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Previous Article | Next Article 
Microbiology and Molecular Biology Reviews, September 2005, p. 393-425, Vol. 69, No. 3
1092-2172/05/$08.00+0 doi:10.1128/MMBR.69.3.393-425.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Life Sciences, Ben Gurion University, Beersheva, 84105 Israel,1 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 306022
SUMMARY INTRODUCTION PROTEIN GLYCOSYLATION Glycosylated Archaeal Proteins S-layer glycoproteins. (i) S-layer glycoproteins reveal unique aspects of archaeal protein glycosylation. Flagellins. (i) Evidence for flagellin glycosylation. Other proteins. Process of Protein N-Glycosylation in Archaea Dolichol carrier. (i) Antibiotics that affect dolichol processing interfere with archaeal protein glycosylation. (ii) Analysis of dolichol-bound glcyans. Enzymes of N-glycosylation. (i) Genomic studies. (ii) Biochemical studies. Subcellular localization of glycosylation. Role of Protein Glycosylation in Archaea Structural roles. Functional roles. Glycosylation as an environmental adaptation. LIPID MODIFICATION Membrane Lipids of Archaea Lipid-Modified Archaeal Proteins Lipoproteins. Isoprenylated proteins. Acylated proteins. GPI-anchored proteins. PROTEIN PHOSPHORYLATION Targets and Functions of Protein Phosphorylation in Archaea Phosphorylation of components involved in signal transduction. Phosphorylation of components involved in DNA replication, cell cycle regulation, and translation. Phosphorylation of other proteins. Archaeal Protein Kinases and Phosphatases Eucaryal protein kinases. Histidine kinases. Protein serine/threonine phosphatases. Protein tyrosine phosphatases. Protein kinases and phosphatases of Thermoplasma acidophilum. PROTEIN METHYLATION Protein Methylation in Response to External Stimuli Methylation of Methyl-Coenzyme M Reductase Methylated Proteins in Thermophilic Archaea Methylation of Archaeal DNA-Binding Proteins Methylation of Archaeal Ribosomal Proteins DISULFIDE BONDS IN PROTEINS Disulfide Bonds in Cytoplasmic Archaeal Proteins Disulfide Bonds in Extracellular Archaeal Proteins Enzymes Involved in Disulfide Bond Formation in Archaea PROTEOLYTICALLY PROCESSED PROTEINS Archaeal Signal Sequences Protein translocation in Archaea. Genomic surveys of archaeal signal sequences. Removal of archaeal signal sequences. Amino-Terminal Methionine Removal Inteins in Archaeal Proteins Carboxy-Terminal Maturation of Archaeal [NiFe] Hydrogenases OTHER POSTTRANSLATIONAL MODIFICATIONS IN ARCHAEA Protein Acetylation Protein Ubiquitination Hypusine-Containing Archaeal Protein PROTEOME-WIDE ANALYSIS OF POSTTRANSLATIONAL MODIFICATIONS IN ARCHAEA CONCLUSIONS ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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Proteins can be modified posttranslationally by covalent attachment of one or more of several classes of molecules, by the formation of intra- or intermolecular linkages, by proteolytic processing of the newly synthesized polypeptide chain, or by any combination of these events. By chemically linking various modifying groups either permanently or temporarily and by allowing for changes in the molecular composition of the modifying moieties, covalent modifications can endow proteins with properties that are very different from those that are predicted by the encoding genes. Examples of such covalent modifications include glycosylation, lipid attachment, phosphorylation, and methylation.
The covalent bonding of pairs of Cys residues to form disulfide bridges not only modulates the three-dimensional conformation of a polypeptide chain but can also be used to maintain proteins in multisubunit complexes. Controlled reduction and reoxidation of protein disulfide bonds is also employed in electron transfer reactions fundamental to many cellular processes. Proteolytic processing of newly synthesized polypeptide chains similarly allows the cell to control the folding and function of a protein. By removing specific targeting sequences or other stretches of amino acid residues, the cell is able to control where, when, and how a protein will act. As such, posttranslational modifications can significantly modulate the physicochemical and biological properties of a protein through effects on protein function, subcellular localization, oligomerization, folding, or turnover. The distribution of posttranslational modifications and their effects on protein chemistry and cell biology become even broader when one also considers the effects of additional, secondary posttranslational modification steps such as the addition of organic (e.g., flavins) or inorganic (e.g., metal groups) cofactors. Such modifications, however, lie beyond the scope of this review.
Long-known to be widespread in Eucarya and Bacteria, it is becoming clear that posttranslational modification of proteins also takes place in Archaea. Best known in their capacities as extremophiles, i.e., microorganisms able to thrive in the harshest environmental conditions on this planet, Archaea express proteins that enable them to succeed in such habitats. Indeed, archaeal proteins are able to remain properly folded and functional in the face of extremes of salinity, temperature, and other adverse physical conditions that would normally lead to protein denaturation, loss of solubility, and aggregation. Although posttranslational modifications may help archaeal proteins overcome the challenges presented by their surroundings, in most cases, the reason for posttranslational modification of a particular archaeal protein remains unclear. Table 1 lists the posttranslational modifications that archaeal proteins may experience.
