar ratio is found in the entire proteome of Halobacterium sp. NRC-1 (21% Lys, 79% Arg). In contrast to predicted signal peptides of M. jannaschii and S. solfataricus (Nielsen et al., 1999 ; Albers & Driessen, 2002 ), there is not an increased isoleucine content in the H-domain. Because archaeal SPases are more closely related to their eukaryotic counterparts (Tjalsma et al., 1998 ), the cleavage sites indicated in Table 1 were based upon the predictions of the eukaryotic dataset in SignalP. The cleavage site of the cell-surface glycoprotein Csg, which is one of the very few that has been determined experimentally (Lechner & Sumper, 1987 ), was correctly predicted.


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Table 1. Predicted Sec-type signal peptides of Halobacterium sp. NRC-1

 
A well-known protein of Halobacterium sp. NRC-1 is bacteriorhodopsin, which is a light-driven proton pump. It was shown that this membrane protein inserts co-translationally (Dale et al., 2000 ), suggesting that bacteriorhodopsin is SRP-dependent. The signal peptide of this protein, which is 13 residues in length, is very unusual as it does not contain a hydrophobic core or any positive charges (Seehra & Khorana, 1984 ). Since the signal peptide is not required for membrane insertion (Xu et al., 1995 ), it is unlikely that it is involved in SRP-dependent targeting and may not even be a true signal peptide.

Tat-type signal peptides
The signal peptide searches revealed 18 putative substrates for the Tat pathway (Table 2). This is 40% of the Class I signal peptides, which is surprisingly high as compared to for instance B. subtilis (8%; Tjalsma et al., 2000 ), E. coli (9%; unpublished data) or S. solfataricus (2·5%; Albers & Driessen, 2002 ). Several of the putative Tat-type signal peptides also contain the remainder of the bacterial RR motif, including the signal peptide of DmsA (DMSO reductase). The latter protein, which is predicted to bind a molybdopterin cofactor, is a typical Tat substrate that is involved in anaerobic respiration. Notably, Halobacterium species have, to my knowledge, never been reported to grow anaerobically using DMSO as the terminal electron acceptor. Several proteins that contain Tat-signal peptides are normally Sec-dependent in bacteria and do not bind cofactors, such as the subtilisin-like protease Sub and the alkaline phosphatase Aph. Interestingly, alkaline phosphatase from the thermophilic bacterium Thermus thermophilus was recently shown to be a Tat-dependent substrate (Angelini et al., 2001 ). In this study, it was speculated that the Tat pathway could be a more suitable transport route for thermophilic enzymes because they are more rigidly folded.


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Table 2. Predicted Tat-type signal peptides of Halobacterium sp. NRC-1

 
Lipoproteins
Surprisingly, several signal peptides that were identified contained a conserved cysteine together with other residues that are diagnostic of bacterial lipoproteins. The H-domains of lipoprotein signal peptides are usually shorter than those of secretory proteins and are not always recognized by the SignalP server (Nielsen et al., 1997 ; Tjalsma et al., 2000 ). Therefore, all proteins from Halobacterium sp. NRC-1 were screened for the presence of the lipobox in the amino-terminal portion of proteins (using ‘pattern search’ in the Pedant database at http://pedant.gsf.de/). Positive scoring proteins were analysed for the presence of a hydrophobic domain, which resulted in several additional putative lipoproteins and bringing the total number of putative lipoproteins in Halobacterium sp. NRC-1 to 51 (Tables 3 and 4). More than 80% of these (44) contained a twin-arginine motif, suggesting that most lipoproteins are exported by the Tat pathway. This is very remarkable for a number of reasons. Firstly, the presence of twin-arginine motifs in lipoproteins in bacteria is very rare as, for example, none of the lipoproteins from E. coli (unpublished data) and only 4% of the lipoproteins from B. subtilis (Tjalsma et al., 2000 ) contain a twin-arginine motif. Secondly, archaea do not contain homologues of any of the proteins involved in bacterial lipo-modification and processing, and there is only limited evidence for the presence of lipoproteins in archaea. One study showed that halocyanin, a blue copper protein from the haloalkaliphilic archaeon Natronobacterium pharaonis, contains a typical lipoprotein signal peptide and that the protein is modified at the cysteine with, most likely, a diphytanyl (glycerol)diether (Mattar et al., 1994 ). Interestingly, Halobacterium sp. NRC-1 encodes at least seven extracellular halocyanin, plastocyanin, and other copper-binding proteins (see Tables 2, 3 and 4). Five of these are predicted to be lipoproteins, and all but one (HcpC) are predicted to be Tat-dependent. Notably, halocyanin from N. pharaonis also contains a twin-arginine motif in its signal peptide. Several putative lipoproteins are also found in other archaea, such as M. jannaschii and Archaeoglobus fulgidus (Tjalsma et al., 2001 ). Since archaea do not contain homologues of bacterial enzymes involved in modification and processing of lipoproteins, it is very likely that they contain a completely novel mechanism for this process. The presence of a twin-arginine motif in more than 80% of putative lipoproteins may be specific to Halobacterium sp. NRC-1 and other halophiles. A preliminary survey of the partially sequenced halophilic archaeon Hf. volcanii genome (http://wit-scranton.mbi.scranton.edu/Haloferax/) indicates that also this halophilic archaeon contains several putative lipoproteins, most of which are putative Tat substrates (data not shown). As mentioned before, Hf. volcanii also contains a TatC-like protein with the same topology as TatC2 (Fig. 1c). Thus it is conceivable that halophiles contain specially adapted Tat pathways that enable the export of a large number of Tat-dependent (lipo)proteins.


