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Keywords:

  • archea;
  • ArsA/GET3;
  • bacteria;
  • bioinformatic screen;
  • membrane proteins;
  • post-translational targeting

Abstract

  1. Top of page
  2. Abstract
  3. Identification of prokaryotic TA proteins
  4. Functions of prokaryotic TA proteins
  5. Hydropathy analysis of tail anchors
  6. Possible pathways for delivery of TA proteins to the prokaryotic cytoplasmic membrane
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

C-tail-anchored (TA) proteins constitute a heterogeneous group of membrane proteins that are inserted into membranes by unique post-translational mechanisms and that play key roles within cells. During recent years, bioinformatic screens on eukaryotic genomes have helped to obtain comprehensive pictures of the number, intracellular distribution and functions of TA proteins, but similar screens had not yet been carried out on prokaryotic cells. Here, we report the results of a bioinformatic screen of the genomes of two bacteria and one archeon. We find that all three of these prokaryotes contain TA proteins in proportions approaching those found in eukaryotic cells, indicating that this protein group is present in all three domains of life. Although some of our hits correspond to proteins of unknown function, others are enzymes with hydrophobic substrates or have functions carried out at the inner face of the cytoplasmic membrane. To generate hypotheses on the insertion mechanisms of prokaryotic TA proteins, we compared the sequences of the prokaryotic and eukaryotic versions of Asna1/Trc40/GET3, a cytosolic ATPase that plays a key role in TA protein post-translational delivery to membranes in eukaryotic cells. We found that hydrophobic residues involved in TA binding by the eukaryotic chaperone (Mateja et al., Nature 2009;461:361–366) are generally replaced with equally hydrophobic amino acids in the archeal homologue (ArsA), whereas this is not the case for the bacterial protein. Thus, eukaryotes may have inherited the GET3 targeting pathway from our archeal ancestor, while the bacterial homologue may be exclusively dedicated to heavy metal resistance.

Because of their unusual biogenesis and because of the fundamental processes they are involved in, tail-anchored (TA) membrane proteins have recently attracted a great deal of interest among investigators in the field of membrane biogenesis (reviewed in 1,2). In TA proteins, a functional N-terminal region located in the cytosol is anchored to the appropriate bilayer by a transmembrane domain (TMD) very close to the C-terminus [at no more than 30 residues (3)]. Because the C-terminal TMD is the only membrane-targeting sequence within the polypeptide and because it emerges from the ribosome only upon completion of translation, TA proteins are obliged to insert into their target membranes by post-translational mechanisms.

Numerous investigations carried out in recent years have begun to shed light on the unique post-translational pathways followed by TA proteins to insert into their target membranes (1,2). In parallel with these studies on TA protein biogenesis, bioinformatic screens, aimed at generating complete catalogues of eukaryotic TA proteins, have been carried out (4–7). The results of these screens have demonstrated the abundance of TA proteins and the variety of important processes they are involved in—e.g. membrane traffic, apoptosis, protein translocation—in animals, plants and fungi. In contrast, similar systematic screens on prokaryotic proteomes have not been carried out so far. Here, we have filled this gap, by applying a bioinformatic approach to identify TA proteins in the proteomes of three different prokaryotes—two bacteria and one archeon. Our results indicate that TA proteins are present in all domains of life and suggest that mechanisms underlying their insertion in modern eukaryotes may have originated in ancestral prokaryotic cells.

Identification of prokaryotic TA proteins

  1. Top of page
  2. Abstract
  3. Identification of prokaryotic TA proteins
  4. Functions of prokaryotic TA proteins
  5. Hydropathy analysis of tail anchors
  6. Possible pathways for delivery of TA proteins to the prokaryotic cytoplasmic membrane
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

For our analysis, we chose one archeon, Methanococcus maripaludis, and two bacteria, Escherichia coli K12 and Rickettsia prowazekii. The latter is a member of the group of α-proteobacteria, which are recognized to be the closest existing relatives to eukaryotic mitochondria (8).