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| PROTEIN GLYCOSYLATION |
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Like eucaryal glycoproteins, archaeal S-layer glycoproteins can undergo both N- and O-glycosylation. In contrast, bacterial S-layer glycoproteins contain only O-linked glycans (285, 445), although examples of N-glycosylation of other bacterial proteins have been shown (107, 425, 456). Analysis of the composition of the N-linked glycan moieties of archaeal S-layer glycoproteins has revealed the wide variety of saccharides available for protein glycosylation in Archaea, including galactofuranose, galactouronic acid, glucose, glucuronic acid, iduronic acid, mannose, N-acetylgalactosamine, N-acetylglucosamine, and rhamnose (204, 280, 335, 421, 456). In many cases, these sugar subunits may themselves be modified by methylation or sulfation. Such diversity in the range of saccharides used in archaeal S-layer glycoprotein N-glycosylation exceeds that seen in the bacterial and eucaryal N-glycosylation processes (425, 456).
(i) S-layer glycoproteins reveal unique aspects of archaeal protein glycosylation. Despite the proposed evolution of the eucaryal N-glycosylation system from a precursor process in Archaea (46, 157), studies of archaeal S-layer glycoprotein glycosylation, and in particular glycosylation of the Halobacterium salinarum S-layer glycoprotein, have revealed differences in N-glycosylation in the two domains. Such differences are reflected, for example, in the failure thus far to detect antennary structures in Archaea similar to those employed in eucaryal protein N-glycosylation (46, 157, 235, 333, 409, 442), or in the identified amino acid sequence motifs recognized by the archaeal N-glycosylation machinery.
It was observed that replacement of the Ser residue of the Asn-2-Ala-3-Ser-4 sequence of the Halobacterium salinarum S-layer glycoprotein with Val, Leu, or Asn did not prevent N-glycosylation at the Asn-2 position (486). By contrast, the eucaryal system almost invariably recognizes the Asn-X-Ser/Thr sequence motif, where X is any residue apart from Pro (46, 157, 235, 333, 409, 442), although a rare exception of N-glycosylation at an Asn-Gly-Gly-Thr motif has been reported (211). The ability of Archaea to glycosylate proteins at Asn residues that are not part of the consensus Asn-X-Ser/Thr motif suggests that predictions of the glycosylation status of archaeal proteins may have overlooked similar or novel N-glycosylation sites. Moreover, the finding that the repeating sulfated pentasaccharide moiety attached at the Asn-2 position of the Halobacterium salinarum S-layer glycoprotein through an N-acetylgalactosamine link is chemically distinct from the sulfated polysaccharide unit attached via glucose subunits found at the other ten N-glycosylation sites in the S-layer glycoprotein (247) implies the existence of two different N-saccharyltransferases in this species. At present, it remains unclear how the cell would distinguish between the different N-glycosylation sites.
Finally, the linkage of glycan moieties to the Halobacterium salinarum S-layer glycoprotein at selected Asn residues through either N-acetylgalactosamine or glucose subunits (335) is in contrast to the N-acetylglucosamine linkage largely employed in eucaryal N-glycosylation (46, 157, 235, 333, 409, 442). In the case of the eucaryal protein laminin, however, N-glycosylation involves a ß-glucosyl-Asn protein linkage (385). It is of note that laminin is a component of the extracellular basement membrane, a structural layer surrounding mammalian cells in a manner reminiscent of the archaeal S-layer.
In addition to N-glycosylation, archaeal S-layer glycoproteins can also be modified by O-glycosylation of selected Ser or Thr residues. In both Halobacterium salinarum and Haloferax volcanii, Thr-rich regions adjacent to the membrane-spanning domain of the protein are decorated at numerous positions with galactose-glucose disaccharides (283, 421). Interestingly, a glycoprotein isolated from a eucaryal basement membrane contains a similar disaccharide (254). Presently, little is known of the steps involved in archaeal O-glycosylation or the relation of such steps to the parallel eucaryal or bacterial processes.
Flagellins. In Archaea, cell motility mediated by flagella has been reported for representatives of the major phenotypic groups, i.e., the halophiles, the methanogens, the thermophiles, and the hyperthermophiles, largely based on microscopic investigation (20, 184, 436). Although fulfilling similar roles, archaeal flagella bear little resemblance to their better-characterized bacterial counterparts (7, 265) in terms of structure or assembly. Such differences become evident when one considers the flagellar filament in the two domains. Ultrastructural studies have shown that, unlike bacterial filaments, archaeal flagellar filaments are not hollow structures (72) and that the archaeal structures are generally thinner than their bacterial counterparts (79, 185, 190, 406).
