REVIEW ARTICLE published: 02 July 2012 doi: 10.3389/fmicb.2012.00207 Diversity and subcellular distribution of archaeal secreted proteins

Zalan Szabo1 and Mechthild Pohlschroder 2*

1 MicroDish BV, Utrecht, Netherlands 2 Department of Biology, University of Pennsylvania, Philadelphia, PA, USA

Edited by: Secreted proteins make up a significant percentage of a prokaryotic proteome and play Frank T. Robb, University of California, critical roles in important cellular processes such as polymer degradation, nutrient uptake, USA signal transduction, cell wall biosynthesis, and motility.The majority of archaeal proteins are Reviewed by: Thijs Ettema, Uppsala University, believed to be secreted either in an unfolded conformation via the universally conserved Sweden Sec pathway or in a folded conformation via theTwin arginine transport (Tat) pathway. Exten- Jerry Eichler, Ben Gurion University of sive in vivo and in silico analyses of N-terminal signal peptides that target proteins to these the Negev, Israel pathways have led to the development of computational tools that not only predict Sec and *Correspondence: Tat substrates with high accuracy but also provide information about signal peptide pro- Mechthild Pohlschroder, Department of Biology, University of Pennsylvania, cessing and targeting. Predictions therefore include indications as to whether a substrate is 201 Leidy Laboratories, Philadelphia, a soluble secreted protein, a membrane or cell wall anchored protein, or a surface structure PA 19104, USA. subunit, and whether it is targeted for post-translational modification such as glycosylation e-mail: [email protected] or the addition of a lipid. The use of these in silico tools, in combination with biochemical and genetic analyses of transport pathways and their substrates, has resulted in improved predictions of the subcellular localization of archaeal secreted proteins, allowing for a more accurate annotation of archaeal proteomes, and has led to the identification of poten- tial adaptations to extreme environments, as well as phyla-specific pathways among the archaea. A more comprehensive understanding of the transport pathways used and post- translational modifications of secreted archaeal proteins will also facilitate the identification and heterologous expression of commercially valuable archaeal enzymes.

Keywords: Tat transport, Sec transport, protein transport, archaea, lipoprotein, pili, cell surface structures, archaeosortase

INTRODUCTION such as glycosylation, and associations with cofactors, must occur During protein translation at the ribosome, the presence of an in an extracytoplasmic environment devoid of ATP, and hence N-terminal signal sequence or a transmembrane helix can tar- ATP-dependent chaperones. These complications are circum- get the corresponding protein for translocation into or across vented by substrates of the twin arginine transport (Tat) path- the cytoplasmic membrane (Driessen and Nouwen, 2008; Natale way, at least in part, because protein folding, cofactor incorpo- et al., 2008; Ellen et al., 2010b; Yuan et al., 2010; Calo and Eichler, ration, and possibly protein modifications, are accomplished in 2011). Archaeal proteins targeted for secretion, including polymer the cytoplasm (Pohlschroder et al., 2005a; Natale et al., 2008). degrading enzymes,antimicrobial porteins,or proteins involved in Regardless of the transport pathway used, archaeal secreted pro- intercellular signaling may be released into the extracellular envi- teins that are not released into the extracellular environment ronment. However, most proteins remain associated with the cell, must be anchored to the cell, the only exception known to requiring either a direct interaction with the membrane, an indi- date being proteins of the archaeal genus Ignicoccus, which has rect association with membrane-anchored proteins, or, as recently an outer membrane that provides a periplasmic space that can predicted, attachment to the cell wall (Figure 1; Gimenez et al., retain secreted proteins (Huber et al., 2002; Nather and Rachel, 2007; Albers and Pohlschroder, 2009; Storf et al., 2010; Haft et al., 2004). 2012). Proteins that remain associated with the cell surface are The results generated by several in vivo studies of archaeal involved in a wide variety of cellular processes, including nutrient secreted proteins and their transport pathways has allowed for uptake, motility, and surface adhesion (Ellen et al., 2010b; Albers the identification of the signal peptides required to target secreted and Meyer, 2011). proteins to a specific transport pathway as well as amino- or In bacteria and archaea, the universally conserved Sec pathway carboxy-terminal motifs within the protein that mediate substrate is commonly used to transport secreted proteins to the cytoplas- interactions with extracytoplasmic structures (Kobayashi et al., mic membrane (Nather and Rachel, 2004; Pohlschroder et al., 1994; O’Connor and Shand, 2002; Rose et al., 2002; Dilks et al., 2005b; Driessen and Nouwen, 2008). Since these proteins are 2005; Gimenez et al., 2007; De Castro et al., 2008; Albers and secreted in an unfolded conformation through a narrow pro- Pohlschroder, 2009; Ng et al., 2009; Kwan and Bolhuis, 2010; Storf teinaceous pore, protein folding, post-translational modifications, et al., 2010; Calo and Eichler, 2011). In turn, the identification of