Individual proteomes (UniProt Taxon identifiers: E. coli strain K12, substrain M61655, ID 511145; M. maripaludis strain C7, ID 426368; R. prowazekii strain Madrid E, ID 272947) were downloaded from the Integr8 database (http://www.ebi.ac.uk/integr8/EBI-Integr8-HomePage.do) as a single file in FASTA format. Each protein was then run through TMpred software accessed through the public web interface (http://www.ch.embnet.org/software/TMpred_form.html) in order to identify transmembrane regions of lengths between 14 and 33 residues. The results were screened for proteins containing a single predicted TMD scoring higher than 1500. This cutoff was chosen in conformity with a previous screen on Homo sapiens(5). From this list we extracted proteins whose TMD was at a distance of less than 30 residues from the C-terminus.

In our screen, we ignored the topology predictions of TMpred. This decision was justified by the observation that the TMD of the β subunit of the SecYEβ complex of M. maripaludis was predicted to be in an outside-in conformation (with the consequent localization of the N-terminal domain in the periplasmic space) in contrast to the known 3D structure of the archeal complex (9). We accepted as bona fide tail anchors also TMDs whose scores exceeded 1500 only in one of the possible topologies, regardless of whether this was inside-out or ouside-in.

The lists were then filtered by analysis with SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/), and proteins with a signal peptide HMM (Hidden Markov Models) probability score greater than 0.65 were eliminated. Proteins of the final list were then divided into two categories: the first category contains members that have a clear TA topology (listed in Tables 1 and 2), whereas the second one is composed of proteins whose TA status appears less certain (listed in Table S1). The main class of potential TA proteins excluded from the first category comprises small polypeptides, in which the TMD satisfies the distance criterion from the C-terminus, but is also at less than 30 residues from the N-terminus. These proteins might be easily inserted into the membrane in inverted orientation as compared to classical C-TA proteins. These small hydrophobic polypeptides are abundant in E. coli (Table S1). Another reason for exclusion from the first category was the possible presence of a second TMD. Although proteins with a potential second TMD scoring more than 1500 in TMpred were excluded from the initial list, a number of candidate TA proteins did present a potential second TMD with lower score (but considered to be significant by the algorithm). We therefore excluded proteins with a potential second TMD scoring above 800.

Table 1.  Proportion of protein-coding genes that specify TA proteins in different organisms
OrganismNumber of protein-coding lociTA proteins
Number% of protein-coding loci
H. sapiens(5)20 3234112.02
A.thaliana(7)32 8244541.3
S. cerevisiae(4)  5861550.94
E. coli chromosome and plasmid [this study]  4812110.23
M. maripaludis [this study]  1787120.67
R. prowazekii [this study]   83570.84
Table 2.  Putative prokaryotic TA proteins
uniprotkb/trembl accession numberNameNumber of residuesNumber of residues downstream to TMDFunction
  1. aThe structure of the formate dehydrogenase-N complex demonstrates that the N-terminal domain of the β subunit faces the periplasmic space (14).