Archaeal and bacterial flagella also differ at the level of flagellin, the major structural component of the flagellar filament. Whereas bacterial flagella are, for the most part, composed of a single type of flagellin, archaeal flagellar filaments are made up of several types of flagellins (with the possible exception of Sulfolobus solfataricus, where genome annotation efforts have reported the existence of only a single flagellin-encoding gene) (20, 184, 436). Indeed, archaeal and bacterial flagellins do not share sequence similarity (19). Moreover, many archaeal flagellins are glycosylated (184), a posttranslational modification that is considered rare for bacterial flagellins (95, 139, 291, 384, 435).
(i) Evidence for flagellin glycosylation. Glycosylation has been reported for flagellins of numerous archaeal strains (112, 184, 196, 197, 389, 436), including Halobacterium salinarum (470), Methanococcus deltae (27), Methanococcus voltae (453), and Methanospirillum hungatei (406). In most of these examples, the evidence for glycosylation comes from studies employing glycan-detecting stains, such as thymol-sulfuric acid or periodic acid-Schiff reagent. Such techniques, however, may not always accurately reflect the glycosylated nature of a protein (222). Hence, additional evidence for glycosylation is desirable.
This has been achieved for the flagellins of Halobacterium salinarum and Methanococcus voltae, for which the chemical compositions of the covalently linked glycan moieties have been elucidated. The Halobacterium salinarum flagellin contains a sulfated glycoconjugate, N-linked through a glucose bridge and based on glucuronic or iduronic acid, similar to the glycan moiety found on the S-layer glycoprotein (420, 468). More recently, Methanococcus voltae flagellins have been shown to contain a novel N-linked trisaccharide (453), despite the fact that earlier glycoprotein staining-based studies had failed to detect flagellar glycosylation in this species (195). Analysis of trypsin-generated peptides derived from the Methanococcus voltae S-layer glycoprotein also revealed modification by the same novel trisaccharide (453), suggesting a common glycosylation process for the two proteins. Support for the glycosylation of Methanospirillum hungatei flagella beyond glycan staining was presented by chemical deglycosylation with trifluoromethansulfonic acid, a treatment that decreased molecular mass, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (406). The same was noted for Halobacterium salinarum flagellins upon similar treatment (247).
The glycosylated nature of Methanococcus deltae flagellins was indicated upon incubation of cultures with bacitracin, an antibiotic that interferes with protein glycosylation (see below) (247). Such treatment resulted in more rapid migration of the protein as reflected by SDS-PAGE analysis (27). Similar bacitracin treatment, however, had no effect on the glycosylation of Halobacterium salinarum flagellins, as gauged by migration in SDS-PAGE, although incubation with EDTA, thought to specifically inhibit an externally oriented Mg2+-dependent oligosaccharidetransferase (420), successfully modified flagellin migration. By contrast, treating cells with EDTA did lead to the appearance of Methanococcus deltae flagellins of lower apparent molecular weight (27). Together, these observations point to differences in the glycosylation machineries of the two species.
Other proteins. While the bulk of attention on archaeal protein glycosylation has focused on S-layer glycoproteins and flagellins, other archaeal glycoproteins have been identified. Of those additional glycoproteins whose identities are known, the majority are membrane associated. In many instances, these are binding proteins involved in nutrient uptake (see below), such as the maltose/trehalose-binding proteins of Thermococcus litoralis, shown to react with glyco-stain (145) and of Pyrococcus furiosus, shown to contain glucose-containing glycan moieties by lectin binding and molecular analysis (231), or the Pyrococcus furiosus cellobiose-binding protein, which reacts with lectins and glyco-stain (230). Glyco-staining also indicated the glycosylated nature of Pyrococcus furiosus CipA and CipB, two ABC transporter binding proteins whose expression is up-regulated in response to cold shock in this hyperthermophile (464). Glycosylation of pyrolysin, a thermostable serine-protease also associated with Pyrococcus furiosus membranes, was proposed on the basis of sequence analysis that revealed the presence of numerous potential N-glycosylation sites and supported by glyco-staining of the protein (455).
Based on lectin binding, a series of glycosylated sugar-binding proteins, apparently containing mannose, glucose, galactose, and N-acetylglucosamine, was detected in Sulfolobus solfataricus membranes (106). Sulfolobus acidocaldarius cytochrome b558/566 was shown to be a highly glycosylated integral membrane protein, containing both O-linked mannose subunits and N-linked hexasaccharides (161). Analysis of the composition of the latter glycan moiety revealed the presence of glucose, mannose, and N-acetylglucosamine in addition to 6-sulfoquinovose (484). 6-Sulfoquinovose (or 6-deoxy-6-sulfoglucose) is a rare acidic sugar, commonly found in the glycolipids of chloroplasts and photosynthetic bacteria (177), but not previously found in a glycoprotein. The glycosylated character of a membrane-associated Sulfolobus solfataricus protein serine/threonine kinase was confirmed through precipitation of a protein with kinase activity using lectin-conjugated agarose beads and by the decreased apparent molecular mass of the protein and resistance to glyco-staining following treatment with chemical deglycosylation agents (262).