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FIGURE 1 | Archaeal protein secretion and subcellular localization. segment (3). In silico data also suggest that Sec substrates may be anchored Proteins that contain Tat signal peptides pass the cytoplasmic membrane to the cell wall in an archaeosortase-dependent manner (4) and a number of (CM) through the Tat translocon after translation and folding. Conversely, type IV pilin-like proteins have been shown to assemble into cell appendages protein translocation through the Sec pore can occur co- and possibly (5). In I. hospitalis, an additional outer membrane (OM) is present and post-translationally. Upon secretion and signal peptide processing, Sec and periplasmic vesicles (6) are thought to play a role in the trafficking of secreted Tat substrates can be released into the extracellular milieu (1), be embedded and outer membrane proteins across the periplasmic space. See text for into the membrane via a lipid anchor (2) or a C-terminal transmembrane details. Cell components are not drawn to scale. these motifs in a large number of substrates has allowed for the Sec AND Tat SUBSTRATE TARGETING, SECRETION, AND development of software programs that facilitate in silico analy- POST-TRANSLATIONAL MODIFICATION ses of secreted protein sequences encoded by a large number In the following section we briefly describe the two main routes and variety of archaeal genomes (Rose et al., 2002; Szabo et al., for archaeal protein transport across the cytoplasmic membrane, 2007a; Bagos et al., 2009; Storf et al., 2010). These in silico analyses the Sec and the Tat pathways, while focusing on the processing have already lead to important insights into the strategies used to and modification of the substrates transported by these pathways. secrete and anchor proteins to the cell surface, which vary in part These systems have recently been more extensively reviewed else- depending on the function of the secreted protein. Ultimately, the where (Pohlschroder et al., 2005b; Ellen et al., 2010b; Yuan et al., trends revealed by in silico analyses may clarify how an organism 2010; Calo and Eichler, 2011). adapts to the selective pressures imposed on it, and may make clear which aspects of the environment had the greatest impact on the Sec SUBSTRATE RECOGNITION AND TRANSPORT evolution of the organism. In silico and in vivo analyses indicate that all species, whether Besides the well-studied protein transport pathways, archaea eukaryotic or prokaryotic, transport proteins by way of the univer- may use additional, poorly understood or currently unknown, sally conserved Sec pathway, which acts as a conduit for inserting means by which to facilitate the egress of proteins across the proteins into the cytoplasmic membrane or secreting them into the membrane. For example, some archaeal species produce vesi- extracytoplasmic environment (Yuan et al., 2010; Calo and Eich- cles that are released into the extracellular environment (Soler ler, 2011). Proteins are targeted to the Sec pathway by conserved et al., 2008; Ellen et al., 2010b), or in the case of Ignicoccus, amino-terminal signal peptides that have a tripartite structure are present in the periplasmic space (Nather and Rachel, 2004). consisting of a charged amino-terminus, a hydrophobic stretch Future studies may clarify the role of these mechanisms in protein and a signal peptidase recognition motif (Bardy et al., 2003; Ng transport. et al., 2007; Zimmermann et al., 2011; Table 1). The Sec pathway In this review, we describe the highly diverse array of archaeal consists of several components, including the signal recognition secreted proteins. We consider the pathways used to trans- particle (SRP). The SRP recognizes either the signal peptide or port these proteins to the cytoplasmic membrane, the ulti- transmembrane segments in the substrate as the nascent peptide mate destinations of substrates, and whether these proteins are chain emerges from the ribosome, which results in a translational secreted into the extracellular environment or are anchored arrest. Subsequently, the SRP–ribosome nascent chain complex to the cytoplasmic membrane or cell wall. We also explore is targeted to the proteinaceous Sec pore where the substrate the mechanisms that facilitate the tethering of secreted sub- is translocated across the membrane co-translationally (Grud- strates to the cell surface, and discuss the types of functions nik et al., 2009). Conversely, SRP-independent post-translational displayed by cell-associated and secreted proteins. Finally, we Sec transport requires chaperones to maintain the precursor in describe the in silico approaches used to predict the subcellu- an unfolded conformation to allow for the secretion through the lar localization of substrates and the post-translational mod- approximately 20 Å Sec pore – a pore just large enough to allow ifications that these substrates undergo and identify potential for transport of an unfolded polypeptide (Van den Berg et al., trends in the use of these pathways and modifications in various 2004; Mori et al., 2010; Yuan et al., 2010). While the key com- microorganisms. ponents required for co-translational transport are universally

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conserved, post-translational transport across the ER membrane depends on a luminal ATPase, Bip/Kar2, whereas bacterial post- translational transport requires the cytoplasmic ATPase, SecA. - - - -

+ There is evidence for archaeal post-translational translocation ++ of Sec substrates (Dale et al., 2000; Ortenberg and Mevarech,

N. maritimus 2000; Irihimovitch and Eichler, 2003). However, as archaea lack homologs of either of the two aforementioned ATPases, the ener- getics of archaeal post-translational transport is not understood - - - + + to date. ++ Other

K. cryptofilum Tat SUBSTRATE RECOGNITION AND TRANSPORT

chaeum equitans, The Tat pathway, which unlike the Sec pathway, is not uni- versally conserved, being limited to prokaryotes, chloroplasts, - - - - + and a few protists, transports proteins in a folded conforma- ++ tion (Pohlschroder et al., 2005a; Natale et al., 2008; Yuan et al., N. equitans 2010; Robinson et al., 2011). Because they are folded prior to transport,Tatsubstrates are too large for the Sec pore to accommo-

opyrum pernix, Nanoa r date. Therefore, it is not surprising that the Tat pathway consists - - - + + Ae r ++ of components and mechanisms that are fundamentally differ-

A. pernix ent from those employed by the Sec pathway. However, just as Sec substrates are targeted to the Sec pathway by a signal pep- tide, a Tat signal peptide targets proteins to the Tat pathway

chaea (Berks et al., 2005; Robinson et al., 2011). Organisms contain- - - - + +

++ ing a functional Tat pathway require at least TatA and TatC, ena r

I. hospitalis which play particularly pivotal roles, with TatC (and TatB in C r many organisms) apparently being involved in substrate target- ing, while multimers of TatA, a single transmembrane spanning protein, allow for the formation of pores of varying sizes to - - - + + ++ accommodate the diverse sizes of secreted substrates (Dilks et al., 2005; Gohlke et al., 2005; Leake et al., 2008; Robinson et al., S. solfataricus 2011). While the Sec and Tat pathways are mechanistically very dif- ferent, the tripartite structures of the signal sequences that target + + + + + ++ substrates to these systems are surprisingly similar (Table 1). How-

A. fulgidus ever, there are some subtle differences between Sec and Tat signal peptides. Among the most significant of these differences is the

chaeoglobus fulgidus, Sulfolobus solfataricus, Ignicoccus hospitalis, highly conserved twin arginine motif that lies within the charged A r chaea region of the Tat signal peptide. The Tat signal peptide also has a - - - - + ++ hydrophobic stretch that is generally less hydrophobic than that

M. kandleri of the Sec signal sequence (Rose et al., 2002; Bendtsen et al., Eurya r 2005).

Sec AND Tat SIGNAL PEPTIDE PROCESSING + + + + ++ ++ In addition to the regions of signal peptides that are criti- H. volcanii cal to targeting secreted proteins to a protein transport path- way, Sec and Tat signal peptides also usually contain a recog- nition sequence that targets it for cleavage by a signal pepti- osopumilus maritimus dase, although unprocessed Sec substrate signal peptides occa- sionally serve as amino-terminal membrane anchors (Eichler, Haloferax volcanii, Methanopyrus kandleri, 2002; Paetzel et al., 2002; Tuteja, 2005; Ng et al., 2007). Sub- Signal Peptide* Signal Peptide* strates having signal peptides that are processed by the univer- sally conserved signal peptidase I (SPase I) are either released

from the membrane or anchored to the cell by way of a

chaeum cryptofilum, Nit r carboxy-terminal membrane anchor or protein–protein inter- action (Rose et al., 2002; Dilks et al., 2005; Tuteja, 2005; at/Spase I at/Spase II Kingdom Sec/Spase I Sec/Spase II Sec/Spase III T T PGF C-term Species abbreviations: Kora r *Spaces between boxes represent SPase processing sites, except for PGF C-term, where it represents predicted archaeosortase pro cessing at PGF-motif (exact site has not yet been determined)

Table 1 | Frequencies of secretion and cell surface-anchoring motifs. Gimenez et al., 2007; De Castro et al., 2008; Uthandi et al., 2010).