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Table 3. Predicted Sec-type lipoprotein signal peptides of Halobacterium sp. NRC-1

 

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Table 4. Predicted Tat-type lipoprotein signal peptides of Halobacterium sp. NRC-1

 
Interestingly, lipid modification of archaeal proteins such as the cell surface glycoprotein (Csg) of Hf. volcanii and Halobacterium salinarum has been reported before (Kikuchi et al., 1999 ; Konrad & Eichler, 2002 ). However, the modification described in these studies appears to be of a different type than signal peptide modification since it involved the modification of the C-terminal domain of the mature protein (Konrad & Eichler, 2002 ). Whether the same components are involved in both types of modification remains an open question.

Type IV prepilin signal peptides
In archaea, flagellin proteins contain a signal peptide that is similar to that of type IV prepilins (see Thomas et al., 2001 ). It is not known how these signal peptides are cleaved, as archaea lack a prepilin-type SPase, or how these flagellin proteins are translocated across the cytoplasmic membrane (Thomas et al., 2001 ). Interestingly, the archaeon S. solfataricus contains several sugar-binding proteins with a similar signal peptide, and for some of these the cleavage site, which is located just before the hydrophobic domain, was experimentally verified (Albers & Driessen, 2002 ). Strikingly, most binding proteins of Halobacterium sp. NRC-1 seem to be retained at the membrane through lipo-modification and not through the hydrophobic domain of a prepilin-like signal peptide (Table 4). The genome of Halobacterium sp. NRC-1 does encode six flagellin proteins, all of which have very similar prepilin-like signal peptides (Table 5; Ng et al., 2000 ). Four genes that are closely located on the chromosome (vng0198, vng0199, vng0200 and vng0204; see Tables 1 and 2) encode proteins containing the same residues as the flagellin proteins in their cleavage site (RGQ). The processing of these proteins should, however, be experimentally determined as they also contain a predicted cleavage site for type I SPase.


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Table 5. Flagellin proteins of Halobacterium sp. NRC-1

 

   Concluding remarks
TOP
Background
The Sec pathway
The Tat pathway
Signal peptides
Signal peptide predictions in...
Concluding remarks
REFERENCES
 
In total, 103 proteins with putative signal peptides were identified in Halobacterium sp. NRC-1, and more than 60% of these contain a twin-arginine motif. This is extremely high since in bacteria and non-halophilic archaea the majority of proteins (>90%) appear to be Sec-dependent. Although it has to be proven experimentally that Halobacterium sp. NRC-1 proteins are indeed Tat-dependent, it suggests that the halobacterial Tat pathway plays a major role in protein translocation. Similar conclusions were reached by Rose et al. (2002 ) through in silico analyses and the observation that an {alpha}-amylase (which is usually a Sec substrate) from the alkalihalophilic archaeon Natronococcus sp. was not exported in Hf. volcanii when the RR motif in its signal peptide was replaced with a KK motif. The question remains why so many proteins in halophilic archaea are Tat-dependent. The reason for this may lie in the environment in which Halobacterium spp. and other haloarchaea thrive. They thrive in conditions with about 4–5 M NaCl, and to maintain an osmotic balance K+ is accumulated inside the cell to a concentration that approximates that of the external Na+ concentration (Lanyi, 1974 ). Proteins from non-halophilic organisms would just aggregate and precipitate under these conditions, but halobacterial proteins have increased negative surface charge that enables the binding of essential water molecules and prevents aggregation through electrostatic repulsion (Elcock & McCammon, 1998 ; Kennedy et al., 2001 ; Madern et al., 2000 ). Newly synthesized proteins will have to fold rapidly in their native conformation to prevent aggregation. Thus many secretory proteins may fold into their 3-dimensional conformation before they reach the membrane. This makes these proteins incompatible with the Sec system and necessitates export via the Tat system. The alternative solution to the problem of aggregation in the cytoplasm is co-translational translocation in which the secretory protein is synthesized at the site of translocation. Therefore, it may be that in Halobacterium spp. post-translational translocation occurs exclusively via the Tat pathway, leaving the Sec pathway with co-translational translocation only.

In conclusion, several novel aspects can be found in the protein transport pathways of Halobacterium sp. NRC-1, some of which may be specific for halophiles. Depending on the environment in which they thrive, specific adaptations may indeed be found in other archaea as well. Nevertheless, because of the genetic amenability and ease of culturing, Halobacterium species are very good model systems to study protein translocation in archaea and provide a fundamental insight into an important aspect of the cell biology of the third domain of life.


   ACKNOWLEDGEMENTS
 
I thank Dr M. Pohlschröder for sharing information prior to publication, Dr V. DelVecchio and other members of the H. volcanii sequencing group for making sequences available, and Dr J. M. van Dijl for stimulating discussions. I also thank two anonymous referees for helpful suggestions and comments. This work is supported by a Royal Society University Research Fellowship.


   REFERENCES
TOP
Background
The Sec pathway
The Tat pathway
Signal peptides
Signal peptide predictions in...
Concluding remarks
REFERENCES
 
Akita, M., Sasaki, S., Matsuyama, S. & Mizushima, S. (1990). SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem 265, 8164-8169.[Abstract/Free Full Text]

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