In addition to membrane proteins, secreted archaeal glycoproteins have also been detected. Lectin binding and chemical deglycosylation confirmed the glycosylated nature of the copper response extracellular proteins secreted by the copper-resistant methanogen Methanobacterium bryantii BKYH (219). Indeed, differential glycosylation is responsible for the appearance of multiple isoforms of the copper response protein. A secreted, inducible alkaline phosphatase purified from Haloarcula marismortui was shown to be glycosylated, in part through the use of radiolabeled glucosamine-containing growth medium (136). Quantitative analysis revealed that glycosylation accounted for 3% of the mass of the protein. Based on glyco-staining, a secreted enzyme possessing thermostable amylopullulanase activity, i.e., capable of hydrolyzing both 
-1,6 linkages in pullulan and -1,4 linkages in amylose and soluble starch, was detected in the growth media of both Pyrococcus furiosus and Thermococcus litoralis (44). Based on aberrant SDS-PAGE migration and sequencing data, it has been proposed that the partially secreted acid protease of Sulfolobus acidocaldarius, thermopsin, is also glycosylated (258).
In addition to these identified membrane and secretory glycoproteins, numerous other glycoproteins, uncharacterized apart from their glycosylated nature, have been reported. Using lectin-based purification techniques, a 152-kDa glycoprotein was isolated from Thermoplasma acidophilum membranes (478). Subsequent analysis of the glycan moiety of the protein revealed it to be a highly branched, mannose-based structure, N-linked to the polypeptide chain through an N-acetylglucosamine subunit. Several lectin-binding proteins have been observed in Methanococcus mazei S-6, with the levels of these glycoproteins related to the adoption of morphologically distinct forms by the cells (481). In Haloferax volcanii, membrane glycoproteins of 150, 98, 58, and 54 kDa, distinct from the S-layer glycoprotein, were identified in lectin-based studies (98). A second study of the same strain noted the presence of glycoproteins of 105, 56, and 52 kDa in whole-cell lysates (489). It remains to be seen whether any of the proteins identified in the two studies are the same and whether the smaller glycoproteins are derived from the heavier polypeptides.
Relying on glyco-staining, lectin-binding techniques, and treatments with inhibitors of glycosylation or deglycosylating agents, the membranes of both Sulfolobus acidcaldarius and Sulfolobus solfataricus were shown to contain unidentified glycoproteins distinct from the S-layer glycoprotein (147, 262). Glycoprotein staining was used to identify a series of glycosylated proteins in Pyrococcus furiosus membranes that are distinct from CipA and CipB and the expression of which is related to growth temperature (464).
If, as proposed (46, 157), the elaborate process responsible for protein N-glycosylation in Eucarya originated from a simpler archaeal system, then many of the fundamental steps and central components involved in eucaryal protein N-glycosylation should also be present in Archaea. As summarized in Table 3 and discussed in the following section, available evidence suggests that this is indeed the case.
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(i) Antibiotics that affect dolichol processing interfere with archaeal protein glycosylation. The use of various antibiotics and other compounds known to prevent protein glycosylation by interfering with the processing of dolichol carriers has provided insight into the role of this lipid in archaeal protein N-glycosylation. Tunicamycin hinders transfer of UDP-N-acetylglucosamine to polysaccharide-loaded dolichol carriers (105). Treatment with this antibiotic interferes with Sulfolobus acidocaldarius S-layer glycoprotein glycosylation (147). In contrast, tunicamycin has no effect on the biosynthesis of the Haloferax volcanii S-layer glycoprotein (99) and accordingly, the glycan moiety of the Haloferax volcanii S-layer glycoprotein does not include N-acetylglucosamine (242, 280). Bacitracin is another drug that interferes with protein glycosylation via an interruption of the recycling of pyrophosphate-containing dolichol species (420). Accordingly, in Halobacterium salinarum, bacitracin interferes with the attachment of the repeating sulfated pentasaccharide found at the Asn-2 position of the S-layer glycoprotein (284, 469), although not with the attachment of the sulfated polysaccharide found at the other N-glycosylation sites of the protein (469).
Bacitracin also inhibits glycosylation of flagellins in Methanococcus deltae (27) and slowed Sulfolobus acidocaldarius growth, possibly through interference with the protein N-glycosylation pathway (286). In contrast, bacitracin had no effect on the glycosylation of the S-layer glycoprotein or a second 98-kDa glycoprotein in Haloferax volcanii (99, 232). The failure of the antibiotic to prevent Haloferax volcanii glycoprotein biogenesis is likely related to the fact that, unlike Halobacterium salinarum, in which both monophosphate- and pyrophosphate-containing dolichol oligosaccharide carriers are present (247), only bacitracin-insensitive monophosphate-containing oligosaccharide-dolichol intermediates are found in Haloferax volcanii (242). Incorporation of glucose from UDP-glucose into Haloferax volcanii glycoproteins was, however, inhibited by amphomycin and two sugar nucleotide analogs, PP36 and PP55 (489), compounds reported to block transfer of nucleotide-conjugated sugars to phosphopolyisoprenols in Eucarya (201, 202, 336).