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Recent in silico data suggest that several euryarchaeal SPase I peptides processed by SPase I or SPase II, the processing site is processed Sec substrates have an additional carboxy-terminal sort- N-terminal to the hydrophobic stretch, which hence remains part ing signal that targets these proteins to the cell wall or to another of the mature protein where it is essential for subunit–subunit cell surface structure (Haft et al., 2012). Originally identified in interactions that allow the formation of an α-helical scaffold Gram-positive bacteria, an enzyme known as a sortase processes facilitating the assembly of type IV pili and pilus-like structures these sorting signals, which are characterized by a signature motif (Bardy and Jarrell, 2002; Albers et al., 2003; Table 1). Although containing a hydrophobic transmembrane α-helix and a cluster of the translocation of archaeal type IV pilin-like proteins into the basic amino acids (Spirig et al., 2011; Table 1). Upon cleavage, sub- cytoplasmic membrane has not been studied, in Gram-negative strate proteins are typically covalently linked to the peptidoglycan bacteria, they were shown to be secreted by the Sec pathway in a cell wall by the sortase. Several types of sortases, each processing signal particle-dependent fashion (Arts et al., 2007; Francetic et al., distinct motifs within the conserved tripartite structure have now 2007). been identified in bacteria, and similar C-terminal tripartite sort- ing signals have also been found in archaea. Furthermore, using N- AND O-GLYCOSYLATION comparative genomics, potential archaeosortases have been iden- Additional post-translational modifications of archaeal secreted tified (Haft et al., 2012). However, sorting signal processing and proteins,other than signal peptide processing,include both O- and modification in archaea awaits in vitro and in vivo confirmation. N-glycosylation (Eichler and Adams, 2005; Calo et al., 2010; Jar- Bacteria and archaea, but to the best of our knowledge not rell et al., 2010). The first non-eukaryotic N-glycosylated protein eukaryotes, express proteins that have signal peptides processed reported was the surface S-layer glycoprotein of the haloarchaeon by signal peptidases distinct from the universally conserved SPase Halobacterium salinarum, a protein that was subsequently also I. Bacterial signal peptides recognized by SPase II are largely shown to be O-glycosylated (Mescher et al., 1974; Sumper et al., similar to those processed by SPase I, containing a charged amino- 1990). While little is known about the O-linked glycosylation terminus and a hydrophobic stretch; however, they also contain process, genetic and biochemical studies of the S-layer, as well as a short motif known as a lipobox that lies subsequent to the of the flagella subunits in two methanogenic archaea and a haloar- hydrophobic stretch (Table 1). The lipobox contains an invariant chaeon, revealed a set of archaeal glycosylation (Agl) enzymes cysteine residue that is acylated by a prolipoprotein diacylglyc- that catalyze the assembly and attachment of N-linked glycans to eryl transferase prior to signal peptidase processing by SPase II, target proteins (Calo et al., 2010; Jarrell et al., 2010). As in eukary- which then cleaves the precursor immediately upstream of the otes, in the final step of glycosylation, an oligosaccharyltransferase lipid-modified cysteine. The acylated cysteine residue anchors the homolog covalently links an assembled oligosaccharide onto the processed substrate to the cytoplasmic membrane (Sankaran and asparagine residue of a target motif (N-X-T/S) in the protein. Wu, 1994; Hutchings et al., 2009; Thompson et al., 2010; Okuda Interestingly, aside from the oligosaccharyltransferase, the Agl and Tokuda, 2011). Although archaea, particularly euryarchaea, components of haloarchaea and methanogens, or even closely express Sec and Tat substrates containing lipobox motifs, neither related methanogens, show no homology to each other, indicating an archaeal SPase II homolog nor a prolipoprotein diacylglyceryl a wide diversity in the specifics of post-translational modification transferase homolog has yet been identified (Mattar et al., 1994; by N-glycosylation across the archaea (Calo et al., 2010; Jarrell Dilks et al., 2005; Falb et al., 2005; Gimenez et al., 2007; Storf et al., et al., 2010). Initial studies on N-glycosylated proteins in crenar- 2010). chaea support this hypothesis; however, much less is known about The highly conserved nature of SPase II processing site the components required for N-glycosylation in these organisms sequences across prokaryotic domains makes the apparent absence (Peyfoon et al., 2010; Meyer et al., 2011). of an archaeal SPase II homolog even more intriguing (Storf et al., DIVERSITY OF SECRETED PROTEINS 2010). Perhaps the mechanisms involved in archaeal lipoprotein Secreted proteins play a crucial role in the interaction of cells biosynthesis are unique to archaea, possibly owing to the distinct with their environment. They fulfill a variety of functions such as membrane lipid composition in the two prokaryotic domains. degradation of complex polymeric substances (e.g.,carbohydrates, Studies performed on bacterial and archaeal lipoprotein mutants proteins), nutrient uptake, signal transduction, or formation of in which the conserved lipobox cysteine was replaced with a ser- surface complexes such as the cell wall or pili (for examples see ine lend credence to the hypothesis that this cysteine is critical in Table 2). While for the majority of secreted proteins no func- both domains. Mutant lipoproteins are not processed in either tion can be predicted, many secreted proteins encoded in model archaea or bacteria, and like in bacteria, unprocessed archaeal archaea or heterologously expressed in a model archaeon have substrates often remain cell-associated while a subset of similar been characterized, leading to a better understanding of the physi- mutant lipoprotein precursors are secreted into the supernatant ology of the organisms, as well as the transport pathways and post- (Hutchings et al., 2009; Storf et al., 2010). translational modification (i.e., some have been used as reporters). SPase III specifically processes the subunits of a wide array of In the following section, several examples are given for the variety type IV pilus-like surface structures; included among these are the of tasks that secreted archaeal proteins fulfill and what is known flagella of archaea (see below; Pohlschroder et al., 2011). Simi- about their secretion and localization. lar to SPase I and SPase II, SPase III recognizes a processing site located adjacent to the hydrophobic stretch of the N-terminal sig- DEFENSE nal peptide that targets the substrate to the transport pathway Several archaea produce peptides or proteins with antimicrobial (Arts et al., 2007; Francetic et al., 2007). However, unlike signal activity that are collectively termed archaeocins. Thus far they have

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Table 2 | Archaeal secreted proteins.