E. coli chromosome and plasmid
P77381Uncharacterized J domain-containing protein djlB4754Co-chaperone
P77359Uncharacterized J domain-containing protein djlC4831Hsc56 co-chaperone of HscC
P765033-ketoacyl-CoA thiolase4363Anaerobic degradation of long- and medium-chain fatty acids
P15286Flagellar regulator flk3311Flagella assembly protein
P0A6U3tRNA uridine 5-carboxymethylaminomethyl modification enzyme mnmG62910Enzyme involved in tRNA modification
P30137Thiamine-phosphate pyrophosphorylase21122Enzyme involved in Cofactor biosynthesis
P311423-mercaptopyruvate sulfurtransferase28030Enzyme that transfers a sulfur ion to cyanide or to other thiol compounds
P0AEH5Protein elaB (DUF883 superfamily)1012Unknown
P0ADQ7Uncharacterized protein ygaM (DUF883 superfamily)1133Unknown
P64581Uncharacterized protein yqjD (DUF883 superfamily)1012Unknown
P08321TraL9129Involved in F pilus formation
P0AAJ3Formate dehydrogenase-N β subunita29417Allows E. coli to use formate as major electron donor during anaerobic respiration
M. maripaludis
A6VHF1Tetrahydromethanopterin S-methyltransferase, subunit B (MtrB family)1089Part of a complex that catalyzes the formation of methyl-coenzyme M and tetrahydromethanopterin. Coupled to Na+ transport
A6VHF2Tetrahydromethanopterin S methyltransferase, subunit A (MtrA superfamily2390As for Tetrahydromethanopterin S-methyltransferase, subunit B
A6VHF3Tetrahydromethanopterin S-methyltransferase, subunit A—MtrA and F superfamilies2535As for Tetrahydromethanopterin S-methyltransferase, subunit B
A6VHF4Tetrahydromethanopterin S-methyltransferase, subunit G—MtrG superfamily747As for Tetrahydromethanopterin S-methyltransferase, subunit B
A6VG28Glycosyltransferase family A26526May be involved in synthesis of dolichol-linked oligosaccharide
A6VH14SBHA superfamily (Sec61 beta)530Protein translocation
A6VH21Sec61 gamma—SecE superfamily796Protein translocation
A6VIP5PHP (polymerase and histidinol phosphatase) domain protein29921Predicted metal-dependent phosphoesterase
A6VFQ3Conserved hypothetical protein815Unknown
A6VHG4Conserved hypothetical protein511Unknown
A6VJ19Uncharacterized protein containing a Zn-ribbon604Unknown
A6VJL3Conserved hypothetical protein9618Unknown
R. prowazekii
Q9ZCH13-Oxoacyl-[Acyl-Carrier-Protein] Synthase III (Fabh)3172Fatty acid elongation
P50054Pre-protein translocase subunit SecE6616Protein translocation
Q9ZCL4Putative glutamine amidotransferase-like protein RP71316412Amine transfer
Q9ZDZ4Uncharacterized protein9018Unknown
Q9ZD41Uncharacterized protein2014Unknown
Q9ZCK6Uncharacterized protein941Unknown
Q9ZD80Uncharacterized protein2766Unknown

As summarized in Table 1, all three of the analysed prokaryotic organisms possess TA protein-coding genes. Compared to eukaryotes, E. coli has a lower percentage of TA protein-coding loci, whereas the proportion in M. maripaludis and R. prowazekii approaches that found in yeast. As explained in the preceding paragraph, the list of Table 1 is quite restrictive, and some of the proteins of Table S1 may well be bona fide TA proteins.

Two considerations support the general validity of the results of our screen. First, the only well-characterized TA proteins of archea, SecE and Secβ(9) were both retrieved in the M. maripaludis proteome. Second, a number of our hits (Table 2) correspond to proteins known to be associated with the inner face of the cytoplasmic membrane: e.g. in E. coli, the flagella assembly protein flk (10) and the TraL polypeptide involved in F pilus formation (11); in M. maripaludis, the A subunit of the Na(+)-translocating methyltransferase complex (12) and a member of the glycosyltransferase family A, involved in dolichol-linked oligosaccharide synthesis (13); in R. prowasekii, the E subunit of the SecYEG translocon. On the other hand, we did find one interesting false-positive hit (last entry for E. coli in Table 2, in italics): the iron–sulfur cluster-containing β subunit of nitrate inducible formate dehydrogenase (FDH-N β), whose N-terminal domain faces the periplasmic space (demonstrated by the crystal structure of the FDH-N complex (14)). This topology initially appeared surprising to us, given that FDH-N β lacks a signal peptide, as dictated by our screen. The apparent paradox is resolved if one considers that translocation of the associated α-subunit of the trimeric FDH-N complex depends on the Twin Arginine Transporter (Tat) (15). This system, exclusive to prokaryotes and to chloroplast thylakoid membranes, has evolved for the translocation of fully folded, cofactor-containing, proteins across energy-transducing membranes (16). Thus, the β subunit, although lacking a Tat recognition sequence, is probably translocated in association with the α-subunit by this transporter. This false-positive hit indicates that caution must be exerted in drawing conclusions on the topology of polypeptides that belong to complexes potentially translocated by the Tat system.