(ii) Analysis of dolichol-bound glcyans. Evidence for the involvement of dolichol phosphate-linked oligosaccharides in archaeal protein N-glycosylation also comes from examination of the carrier-bound glycan moieties. The transfer of radiolabeled glucose from UDP-[3H]glucose to Haloferax volcanii glycoproteins proceeds through a glucose-containing phosphopolyisoprenol intermediate (489). The dolichol-linked sulfated polysaccharide moiety found in Halobacterium salinarum is identical to glycan moieties found on the S-layer glycoprotein and flagellin in this species (248, 470). On the other hand, the sulfated polysaccharide is methylated at the dolichol-linked stage, whereas no 3-O-methylglucose is detected in the protein-linked polysaccharide (249).
The importance of this transient methylation is illustrated by the detrimental effect of inhibiting S-adenosylmethionine-dependent methylation. Such treatment interfered with glycoprotein biosynthesis but did not affect either general protein biogenesis or the biosynthesis of sulfated phosphodolichol-bound oligosaccharides. It thus appears that methylation is an essential step in the biosynthesis of the sulfated oligosaccharide moiety prior to being transferred to its nascent polypeptide target. By contrast, the hexasaccharide moiety attached to the Methanothermus fervidus S-layer glycoprotein retains its methylation (204). It is not clear whether such methylation is involved in the translocation of the sulfated oligosaccharide phosphodolichol across the membrane or the subsequent transfer of the glycan moiety to the nascent polypeptide chain. In Eucarya, chemical modification of glycoprotein glycan moieties occurs only after the oligosaccharide has been transferred to the nascent polypeptide (449).
Enzymes of N-glycosylation. Just as archaeal N-glycosylation relies on the dolichol carriers implicated in eucaryal protein glycosylation, Archaea also contain homologues of many of the enzymes involved in eucaryal N-glycosylation. These include those involved in oligosaccharide charging of the lipid carrier, translocation of the dolichol carrier across the membrane, and transfer of the oligosaccharide entity to the nascent polypeptide chain (Fig. 2).
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A close examination of the Archaeoglobus fulgidus genome sequence revealed genes encoding STT3-like proteins within two gene clusters encoding putative homologues of other enzymes involved in yeast protein glycosylation (Fig. 3) (46). One of these clusters contains three adjacent open reading frames (ORFs), one of which encodes a polypeptide that appears to contain a motif present in the yeast Alg1p and Alg2p glycosyltransferase proteins. In the yeast proteins, this motif is involved in the transfer of nucleotide sugars to the phosphodolichol carrier (46). The other two ORFs putatively encode a dolichyl-phosphoglucose synthase homologue and a homologue of Stt3p. Other ORFs in this cluster show high sequence similarity to RfbA and RfbB, components of a transporter family presumably involved in the flipping of bacterial O-antigen (467) and lipopolysaccharides (364) across the plasma membrane. While the functions of these putative gene products remain to be shown, it has been postulated that this Archaeoglobus fulgidus gene cluster encodes a functional unit involved in the assembly, translocation, and transfer of dolicholphosphate-linked oligosaccharides to protein targets (46). The second gene cluster in Archaeoglobus fulgidus includes ORFs also encoding putative glycosyltransferase, dolichyl-phosphoglucose synthase, and STT3 proteins, and lies near six ORFs bearing similarity to genes encoding proteins involved in bacterial lipopolysaccharide biosynthesis (46).
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-D-glucose pyrophosphorylase, responsible for UDP-glucose synthesis, represents the first analysis of an archaeal sugar nucleotidyltransferase (290). An N-acetylglucosamine transferase was also partially characterized from membranes of Halobacterium salinarum (281). Dolichylphosphate mannose synthase, which is able to transfer GDP-mannose to a dolichol phosphate carrier, was purified from Thermoplasma acidophilum (490). Amphomycin, an inhibitor of dolichylphosphate mannose synthases (202), blocked the activity of the enzyme (490). Using 5-azido-[32P]UDP-glucose in a photoaffinity approach, a single 45-kDa species was identified in Haloferax volcanii homogenates that is thought to correspond to dolichylphosphate glucose synthase (489). Pyrophosphatases with their active site oriented towards the cell exterior have been purified from the membranes of two different Sulfolobus acidocaldarius strains (8, 286). The pyrophosphate-hydrolyzing activity of the enzymes, proposed to participate in the hydrolysis of dolicholpyrophosphate-linked oligosaccharides during protein glycosylation, was stimulated in the presence of Sulfolobus membrane lipids. Sequence analysis of one of these pyrophosphatases has led to the identification of putative homologues in the genome sequences of Sulfolobus tokodaii and Solfolobus solfataricus as well as in Methanobacterium thermoautotrophicum (294). This study also revealed the presence of a strongly conserved phosphatase tripartite sequence motif, Lys-XXXXX-Arg-Pro-X12-54-Pro-Ser-Gly-His-X31-54-Ser-Arg-XXXXX-His-XXX-Asp, also detected in Lpp1p and Dpp1p, Saccharomyces cerevisiae proteins showing hydrolytic activity towards dolichylphosphate, dolichylpyrophosphate, and other isoprenoid phosphates/pyrophosphates (116).