Secreted protein Secretion Signal peptide Localization pathway processing

Extracellular CW-anchor TM-anchor Lipid anchor Peripheral#

ARCHAEOCINS HalH4, Hfx. mediterranei R4 Tat SPase I + HalC8, haloarchaeal stain AS7092 Tat SPase I + SulAB, S. acidocaldarius Sec SPase I + ENZYMES Pyrolysin, P.furiosus Sec SPase I + α-Amylase, S. solfataricus Sec SPase I + + Protease, S. marinus Sec SPase I + Protease (Nep), N. magadii Tat SPase I + Halocyanin I, Hfx. volcanii Tat SPase I + Halocyanin, Hbt. salinarum Tat SPase II + Laccase, Hfx. volcanii Tat SPase I + Alkaline phosphatase, Hbt. salinarum Tat SPase I + SUBSTRATE-BINDING PROTEINS Glucose-binding protein, S. solfataricus Sec SPase III + Iron-binding protein, Hfx. volcanii Tat SPase II + Trehalose-binding protein, T. litoralis Sec SPase II + Oligopeptide-binding protein, A. pernix Sec SPase I + SURFACE STRUCTURES S-layer glycoprotein Hfx. volcanii Sec SPase I + S-layer glycoprotein, S. solfataricus Sec SPase I + S-layer glycoprotein, M. fervidus Sec SPase I + Flagellin, M. maripaludis Sec SPase III + Type IV pilins, S. acidocaldarius Sec SPase III + Mth60 pilin, M. thermoautotrophicus Sec SPase I +

Information based on in vivo and/or in silico studies (see text for details and references); #mode of surface-anchoring not known. been identified in halophiles (halocins) and crenarchaea from the tokodaii, sulA and sulB, both of which are necessary for antimicro- genus Sulfolobus (sulfolobicins; Ellen et al., 2011). Halocins are bial activity (Figure 2A). The gene products carry putative SPase I plasmid-encoded, secreted antimicrobials that can be subdivided processed Sec signal peptides and were suggested to be peripheral into protein or peptide (micro) halocins (O’Connor and Shand, membrane proteins (Ellen et al., 2011). 2002). HalH4 from Haloferax mediterranei is the best-characterized SECRETED ENZYMES protein halocin. It is a 35 kDa protein predicted to be secreted by Nutrient sources in the environment are often in a form that the Tat pathway that is active against other haloarchaea (Cheung cannot be directly taken up by cells. Polymeric molecules such et al., 1997). Microhalocins are peptides ranging from about 3 as carbohydrates must be cleaved into smaller subunits, proteins to 8 kDa in mass that are released from a larger prepro-protein degraded into peptides, and inorganic phosphate liberated from that also contains a predicted Tat signal peptide (Price and Shand, organic compounds (Worthington et al., 2003; Wende et al., 2010; 2000; O’Connor and Shand, 2002). They are effective against other Xu et al., 2011). Many archaeal enzymes that perform these tasks haloarchaea, and in some cases, against thermophilic crenarchaea have been identified and characterized, either by sequence homol- (Haseltine et al., 2001). In the case of halocin 8, the C-terminal ogy search and heterologous expression, or by purification of the 76 amino acid peptide carries antimicrobial activity while the N- native enzymes from cell culture supernatants of the host (Moracci terminal protein portion termed HalI, which remains membrane- et al., 2007). In the case of native enzymes, protein identifica- associated, confers immunity against the halocin, probably by tion by N-terminal sequencing provided valuable information sequestration (Sun et al., 2005). regarding the signal peptidase cleavage site and in some cases post- Sulfolobicins, proteins (about 20 kDa) associated with the translational modifications by glycosylation or lipid modification. membrane and extracellular vesicles, were first identified in Sul- In addition, functional assays can also be used as reporters for folobus islandicus (Prangishvili et al., 2000). The genes encoding in vivo studies on secretion, using either plate assays or measuring sulfolobicins were recently identified in S. acidocladarius and S. enzyme activity in cell culture supernatant (Figure 2B; Rose et al.,

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membrane and the S-layer (Protze et al., 2011). More recently, another tetrathionate hydrolase from A. hospitalis was found to be assembled into“zipper-like particles”on the cell surface and extra- cellular space (Krupovic et al., 2012)(Figure 3A). The secretion mechanism of enzymes has only been addressed in haloarchaea. Secretion by the Tat pathway was demonstrated for the protease SptA from Natrinema sp. J7 (Shi et al., 2006), arabinanase, halo- cyanin 2 and 3, a DsbA-like protein, and two hypothetical proteins from Haloferax volcanii (Gimenez et al., 2007; Storf et al., 2010) as well as α-amylases from Natronococcus sp. strain Ah36 and Haloarcula hispanica (Rose et al., 2002; Hutcheon et al., 2005). Enzymes, especially those of archaeal hyperthermophiles, have potential industrial applications, in particular hydrolytic enzymes that degrade complex polymers. At high temperatures, starch and cellulose are more accessible and the risk of microbial contam- ination is low. Most known hyperthermophiles (cells that grow at 80˚C and above) are archaea and their enzymes can withstand the harsh conditions employed in industrial processes (Egorova and Antranikian, 2005). For example, an α-amylase with high homology to a protein from Thermococcus kodakarensis, which was identified in an environmental DNA library, is stable and highly active under the conditions applied for starch liquefaction (Richardson et al., 2002).

SUBSTRATE BINDING PROTEINS FIGURE 2 | Detection of extracellular protein activities. (A) Overlay Nutrients such as peptides and oligosaccharides produced by assay with colonies of S. acidocaldarius wt (right) and ∆sulAB mutant (left) grown on a lawn of S. solfataricus strain P2. Clear halos surrounding the extracellular enzymes are taken up by dedicated transporters. colonies indicate killing of archaea by the sulfolobicins. Image reproduced Archaea make extensive use of binding protein-dependent ATP with permission from Ellen et al.(2011). (B) Iodine vapor staining of H. binding cassette (ABC) transporters. Binding proteins have high volcanii expressing wild-type (left) or signal sequence mutated (right) affinity for their substrates. Although substrate binding proteins α-amylase grown on rich medium supplemented with 0.2% soluble starch. of Gram-negative bacteria reside in the periplasmic space, they Clear halos surrounding colonies indicate starch hydrolysis by extracytoplasmic α-amylase (Rose et al., 2002). are usually associated with the cytoplasmic membrane in archaea. Different mechanisms are employed for anchoring. The class of oligopeptide-binding proteins characterized in the 2002; Worthington et al., 2003; Hutcheon et al., 2005; Wende et al., crenarchaeal species S. solfataricus and Aeropyrum pernix contains 2010; Xu et al., 2011). an N-terminal secretory signal sequence with an SPase I cleav- Examples of secreted enzymes include proteases such as age site and an additional C-terminal transmembrane segment halolysins and pyrolysins (Kamekura et al., 1992; de Vos et al., (Elferink et al., 2001; Gogliettino et al., 2010; Balestrieri et al., 2001; Shi et al., 2006; De Castro et al., 2008), proteins involved 2011). The membrane anchor is preceded by a stretch of glycosy- in starch degradation such as α-amylase and cyclodextrin lated serine and threonine residues. Several euryarchaeal binding glycosyltransferase (Kobayashi et al., 1994; Hutcheon et al., 2005; proteins were shown to be lipid anchored after processing by the Bautista et al., 2012), and a copper-containing oxidase (laccase; yet to be identified SPase II. These lipobox-containing proteins Uthandi et al., 2010). In some cases, secretion and localization are associated with transport of maltose in Thermococcus litoralis have been studied in more detail. These studies showed that not (Horlacher et al., 1998), or predicted to bind iron and maltose, all secreted enzymes are soluble extracellular proteins, with some respectively, in H. volcanii (Gimenez et al., 2007). In addition, being anchored to the membrane via a lipid anchor (Mattar et al., a putative phosphate binding protein from Hfx. volcanii was pre- 1994; Gimenez et al., 2007; Storf et al., 2010) or a C-terminal dicted to be processed by an archaeosortase and may therefore have transmembrane segment (Gimenez et al., 2007). Interestingly, an a C-terminal lipid anchor (Haft et al., 2012). Interestingly, BasA α-amylase from S. solfataricus that contains a C-terminal mem- and CosA from Hbt. salinarum are two lipid anchored binding brane anchor in addition to an SPase I processed signal sequence proteins shown to be involved in chemotaxis rather than substrate was detected in both membrane and extracellular fractions (Wor- uptake despite their homology to ABC transporter solute binding thington et al., 2003; Ellen et al., 2010a). Apparently, part of proteins (Kokoeva et al., 2002). the protein pool is released by an unknown mechanism, which A third class of binding proteins contains a prepilin-like sig- may contribute to more efficient substrate degradation. Direct nal sequence that is cleaved by SPase III. Removal of the short attachment to the S-layer was demonstrated for a protease from positively charged leader peptide has been shown for S. solfatar- Staphylothermus marinus (Mayr et al., 1996), and tetrathionate icus glucose and arabinose binding proteins (Albers et al., 1999, hydrolase from Acidianus ambivalens was suggested to be localized 2003; Elferink et al., 2001). In silico analyses predicted substrate to the pseudo-periplasmic space, i.e., between the cytoplasmic binding proteins with SPase III processing sites in many Eury-