A recent study on the evolution of biological membranes included a list of 14 E. coli TA proteins (17). Six of the proteins identified by this author are also contained in our list in Table 1, while two of them were assigned by us to our second category (Table S2), because of the possible presence of a second TMD. Another six do not satisfy the criteria of our screen, either because of the distance of the TMD from the C-terminus or because of the probable presence of an N-terminal signal peptide, or because of a second predicted TMD scoring above 1500 with TMpred.

Functions of prokaryotic TA proteins

  1. Top of page
  2. Abstract
  3. Identification of prokaryotic TA proteins
  4. Functions of prokaryotic TA proteins
  5. Hydropathy analysis of tail anchors
  6. Possible pathways for delivery of TA proteins to the prokaryotic cytoplasmic membrane
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

As shown in Table 2, the identified putative TA proteins carry out a variety of functions, and, with the exception of the E (γ) subunit of the Sec YEG (β) complex, there is little overlap between the three prokaryotic organisms that we analysed. Our screen identified several proteins known to function at the inner face of the cytoplasmic membrane (see previous section) and a number of enzymes with hydrophobic substrates that are expected to be favoured by membrane anchorage. Among these are E. coli 3-ketoacyl-CoA thiolase, involved in the anaerobic degradation of fatty acids, and R. prowazekii 3-Oxoacyl-[Acyl-Carrier-Protein] Synthase III. The latter is a member of the FabH family, which contains enzymes that initiate elongation in type II fatty acid synthase systems found in bacteria and plants. Another enzyme with a hydrophobic substrate is M. maripaludis family A glycosyltransferase. The A family glycosyltransferases are involved in the production of oligosaccharyl-dolichol, the donor for protein N-glycosylation (13). This metabolic pathway has been inherited by modern eukaryotes. The protein identified in our screen belongs to the glycosyltransferase 2 superfamily, of which an additional seven members in the C7 strain of M. maripaludis are known (13). None of these qualify as TA proteins, and only one (MmarC7_1365) is predicted to be a single-spanning membrane protein.

Another function worth noting is that of the co-chaperone (DnaJ—Hsp40), found in two E. coli TA proteins. Interestingly, the published screen on plants identified five TA proteins with N-terminal J domains (7). Instead, no TA homologues were identified in the human proteome (5), although several J domain-containing proteins lacking signal peptides and predicted to have internal membrane spanning helices are present (our unpublished observation). To investigate whether TA J domain-containing proteins are present in yeast, we extracted Saccharomyces cerevisiae Hsp40 proteins (IPR003095 signature) from the InterPro data base. Out of 70 proteins, we found one with predicted TA topology (UniProtKB/TrEMBL Accession Number P48353). Thus, tail anchoring seems to be a relatively common way to localize J domains to the cytosolic face of membranes. Membrane anchorage could facilitate the activity of the Hsp70–Hsp40 chaperone system directed towards clients destined for association with the lipid bilayer.

Hydropathy analysis of tail anchors

  1. Top of page
  2. Abstract
  3. Identification of prokaryotic TA proteins
  4. Functions of prokaryotic TA proteins
  5. Hydropathy analysis of tail anchors
  6. Possible pathways for delivery of TA proteins to the prokaryotic cytoplasmic membrane
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

We next analysed the hydrophobicity of all the predicted TMDs of the TA proteins of Table 1, applying commonly used hydrophobicity scales. The values for the single amino acid residues contained in the TMD predicted by TMpred were added to give total hydrophobicity values. As shown in Table S2, by all the scales used, E. coli TA protein TMDs were of low hydrophobicity compared to the other two analysed prokaryotes and mammals (5). The results obtained with the Zhao-London scale (18) are graphically illustrated in Figure 1. Notably, out of 11 E. coli putative TA proteins, 5 had total hydrophobicity lower or equal to that of mammalian cytochrome b(5) and close to that of protein tyrosine phosphatase (PTP)1B. Both of these are well-characterized TA proteins with TMD of low hydrophobicity localized to the endoplasmic reticulum (ER) (19).