Subcellular localization of glycosylation. Several lines of evidence suggest that archaeal glycosylation occurs at the outer cell surface, the topological equivalent of the luminal-facing leaflet of the endoplasmic reticulum membrane bilayer, the site of N-glycosylation in Eucarya (46, 157, 235, 333, 409, 442). Despite its inability to cross the plasma membrane of haloarchaea (284), bacitracin is nonetheless able to interfere with Halobacterium salinarum protein glycosylation by preventing transfer of sulfated oligosaccharides to the S-layer glycoprotein (284, 469). The external orientation of the archaeal glycosylation apparatus is further supported by the decoration of exogenously added, soluble cell-impermeable hexapeptides containing the Asn-based N-glycosylation motif with sulfated oligosaccharides by living Halobacterium salinarum cells (248). Other observations also favor the assignment of archaeal protein glycosylation to the cell's outer surface. These include the ecto-enzymatic nature of a Sulfolobus acidocaldarius pyrophosphatase (8, 286), the proposed specific inhibition of an externally oriented Mg2+-dependent oligosaccharidetransferase by EDTA, a non-cell-permeant reagent, and subsequent interference with Halobacterium salinarum flagellin glycosylation (420), as well as studies supporting the cotranslational mode of membrane protein insertion in Archaea (360).
Functional roles. The glycosylation of archaeal proteins has also been implicated in protein assembly and function. In archaeal flagellins, glycosylation is associated with proper flagellar assembly, since upon bacitracin-mediated interference with flagellin glycosylation, a loss of Methanococcus deltae flagellation was observed microscopically (196). In a mutant Halobacterium salinarum strain in which underglycosylated flagellins are overproduced, increased levels of flagella were detected in the growth medium, suggesting proper flagellin glycosylation to be important for correct flagellar incorporation into the plasma membrane (470). This explanation is, however, inconsistent with the apparent nonglycosylated nature of other archaeal flagellins (184) or the glycosylation of Methanospirillum hungatei flagellins, which only occurs in low-phosphate media (406). Similarly, evidence against glycosylation's playing a role in protein function comes from bacterial expression of archaeal binding proteins. Normally glycosylated in their native hosts, nonglycosylated heterologously expressed versions of these proteins were also capable of substrate binding (170, 230, 231). Nevertheless, glycosylation could play a role in stabilization against proteolysis or could affect the interaction of binding proteins with the cell membrane or envelope (4).
Glycosylation as an environmental adaptation. Coping with the often harsh environmental conditions encountered by Archaea serves as the basis for yet another hypothesized role for archaeal protein glycosylation. In a comparison of the glycosylation profiles of S-layer glycoproteins from the moderate halophile Haloferax volcanii and the extreme halophile Halobacterium salinarum, it was noted that the latter experiences a higher degree of glycosylation than the former (280). Moreover, the glycan moieties of the extreme halophile were enriched in sulfated glucuronic acid subunits as opposed to the neutral sugars found in the moderate halophile. These properties endow the Halobacterium salinarum S-layer glycoprotein with a drastically increased surface charge density relative to its Haloferax volcanii counterpart.
The enhanced negative surface charges are thought to contribute to the stability of haloarchaeal proteins in the face of molar salt concentrations (266). Accordingly, the Halobacterium salinarum S-layer glycoprotein also contains 20% more acidic amino acid residues than does the corresponding protein in Haloferax volcanii (246, 421). The enhanced negative surface charge associated with protein glycosylation and the resulting protection that this would afford in the face of acidic conditions have been offered as the role of Sulfolobus acidocaldarius cytochrome b 558/566 glycosylation (161, 484). It has also been suggested that a significant amount of the protein surface is shielded from the 
pH 2 environment by the high degree of glycosylation (484). Finally, glycosylation has also been implicated in the stabilization of thermophilic archaeal glycoproteins (4, 258, 455).
| LIPID MODIFICATION |
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Lipid modification of proteins is largely a posttranslational event (115). It serves a variety of roles, the most obvious being to enhance the membrane affinity of the modified protein. Accordingly, amino-terminal acylation leads to the localization of numerous proteins to the outer membrane of gram-negative Bacteria (156, 379), as exemplified by Braun's lipoprotein in Escherichia coli (40). Similarly, otherwise soluble eucaryal proteins also become membrane associated upon the covalent attachment of one or more lipid moieties (102, 153, 194, 462). Lipid modification can also modulate protein-protein interactions in Eucarya, as shown by the effects of myristylation or prenylation upon trimeric G protein subunit affinity (124, 178, 462), and in viruses, exemplified by the involvement of myristylation of the capsid proteins of human immunodeficiency virus type 1 and picornavirus in virion particle assembly and secretion (65, 142).