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and Crenarchaea, however these predictions await experimental efficient repair of the damaged DNA (Frols et al., 2008; Ajon et al., confirmation (Szabo et al., 2007b). Similar to pilins and archaeal 2011). Interestingly, as noted above, S. solfataricus also contains flagellins that are assembled into cell appendages, sugar binding sugar binding proteins that are processed by the same SPase III as proteins are thought to be incorporated into a macromolecular the flagellin/pilin subunits. It is believed that these proteins are also structure called the bindosome (see below). assembled into a large pilus-like structure, the bindosome, since homologs of pilus-biosynthesis genes are also required for bindo- CELL SURFACE STRUCTURES some function, i.e., the growth of cells on certain sugars (Zolghadr Most archaeal cells are surrounded by a membrane-anchored, et al., 2007). crystalline S-layer that provides stability and osmoprotection to Most archaea appear to only have one SPase III homolog; some, the cells (Engelhardt, 2007a,b; Albers and Meyer, 2011). Known however, contain additional paralogs with specialized functions. S-layers are composed of a single protein or two subunits that M. maripaludis expresses a prepilin-peptidase, EppA, that rec- are secreted by the Sec pathway and whose signal peptides are ognizes type IV pilins with a specific SPase III processing site processed by SPase I. In Sulfolobales, anchoring to the cell mem- (previously designated as domain of unknown function 361), brane occurs via a C-terminal transmembrane segment, which is along with FlaK/PibD (Szabo et al., 2007b; Wang et al., 2008; Ng preceded by a stalk-like structure creates a pseudo-periplasmic et al., 2011)(Figure 3B). space. While haloarchaeal S-layer proteins also contain a C- The only known archaeal pili that do not contain subunits with terminal hydrophobic stretch, it has recently been proposed that a type IV pilin signal peptide are the Mth60 fimbriae produced by it is part of a C-terminal conserved tripartite sorting signal that is Methanothermobacter thermoautotrophicus, which are involved in cleaved and lipid-modified by a yet uncharacterized system pos- adhesion to solid surfaces (Thoma et al., 2008; Figure 3C). Mth60 sibly involving the predicted archaeosortase enzyme (Haft et al., is a small (16 kDa) glycoprotein with no known homologs in any 2012). Consistent with this observation,Hfx. volcanii and Hbt. sali- other species. It contains a predicted SPase I processed Sec signal narum S-layer glycoproteins contain lipid modifications (Kikuchi peptide and while it is not known how Mth60 fimbriae are assem- et al., 1999; Konrad and Eichler, 2002). Conversely, the S-layer bled, the recombinant protein was shown to polymerize into fibers glycoprotein of Methanothermus fervidus does not contain a C- at the optimal growth temperature of M. thermoautotrophicus. terminal transmembrane segment and the mechanism of cell Halomucin is the largest known archaeal protein, produced surface attachment is elusive (Brockl et al., 1991). by the extremely halophilic archaeon Haloquadratum walsbyi Archaea also produce a diverse array of cell appendages that (Figure 3D). The 9195 amino acid protein with homology to provide functions such as motility and surface adhesion. Most eukaryotic mucin, is predicted to be secreted via the Sec pathway characterized cell appendage subunits have an N-terminal type IV and its likely function is to provide a hydration shell in their low pilin-like signal peptide (see above; Albers et al., 2003; Bardy et al., water activity environment (Bolhuis et al., 2006; Sublimi Saponetti 2003; Szabo et al., 2007b; Tripepi et al., 2010). et al., 2011). The best-characterized archaeal cell surface structure is the Other unusual cell surface structures have been described, but flagellum, which is required for swimming motility. In contrast are not yet characterized in molecular detail. One fascinating to bacterial flagellar biogenesis, which uses a type III secretion- example is the hamus produced by the euryarchaeon SM1, which like mechanism, the archaeal flagellum biosynthesis machinery grows in cold sulfidic springs (Rudolph et al., 2001; Moissl et al., includes homologs of the bacterial type IV pilus biogenesis 2005). Each cell of strain SM1 produces about 100 hami. The components, including the archaeal prepilin-peptidase homolog hami are highly complex structures having a helical basic struc- PibD/FlaK (Journet et al., 2005; Ghosh and Albers, 2011). In addi- ture from which prickles emanate at regular distances, forming tion to conferring swimming motility, flagella also play a role in a barbwire-like appearance. These structures consist of large sub- surface adhesion in S. solfataricus, M. maripaludis, and Pyrococcus units of about 120 kDa. The N-terminal sequence of this protein is furiosus (Nather et al., 2006; Zolghadr et al., 2010; Jarrell et al., not known and therefore the mode of secretion cannot be assessed 2011). In fact, the thermal stability and adhesive properties led to (Moissl et al., 2005). Finally, cannulae from the crenarchaeaon the suggestion that Pyrococcus flagella be used as “molecular glue” Pyrodictium abyssi are tube-like extracellular structures that cross (US Patent US 2008/0305524). Finally, motility assays developed the S-layer and the pseudo-periplasmic space, but do not pro- for several flagellated archaea have been used to monitor suc- trude into the cytoplasm. Cannulae are composed of at least three cessful secretion and post-translational modification of flagellins related glycoproteins, but their sequences have not been published (Chaban et al., 2007; Tripepi et al., 2010; Lassak et al., 2012). (Mai et al., 1998). While most currently characterized archaeal Additional genes encoding archaeal type IV pilin-like proteins surface appendages appear to follow the type IV pilin assembly were identified, using a program trained on flagellin subunits route, a more diverse variety of specialized secretion systems can (see below), ultimately leading to the identification and charac- be expected from the molecular analysis of structures that have so terization of non-flagellar pilus-like structures. For example, S. far only been studied on a morphological level. acidocaldarius,use Aap and Ups-pili,along with flagella,for surface adhesion and biofilm formation (Henche et al., 2012). DNA dam- SUBCELLULAR LOCALIZATION PREDICTION age such as that caused by UV light irradiation strongly induces As described above, the fate of a secretory protein upon translo- expression of the subunits of the Ups-pili, cell surface struc- cation is determined by specific sequence motifs within the signal tures that subsequently facilitate cell aggregation. Once aggregated, peptide, such as those for signal peptidase processing and lipid these cells are thought to exchange chromosomal DNA, allowing modification (Bardy et al., 2003; Ng et al., 2007). Additionally, the