image

Figure 1. Hydrophobicity of TMDs of prokaryotic TA proteins. The hydrophobicity values according to the Zhao-London scale (18) for each residue in the TMD (as predicted by TMpred) of the proteins listed in Table 2 were summed. Averages for each prokaryote are indicated by the horizontal lines. For comparison, the average TMD hydrophobicity ± SD of human TA proteins calculated with the Zhao-London scale from the compilation of Kalbfleisch et al. (5) is shown on the right. The arrow and arrowhead on the left indicate the TMD hydrophobicity of mammalian cytochrome b(5) and PTB1B, respectively.

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Possible pathways for delivery of TA proteins to the prokaryotic cytoplasmic membrane

  1. Top of page
  2. Abstract
  3. Identification of prokaryotic TA proteins
  4. Functions of prokaryotic TA proteins
  5. Hydropathy analysis of tail anchors
  6. Possible pathways for delivery of TA proteins to the prokaryotic cytoplasmic membrane
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

Recent work on TA protein biogenesis in eukaryotes has led to the conclusion that different pathways operate to deliver TA substrates to their target membranes (1,2). TA proteins with moderately hydrophobic TMDs can insert into pure lipid bilayers without assistance from any protein, although, in vivo, chaperones may be required for faithful targeting (20). Other TA proteins are incapable of inserting into pure phospholipid bilayers, and instead require cytosolic chaperones and membrane receptors for their transmembrane integration. In vitro studies have implicated three chaperone systems: (i) Hsp40/Hsc70 (for a subset of TA proteins with not very hydrophobic TMDs—(21)); (ii) Signal recognition particle (SRP), functioning in an unusual post-translational mode for TA proteins with very hydrophobic TMDs (22); (iii) a novel cytosolic ATPase, called Asna1 (arsA arsenite transporter, ATP-binding, homologue 1) or TRC40 (TMD recognition complex subunit of 40 kDa) in mammals (23), and GET3 (guided entry of tail-anchored proteins-3) in yeast (24). Investigations in yeast deletion strains have demonstrated the importance of the GET3 system in vivo(24,25).

Recent X-ray structures of GET3 (26–30) have revealed that it consists of a symmetric homodimer stabilized by the co-ordination of a zinc atom by two essential Cys residues (Cys-285/Cys-288). Each monomer comprises a core ATPase subdomain and an α-helical subdomain. In the ATP-bound state, the dimer interface exposes a large hydrophobic groove that mutational analysis has implicated in tail-anchor binding (26,27).

GET 3 is homologous to prokaryotic ArsA, a protein that in bacteria, together with its transmembrane partner ArsB, is involved in arsenite transport (31). At variance with the situation in eukaryotes, in bacterial ArsA two ATPase domains are repeated in tandem within a single polypeptide chain. In contrast, the archeal homologous polypeptide has a single ATPase domain, and the conservation of the two Cys residues involved in Zinc co-ordination (see Figure 2) suggests that it, like its eukaryotic counterpart, forms a dimer.

image

Figure 2. Alignment of bacterial and archeal ArsA proteins with eukaryotic Asna1/TRC40/GET3 homologues shows conservation of residues involved in TA protein targeting between eukaryotes and archea. The five indicated sequences (Sc, S. cerevisiae; Hs, H. sapiens; Mm, M. maripaludis; Ec, E. coli) were aligned by ClustalW, using the Blosum Series matrix with the default parameters of MacVector 6.0 software. Conserved residues in at least three sequences are boxed and conserved substitutions are in bold. Four conserved ATPase sequence motifs are highlighted in light blue. The TRC40/GET3 insert present also in the M. maripaludis protein is highlighted in grey. The two Cys residues involved in Zn co-ordination in the yeast protein are highlighted in yellow. Hydrophobic residues demonstrated by mutagenesis (21) to be involved in tail anchor binding in S. cerevisiae are shown in bold red, and in pink where replaced with hydrophobic residues in other species. The four vertical arrows indicate residues whose hydrophobicity is conserved in M. maripaludis but not in E. coli.