Lipid modifications of eucaryal proteins has also been implicated in a variety of other cellular events. These include signal transduction (287), embryogenesis and pattern formation (271), protein trafficking through the secretory pathway (297), and evasion of the immune response by infectious parasites (369, 461). Yet another role for lipid modification is exemplified by the bacterial toxin hemolysin A, which requires fatty acid acylation on an internal Lys residue for its activation (414).
Given the ubiquitous distribution and numerous functions of lipid modifications in eucaryal and bacterial proteins, it is not surprising that lipid-modified proteins have also been identified in Archaea.
Whereas phospholipids and other polar lipids (phosphoglycolipids, glycolipids, and sulfolipids) account for the vast majority of archaeal membrane lipids, archaeal membranes also contain acetone-soluble nonpolar lipid species, primarily neutral squalenes and other isoprenoid-based polymers (206, 207, 334, 439, 440). In halophilic Archaea, in which membrane lipid composition has been most studied, pigmented carotenoids, in particular bacterioruberins, are major components of the nonpolar lipid pool (243, 438). These have been implicated in affording protection from UV-induced damage (390). In addition, many halophilic Archaea also contain retinal as part of bacteriorhodopsin, the purple retinal-containing protein complex that functions as a light-driven proton pump (244).
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In gram-positive bacteria, it is accepted that substrate-binding proteins, components of multisubunit ABC transporters responsible for cellular uptake of substrates, are lipoproteins (131, 422, 430). The same may well be true in Archaea. The trehalose/maltose-binding protein of the hyperthermophile Thermococcus litoralis contains a lipobox-like sequence motif and requires detergent for its solubilization (170). Similar motifs have been identified in other ABC sugar transporter binding proteins identified in Archaea, suggesting that amino-terminal lipid modification of binding proteins takes place in other species (4, 228).
Lipid modification is not, however, the sole mode of membrane association for archaeal sugar-binding proteins. For example, a membrane-spanning domain is predicted to anchor the glucose-binding protein of Sulfolobus solfataricus (3). It should be noted, however, that binding proteins in this organism differ from those in other Archaea in terms of amino-terminal sequence and subsequent posttranslational processing (see below). In Halobacterium salinarum, BasB and CosB, the first examples of binding proteins involved in chemotaxis in Archaea, are also thought to be lipoproteins due to their membrane localization and bearing of the lipobox sequence motif (228). Indeed, sequence analysis of putative substrate-binding proteins in Halobacterium salinarum, be they involved in nutrient uptake or chemotaxis, suggests that all are lipoproteins (228). Finally, in the case of Pyrococcus species peptide-binding proteins, a conserved Gly-Cys motif reminiscent of the lipobox sequence located near the carboxy terminus may also be a target for lipid modification (4).
Despite the proposed presence of lipoproteins in Archaea, no archaeal homologue of signal peptidase II, one of the enzymes involved in lipoprotein precursor maturation, has been observed. Whether this is because there is no such enzyme in Archaea or because its sequence differs beyond recognition from that of its bacterial homologues, possibly in adaptation to the ether-based phospholipids of the archaeal membrane, remains unknown.
Isoprenylated proteins. Growth of Halobacterium cutirubrum, Halobacterium salinarum, and Haloferax volcanii in the presence of radiolabeled mevalonate, a precursor of the isoprene building block used to synthesize archaeal lipids (38, 398), led to the appearance of several proteins radiolabeled through the covalent attachment of a lipid entity (233, 376). Subsequent chemical analysis of the modifying lipid moiety in Halobacterium salinarum revealed a novel diphytanylglycerol methyl unit, linked to Cys residues of the modified proteins by a thioetheric bond (376). Further analysis of isoprenoid-modified proteins in Halobacterium salinarum using other radiolabeled isoprenyl derivatives revealed that the S-layer glycoprotein is modified by a second novel group, diphytanylglyceryl phosphate, which is attached through an as yet uncharacterized linkage (218). Amino acid sequencing places the modification near an O-glycosylated Thr-rich stretch found in the C-terminal region of the protein, upstream of the single transmembrane domain (218). In Haloferax volcanii, lipid modification of the S-layer glycoprotein was also shown, although the chemical composition of the attached lipid is unknown, as is the site of attachment (99, 233).
Since haloarchaeal S-layer glycoproteins include a membrane-spanning domain (246, 421, 457), it is unclear why an additional membrane anchor in the form of a lipid would be required. Nonetheless, the attachment of the lipid moiety that takes place on the external surface of Haloferax volcanii and Halobacterium salinarum cells is responsible for the posttranslational, posttranslocational maturation of the S-layer glycoprotein in these strains, as detected through pulse-chase radiolabeling studies (99, 233). Furthermore, since other haloarchaeal S-layer glycoproteins also contain a sequence similar to that modified in Halobacterium salinarum (246, 421, 457), it would appear that such isoprenoid-based lipid modification of S-layer glycoproteins is a general trait of halophilic Archaea (218).