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FIGURE 3 | Archaeal surface structures. (A) Tetrathionate hydrolase from A. et al., 2008). (C) Non-type IV Mth60 fimbriae of planktonic M. hospitalis YS8 assembles into zipper-like particles on the cell surface. Image thermoautotrophicus cells by staining with AlexaFluor®-488 (Thoma et al., reproduced with permission from Krupovic et al.(2012). (B) Electron 2008). Image courtesy of R. Wirth, University of Regensburg, Germany. (D) micrographs of M. maripaludis cells expressing type IV pilus-like structures. Secreted halomucin complexes (stained green with specific antibody coupled Arrows indicate EppA-processed pili, while additional, thicker structures, are to fluorescein) surrounds quadratic Haloarcula marismortui cell (stained red PibD-processed flagella. Samples were negatively stained with 2% by Nile blue for polyhydroxy butyrate). Unpublished image, courtesy of D. phosphotungstic acid. Image reproduced with permission from (VanDyke Oesterhelt, Max Planck Institute of Biochemistry, Martinsried, Germany. presence of a C-terminal membrane anchor or a putative sorting as well as bacteria, suggesting that Tat signal peptide sequences are signal is a determinant for the localization of the mature pro- reasonably similar across bacterial and archaeal species (Rose et al., tein. Because most of these motifs are relatively well conserved, as 2002; Dilks et al., 2003, 2005; Gimenez et al., 2007; McDonough has been confirmed by numerous in vivo studies, it was possible to et al., 2008; Bagos et al., 2009; Storf et al., 2010; Uthandi et al., develop in silico programs that can provide more detailed informa- 2010). Consistent with this finding, Coulthurst et al.(2012) iden- tion indicating whether a substrate is secreted into the extracellular tified a Tat signal peptide recognition system in the euryarchaeon environment, is tethered to a membrane by an amino-terminal Archaeoglobus fulgidus resembling that of Escherichia coli. transmembrane or lipid anchor, or is incorporated into a surface Lipoprotein prediction programs such as LipoP (Juncker et al., structure. Predictions such as these, in conjunction with in vivo 2003) and pred-lipo (Bagos et al., 2008), the latter being trained and in vitro confirmations, can also deepen our understanding of specifically on Gram-positive bacterial lipoproteins, were devel- the extent to which specific transport pathways and signal pep- oped to identify bacterial Sec signal peptides containing SPase tidases are used, which may point to the evolutionary pressures II processing sites. In silico and limited in vivo analyses suggest encountered by a particular species (see below). that the bacterial and archaeal lipoboxes are similar, and there- A variety of software programs have been developed to predict fore, these programs can be used to identify archaeal lipoproteins specific signal peptide classes. Phobius, for example, is a combined (Storf et al., 2010). Using these programs in tandem gives the transmembrane helix and SPase I processed Sec signal peptide pre- best results since although both programs identify most puta- diction program (Kall et al., 2004). As laid out above, prokaryotic tive lipoproteins, each misses a subset. When used consecutively, Sec substrates can contain signal peptides cleaved by either SPase I, TatFind and lipoprotein prediction programs can also predict SPase II, or SPase III, and in many prokaryotes structurally similar Tat lipoproteins. However, the only prediction program trained signal peptides can differentially target proteins to the Sec or Tat on Tat substrates processed by SPase II is TatLipo, which was pathway (Bardy et al., 2003; Ng et al., 2007; Calo et al., 2010). trained on haloarchaeal Tat substrates and is not yet fully tested on Hence, to accurately predict the pathways employed to trans- non-haloarchaeal proteins (Storf et al., 2010). Tat substrates not port specific prokaryotic proteins, and their ultimate destinations, identified by LipoP or pred-lipo are assumed to be processed by Phobius needs to be used in conjunction with programs that SPase I. predict whether signal peptides contain a lipobox or a prepilin- Unlike the other types of signal peptides discussed here, signal peptidase processing site, as well as programs that can distinguish peptides processed by SPase III show readily apparent differences between Tat and Sec substrates. in bacteria and archaea, requiring independent prediction pro- TatFind and TatP are two programs that were specifically grams be developed to specifically detect archaeal (FlaFind; Szabo trained to identify Tat substrates (Rose et al., 2002; Bendtsen et al., 2007b) and bacterial type IV pilin-like proteins (PilFind; et al., 2005). Although structurally conserved, Tat signal pep- Imam et al.,2011). In archaeal SPase III substrates,the most impor- tide sequences vary somewhat across prokaryotic species. In some tant residues for cleavage to occur are the amino acids at positions species, a small fraction of Tat substrates tolerate replacement of −2, −1, and +1, relative to the cleavage site. In particular, a pos- one of the arginines in the twin arginine motif with a lysine, while itively charged amino acid residue at position −2 appears to be in others replacing either arginine disrupts transport (Berks et al., specific to archaeal SPase III recognition sites. Conversely, many 2005). There is also some variability in the specificity of the amino archaeal SPase III substrates do not contain a glutamate residue acid residues allowed near the twin arginines, both among bacter- at the +5 position, which appears to be a nearly universal fea- ial species and between prokaryotic domains (McDonough et al., ture of the signal peptides of type IV pilin-like proteins in bacteria 2008). However, TatFind, which was trained on a large set of pre- (Imam et al., 2011). As noted above, Tat signal peptides containing dicted haloarchaeal Tat substrates, and TatP, have been validated SPase III processing sites have not been identified. Clearly,applying by in vivo verification of a large number of substrates in archaea prediction programs in concert can clarify subcellular localization