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To investigate a possible involvement of prokaryotic GET3 homologues in TA protein targeting, we aligned the sequences of the yeast and human proteins with the M. maripaludis homologue and the two halves of E. Coli ArsA. As shown in Figure 2, and as is often the case, the eukaryotic proteins are more similar to the archeal than to the bacterial homologue (59.5%, 42.4% and 26.2% identity between S. cerevisiae GET3 and M. maripaludis, E. coli 1–321 and E. coli 322–583 ArsA, respectively). We then focussed our attention on the putative TA binding region of the GET3 homologues. Mateja et al. (26) identified nine hydrophobic residues predicted to be in the TA binding region of S. cerevisiae GET3, whose mutation to charged residues (alone or in combination) severely reduced TA binding. Mutation of three of these (I133, L183, L187) also reduced ATPase activity, whereas replacement of the other six with charged residues exclusively affected TA binding. Quite strikingly, in M. maripaludis all but one of these nine residues are either conserved (in one case) or replaced with alternative hydrophobic residues (Figure 2). In contrast, two of these residues (sc GET3 F190 and I193) are replaced by hydrophilic aminoacids in the two halves of E. coli ArsA, and two (sc GET3 F204 and M205) are present in a region (called TRC40 or GET3 insert) which is absent in the E. coli protein. We also aligned all the archeal ArsA polypeptides retrieved by blasting the NCBI database, and found that in 21 out of 24 sequences the hydrophobic residues are conserved or replaced with uncharged residues (in most cases hydrophobic—Figure S1). Interestingly, in the three species in which Met198 is replaced with Lys (Thermoproteus neutrophilus Pyrobaculum arsenatocum, Pyrobaculum calidifontis), also the Cys residues presumably involved in Zinc co-ordination are not conserved, whereas they are present in 20 of the 21 other analysed sequences.

The observations described in the preceding paragraph are compatible with the idea that in most archeal species the GET3 homologue ArsA might be involved in TA protein targeting, as it is in eukaryotes. In contrast, the bacterial homologue appears to lack the features required for this task, and might, in conjunction with its partner ArsB, be exclusively dedicated to heavy metal resistance (32).

Conclusions

  1. Top of page
  2. Abstract
  3. Identification of prokaryotic TA proteins
  4. Functions of prokaryotic TA proteins
  5. Hydropathy analysis of tail anchors
  6. Possible pathways for delivery of TA proteins to the prokaryotic cytoplasmic membrane
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

Our bioinformatic screen indicates that TA proteins are present in three different classes of prokaryotic cells—namely, α-proteobacteria, γ-proteobacteria and methanococci—where they carry out a variety of functions known to occur at the inner face of the cytoplasmic membrane or where they are involved in metabolic reactions that are reasonably expected to benefit from membrane anchorage of the responsible enzyme. Thus, these simple transmembrane proteins, characterized by unique biogenetic pathways, are present in all three domains of life. A smaller proportion of protein-coding loci specify TA proteins in prokaryotes than in eukaryotes. The latter presumably adapted TA topology to a wide spectrum of proteins with functions unique to compartmentalized cells.