Acylated proteins. Since some Archaea contain significant amounts of fatty acids (51, 127, 191) and completed archaeal genome sequences reveal the presence of genes involved in fatty acid biosynthesis and ß-oxidation (342), it should not come as a surprise that the acylation of archaeal proteins has been reported. In Halobacterium cutirubrum and Methanobacterium thermoautotrophicum, subcellular fractionation and analytic chemical techniques were employed to show the acylation of several proteins (350). Chromatographic analyses identified palmitic and stearic acids as the main modifying agents, although lower levels of modification by myristic acid and other fatty acids were also observed. These acyl groups are thought to be linked to the protein via amide or ester bonds.
GPI-anchored proteins. Glycosylphosphatidylinositol (GPI) anchors represent a carboxy-terminal posttranslational lipid-based modification used to tether eucaryal proteins to various membranes (176). The GPI anchor is added to target proteins using a preformed GPI-anchoring moiety which consists of a molecule of phosphatidylinositol linked at its myoinositol headgroup to ethanolamine phosphate through an aminoglycan bridge. This lipid is transferred to the newly exposed carboxy terminus of a nascent polypeptide. The modified protein is first synthesized as a membrane-anchored precursor that undergoes proteolytic processing upstream of its carboxy-terminal transmembrane domain. The cleaved protein is thus attached to the ethanolamine end of the preassembled GPI moiety.
Although widespread in the eucaryal domain, GPI-anchored proteins have not been observed in Bacteria (103). They have, however, been detected in Archaea. In Sulfolobus acidocaldarius, three proteins were identified that incorporate radiolabeled precursors of the GPI anchor moiety (224). One of these, a 185-kDa species, was also solubilized by the actions of a bacterial phosphatidylinositol-specific phospholipase C, a characteristic of GPI-anchored proteins (175). Although the other two Sulfolobus proteins were not released by the phospholipase, this is not inconsistent with GPI anchoring as phosphatidylinositol-specific phospholipase C-resistant GPI-anchored proteins have been reported (122, 365). Similarly, a typical archaeal ether-based phospholipid bearing the identical GPI anchor moiety head group as found in Eucarya was identified in Methanosarcina barkeri (310). Incubation of this lipid species with phosphatidylinositol-specific phospholipase C led to the release of the polar head group.
In addition to these biochemical studies, a bioinformatic analysis of available archaeal genome sequences predicts the presence of GPI-anchored proteins in other archaeal species (103). Moreover, many of the 19 enzymes known to participate in the biosynthesis of GPI anchors have been detected in archaeal genome sequences (104).
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Although discovered in the 1950s (240), it took approximately 25 years for the first reports of phosphorylated proteins in Bacteria to appear (126, 459). Shortly thereafter, in 1980, the presence of phosphorylated proteins in Halobacterium salinarum was reported (413), confirming that Archaea too are capable of performing this posttranslational modification. With the subsequent availability of genome sequences, it became clear that Archaea also contain numerous kinases and phosphatases, enzymes responsible for protein phosphorylation and dephosphorylation, respectively (214, 215, 253).
Although other phosphorylated proteins have been identified in Archaea (475), the observed phosphorylation cannot usually be attributed to a regulated protein kinase (see below), but rather reflects phosphorylated intermediates that appear during an enzyme's catalytic cycle. Such enzymes apparently include the alpha subunit of succinyl-coenzyme A synthase in Sulfolobus solfataricus (403) and Sulfolobus acidocaldarius glycogen synthase (52, 397). Nevertheless, examples of regulated protein phosphorylation in Archaea have been reported (Table 5) and are discussed below.
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Protein phosphorylation in response to environmental change has also been observed in other archaeal species. Growth of Sulfolobus acidocaldarius in the presence of radiolabeled phosphate under limited-phosphate conditions revealed the existence of numerous phosphoproteins (319). In particular, the phosphorylation of a 36-kDa protein was augmented under phosphate starvation, hinting at a regulatory role in a cellular response pathway for this protein. In Haloferax volcanii, growth at elevated salt concentrations may lead to the appearance of several serine-phosphorylated proteins not detected during growth under optimal salt conditions (32). A threonine-phoshorylated 67-kDa membrane protein displaying serine kinase activity has been found in Sulfolobus solfataricus, although the pathway in which this protein participates remains to be defined (261, 264).
Phosphorylation of components involved in DNA replication, cell cycle regulation, and translation. In addition to playing a role in signal transduction, protein phosphorylation has also been implicated in eucaryal DNA replication, cell cycle regulation, and protein translation (313, 314, 349). Similar roles for protein phosphoryation have also been observed in Archaea. In Methanobacterium thermoautotrophicum, Pyrobaculum aerophilum, and Sulfolobus solfataricus (89, 144), DNA-dependent serine autophosphorylation has been reported for the Cdc6 protein, an intiator protein that fulfills an essential role in DNA replication<