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of processed substrates by ensuring more accurate predictions of possibly because lipid anchoring avoids the need to insert a the class of the signal peptide that each substrate contains. hydrophobic stretch of the secreted protein into the cytoplasmic membrane (Storf et al., 2010). For haloarchaea, transporting a DIVERSE USE OF PROTEIN TRANSPORT PATHWAYS AND carboxy-terminal hydrophobic anchor across a membrane and SURFACE-ANCHORING STRATEGIES inserting it into that membrane in an unfolded conformation When applied to the large number of genome sequences completed is problematic since the solubility of hydrophobic amino acids in recent years, the prediction tools discussed above have enabled is low in a high salt environment – it is unlikely that the Tat us to confirm trends first observed several years ago, and have also pathway is able to laterally insert the transmembrane segments helped identify patterns in the usage of secretion pathways and of their substrates as is observed for Sec substrates. Under these protein anchoring mechanisms. These patterns may have signifi- conditions, using chaperones to maintain the solubility of the cant implications for the evolutionary history or relationships of long hydrophobic stretch of amino acids might be counterproduc- the organisms in which they are observed. tive since hydrophobic interactions between the carboxy-terminal Genomic analyses indicate that the Sec protein transport path- anchor and the chaperones may be enhanced, ultimately result- way is conserved, and very likely essential, in all organisms ing in the chaperones becoming affixed to the secreted substrate. (Pohlschroder et al., 2005b). The requirement of the Sec path- The observation that only a small subset of non-haloarchaeal Tat way for protein transport across the membrane as well as for the substrates contain a predicted lipobox also supports the notion insertion of membrane proteins, strongly suggests that it evolved that the extensive use of lipid anchors by Tat substrates is likely early in the history of living organisms with the insertion of mem- an adaptation to high salt. Conversely, similar portions of non- brane proteins preceding the transport of proteins with a SPase haloarchaeal and haloarchaeal Sec substrates appear to have signal I-cleavable signal peptide. The Tat pathway likely evolved later, peptides that contain lipoboxes. rerouting Sec substrates to this pathway, hence the similarities of The genomes of most euryarchaeal species encode proteins hav- their signal peptides. The fact that a highly diverse set of bacter- ing lipoboxes, again indicating the presence of lipid anchoring ial and archaeal species use the Tat pathway to transport secreted in the common ancestor of bacteria and archaea. The absence proteins suggests that this pathway was also present before the of archaeal homologs of the bacterial lipoprotein biosynthesis divergence of bacteria and archaea from the common ancestor components may be due to the distinct cytoplasmic membrane (Dilks et al., 2003; Storf et al., 2010). The lack of this pathway in compositions of bacteria and archaea. Interestingly, to date, in sil- most eukaryotes is likely due to the transport of proteins into the ico analyses of crenarchaeal genomes suggest that the crenarchaea lumen of the ER, an ATP-containing compartment that lacks many lack lipoproteins (Storf et al., 2010). Perhaps crenarchaeal secreted of the challenges of the extracytoplasmic environment prokaryotic proteins, compared to their euryarchaeal counterparts, are more secreted proteins face. Most prokaryotic species are predicted to frequently anchored to the membrane via amino- or carboxy- use the Sec pathway to transport a large majority of their secreted terminal transmembrane segments, or perhaps crenarchaea use as proteins (Dilks et al., 2003; Storf et al., 2010). Interestingly, in vivo yet unidentified mechanisms to anchor or retain proteins, pos- studies strongly support in silico analyses of nearly 20 haloarchaeal sibly by forming a periplasmic space (Ellen et al., 2010a). This genomes showing that the species of this euryarchaeal class trans- is consistent with the fact that crenarchaea also appear to lack port nearly half of their secreted proteins through the Tat pathway both archaeosortase homologs and substrates that contain the (Bolhuis, 2002; Rose et al., 2002; Dilks et al., 2005; Falb et al., 2005; carboxy-terminal motifs targeted by an archaeosortase (Haft et al., Gimenez et al., 2007; Storf et al., 2010). Similar analyses performed 2012). on over a 100 non-haloarchaeal genomes indicated that the use of Conversely, species in nearly all archaeal kingdoms express type the Tat pathway to transport such a large percentage of secreted IV pilin-like proteins, although not every species within a given proteins may be unique to the halophilic archaea (Dilks et al., kingdom necessarily does, strongly suggesting that type IV pili 2003). Perhaps, the extensive use of the Tat pathway is an adapta- are ancient surface structures (Szabo et al., 2007b; Pohlschroder tion to the high salt environments that haloarchaea inhabit. Highly et al., 2011). Interestingly, even the extraordinarily small genome negatively charged surfaces, providing them with a hydration shell, of the symbiont Nanoarchaeum equitans, a species that cannot are a prerequisite for the stability of proteins exposed to high salt even produce its own lipids, encodes homologs of type IV pilus- concentration and hence require rapid, efficient protein folding, biosynthesis pathway as well as FlaFind positive substrates (Huber a process that may be best accomplished in the cytoplasm, which et al., 2002; Szabo et al., 2007b). It is possible that these pro- contains ATP-dependent chaperones (Frolow et al., 1996; Kennedy teins play an integral role in their attachment to their symbiotic et al., 2001). It should be noted that halophilic bacteria appear to host I. hospitalis (Moissl-Eichinger and Huber, 2011). The lack of use the Tat pathway for transporting only a very limited number both lipobox-containing proteins and type IV pilins in eukary- of substrates. However, although they face the same high salt con- otes is not surprising, again because proteins are transported centration challenges as haloarchaea, as noted above, halophilic into the ER-lumen, rather than directly across the cytoplasmic bacteria express a Sec transport designatedATPase (SecA),possibly membrane. resulting in fundamental differences in Sec transport in these two To date, archaea adapted to extreme conditions other than high prokaryotic domains that might have lead to distinctly different salt, such as high temperature or low pH, have not shown a pref- adaptations to high salt (Mongodin et al., 2005). erence for either of the known protein transport pathways, nor do Analysis of the signal peptidase processing sites also suggest that they appear to have a preferred mechanism for anchoring secreted the vast majority of haloarchaeal Tat substrates are lipoproteins, proteins to the cell surface (Storf et al., 2010).