Hydropathy profiles of the tail anchors revealed that E. coli TA proteins on the average have less hydrophobic TMDs than eukaryotes and than the other two analysed prokaryotes. Based on sequence analysis, E. coli also seems to lack a GET3 homologue capable of chaperoning TA proteins. Because of the lower hydrophobicity of their TMDs, E. coli's TA proteins could be inserted either by the unassisted pathway (19,20,33) or with the help of chaperones of the DnaK/DnaJ (hsp70/hsp40) families (21). These insertion mechanisms could represent a primitive mode of membrane assembly that could have been important during the early evolution of biological membranes. On the other hand, the sequence of archeal GET3 homologues is compatible with a function in TA protein chaperoning, and eukaryotes may have inherited this important targeting pathway from our archeal ancestors. Interestingly, and as is true for a large number of archeal species (34), the genome of M. maripaludis does not contain loci coding for Hsp70/Hsp40 chaperones. Finally, it should be mentioned that a search in the InterPro database (using S. cerevisiae GET3—entry IPR003348—as source) failed to reveal the existence of an ArsA/GET3 homologue in R. prowazekii. One might therefore speculate that Rickettsia inserts its TA substrates that have highly hydrophobic TMDs via the SRP post-translational pathway described in eukaryotes (22). Thus, while our observations indicate the ubiquitous nature of TA proteins in all domains of life, they also illustrate once more the versatility of these proteins in accessing different pathways to achieve their final transmembrane integration.

References

  1. Top of page
  2. Abstract
  3. Identification of prokaryotic TA proteins
  4. Functions of prokaryotic TA proteins
  5. Hydropathy analysis of tail anchors
  6. Possible pathways for delivery of TA proteins to the prokaryotic cytoplasmic membrane
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Identification of prokaryotic TA proteins
  4. Functions of prokaryotic TA proteins
  5. Hydropathy analysis of tail anchors
  6. Possible pathways for delivery of TA proteins to the prokaryotic cytoplasmic membrane
  7. Conclusions
  8. Acknowledgments
  9. References
  10. Supporting Information

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Table S1: Possible TA proteins excluded from Table 1.

Table S2: Hydropathy analysis of TA protein TMDs*.

Figure S1: Alignment of archeal ArsA proteins. Archeal ArsA proteins were retrieved by blasting the NCBI database using M. maripaludis C7 ArsA as bait. The 24 retrieved sequences were aligned by ClustalW, using the Blosum Series matrix with the default parameters of MacVector 6.0 software. Conserved residues are boxed. The two Cys residues involved in Zn co-ordination in the yeast protein are highlighted in yellow. Conserved hydrophobic residues of the M. maripaludis protein, corresponding to residues involved in TA binding in S. cerevisiae, are in red. A lysine residue that substitutes one of these hydrophobic residues (M198 of M. maripaludis) in three organisms is shown in green. These same three organisms (Thermoproteus neutrophilus, Pyrobaculum arsenatocum, Pyrobaculum calidifontis) lack the two Cys residues involved in Zn co-ordination in the fungal protein. ArsA sequences are from the following organisms: Methanococcus maripaludis C7 (Mm7), Methanococcus maripaludis C5 (Mm5), Methanococcus maripaludis S2 (Mm2), Methanocaldococcus fervens AG46 (Cf), Methanocaldococcus jannaschii DSM 2661 (Cj), Methanocaldococcus sp. FS406-22 (Cs), Ferroglobus placidus DSM 10642 (Fp), Halorubrum lacusprofundi ATCC 49239 (Hl), Halomicrobium mukohataei DSM 12286 (Hm), Haloarcula marismortui ATCC 43049 (Hmm), Halobacterium sp. NRC-1 (Hs), Haloterrigena turkmenica DSM 5511 (Ht), Halorhabdus utahensis DSM 12940 (Hu), Haloquadratum walsbyi DSM 16790 (Hw), Methanococcus aeolicus Nankai-3 (Ma), Methanocaldococcus vulcanius M7 (Mcv), Methanopyrus kandleri AV19 (Mk), Methanocaldococcus infernus ME (Mi), Methanobrevibacter smithii ATCC 35061 (Ms), Methanosphaera stadtmanae DSM 3091 (Mss), Methanothermobacter thermautotrophicus str. Delta H (Mt), Methanococcus vannielii SB (Mv), Natronomonas pharaonis DSM 2160 (Np), Pyrobaculum arsenaticum DSM 13514 (Pa), Pyrobaculum calidifontis JCM 11548 (Pc) and Thermoproteus neutrophilus V24Sta (Tn).

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