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OUTLOOK Vesicles may be one possible mechanism for transporting The combination of in vivo and in vitro experiments with in silico proteins across the hydrophobic membrane in a Sec and Tat- predictions has greatly extended our understanding of the diver- independent manner. Extracellular vesicles have been detected in sity of archaeal protein secretion and the localization of Sec and various archaea, including Thermococcales and Sulfolobus species Tat substrates. However, additional strategies may have evolved for as well as I. hospitalis (Prangishvili et al., 2000; Reysenbach et al., the export of proteins and their targeting. One possible approach 2006; Soler et al., 2008; Ellen et al., 2009). Proteomic analysis toward the exploration of alternative secretion routes involves of Sulfolobus derived S-layer coated vesicles revealed the pres- the proteomics of secreted proteins. Proteomic studies aim to ence of homologs of the eukaryotic endosomal sorting complex generate a global view of all proteins present in a sample, and (ESCRT-III) proteins (Lindas et al., 2008; Samson et al., 2008). because quantitative methods are available, the relative expression These proteins play an important role in cell division of Sulfolobus of certain gene products under a variety of conditions can also be (Samson and Bell, 2011) and may thus have an additional role evaluated. in eukaryotic-like vesicle budding and release. Unfortunately, the In recent years, there has been some progress on the proteomics physiological roles of extracellular vesicles in archaea are not yet of archaeal extracellular, surface exposed and membrane proteins clear, although sulfolobicins are associated with these structures (Chong and Wright, 2005; Saunders et al., 2006; Burghardt et al., (Prangishvili et al.,2000; Ellen et al.,2011). Interestingly, Sulfolobus 2008; Ellen et al., 2009, 2010b; Williams et al., 2010a,b). One turreted icosahedral virus (STIV) particles released from the host typical observation was that proteins expected to be cytoplas- cells contain ESCRT-III protein homologs, and the expression of mic, based on functional homology to cytoplasmic proteins, or these proteins in the host cell is up-regulated during viral infec- the lack of a predicted Tat or Sec signal peptide are frequently tion. It is tempting to speculate that STIV hijacks the cellular vesicle recovered from the extracellular fractions. These proteins may release mechanism for its own biogenesis (Snyder andYoung,2011; originate from lysed cells, but in some cases could indicate alterna- Maaty et al., 2012). tive secretion strategies. Difficulties in determining which secreted The vesicles present in the periplasmic space of I. hospitalis bud proteins are transported through as yet undefined secretion path- off the inner membrane and probably fuse with the outer mem- ways include an inability to identify motifs that target proteins brane (Figure 4A). Whether this is a strategy by which I. hospitalis to these pathways. Furthermore, although many predicted pro- outer membrane proteins are transported across the periplasmic teins are homologous to proteins having known functions, making space is not yet known. In fact, I. hospitalis requires the trans- the qualifier “hypothetical” obsolete, their functions have not yet port of many outer membrane proteins as this crenarchaeon is the been characterized. While computational analyses do allow us to only reported organism with an energized outer membrane and make predictions, based on homology, concerning the general ATP synthesis within the periplasmic space (Kuper et al., 2010; functions of these proteins, thereby indicating new directions for Figure 4A). The presence of the outer membrane of the only further investigations of their physiological function, homologous diderm archaeon identified to date raises numerous intriguing proteins sometimes have very different functions and hence pre- questions such as why the type IV Iho670 pili required for I. hos- dictions based on homology may be misleading with regard to pitalis surface adhesion are secreted across the outer membrane subcellular localization. Most significantly though, the majority (Muller et al., 2009), or how secreted outer membrane proteins of predicted proteins show no homology to known proteins, mak- that are likely to be required for cell–cell adhesion with the I. hos- ing investigations of their functions and subcellular localization pitalis symbiont N. equitans are being targeted to and transported much more complicated. across this outer membrane (Junglas et al., 2008; Figure 4B).

FIGURE 4 | Ignicoccus hospitalis protein transport into and across the Regensburg, Germany. (B) Electron micrographs of I. hospitalis ultrathin

outer membrane. (A) Localization of A1AO ATP synthase on I. hospitalis outer sections. Outer membrane of I. hospitalis and the cell surface of N. equitans membrane by EM of ultrathin sections and labeled with antibodies specifically in direct contact (Junglas et al., 2008). Image courtesy of T. Heimerl, H. Huber, raised against the purified 440-kDa ATPase complex (Kuper et al., 2010). and R. Rachel, and image (B) courtesy of H. Huber and R. Rachel, University Image courtesy of T. Heimerl, H. Huber, and R. Rachel, University of of Regensburg, Germany.

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In conclusion, further experimental studies, including pro- approaches combine in vivo and in silico analyses. Finally, teomic analyses, will lead to the improvement of existing predic- researchers have identified a plethora of prokaryotic pro- tion programs and may allow the identification of phyla-specific teins having useful, commercially valuable enzymatic activi- sequences. However, along with a better understanding of “clas- ties. Many of these enzymes are secreted proteins. Knowl- sical” secretory routes via the Tat and Sec pathways, the recent edge about the secretion pathways as well as signal peptide development of several eury- and crenarchaeal models (Leigh et al., processing and other post-translational modifications will be 2011) combined with in silico analyses, such as the recent compar- invaluable in designing strategies for efficiently producing these ative genomics that identified the putative archaeosortases (Haft proteins. et al., 2012), may lead to the discovery of novel protein transport and anchoring strategies. ACKNOWLEDGMENTS Exciting new trends have emerged from recent analy- Research at Microdish is in part supported by the EU Seventh ses of archaeal genomes that are shaping studies designed Framework Large Scale Integrated Project “HotZyme”. Mechthild to identify and characterize the secreted proteins and the Pohlschroder was supported by a National Science Foundation mechanisms underlying the transport, processing, and mod- grant MCB02-MT and by a National Aeronautics and Space ification of secreted proteins. The most promising of these Administration grant NNX10AR84G.

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Primary structure a highly stable glycoprotein into the amylase gene in the hyperther- a specialty of Frontiers in Microbiology. and glycosylation of the S-layer pro- extracellular medium by Haloferax mophilic archaeon Sulfolobus sol- Copyright © 2012 Szabo and tein of Haloferax volcanii. J. Bacte- volcanii. Appl. Environ. Microbiol. 76, fataficus. J. Bacteriol. 185, 482–488. Pohlschroder. This is an open-access riol. 172, 7111–7118. 733–743. Xu, Z., Du, X., Li, T., Gan, F., article distributed under the terms of Sun, C., Li, Y., Mei, S., Lu, Q., Zhou, L., Van den Berg, B., Clemons, W. M. Jr., Tang, B., and Tang, X.-F. (2011). the Creative Commons Attribution Non and Xiang, H. (2005). A single gene Collinson, I., Modis, Y., Hartmann, Functional insight into the C- Commercial License, which permits directs both production and immu- E., Harrison, S. C., and Rapoport, terminal extension of halolysin non-commercial use, distribution, and nity of halocin C8 in a haloarchaeal T. A. (2004). X-ray structure of a SptA from haloarchaeon Natrinema reproduction in other forums, provided strain AS7092. Mol. Microbiol. 57, protein-conducting channel. Nature sp J7. PLoS ONE 6, e23562. the original authors and source are 537–549. 427, 36–44. doi:10.1371/journal.pone.0023562 credited.

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