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Analysis of the twin-arginine motif of a haloarchaeal Tat substrate

Daniel Kwan , Albert Bolhuis
DOI: http://dx.doi.org/10.1111/j.1574-6968.2010.02001.x 138-143 First published online: 1 July 2010


The twin-arginine translocase (Tat) is a system specific to the transport of fully folded proteins. In contrast to most prokaryotes, the Tat pathway is the main route for export in halophilic archaea (haloarchaea). The haloarchaeal Tat system also seems to differ in a number of other aspects from the nonhalophilic counterparts, such as the constituents of the translocase and bioenergetic requirements. Therefore, it was important to test which features in haloarchaeal Tat substrates were important for transport, as these might also be different from those of nonhalophilic organisms. Here, we analysed residues in the so-called Tat motif, which is found in the amino-terminal signal peptide of all Tat substrates. Bioinformatics analysis showed that in haloarchaea, the consensus sequence of this motif is (S/T)RRx(F/L)L. Using the model protein AmyH, we found that both arginines and both hydrophobic residues were essential to translocation: either replacing one or both of the arginine residues with lysine, or replacing one of the hydrophobic residues with alanine, led to a block in translocation. Other residues in or around the motif were found not to be essential for transport.

  • twin-arginine translocation
  • protein secretion
  • halophilic archaea
  • Tat motif
  • signal peptide


The twin-arginine translocase (Tat) is a protein translocation system that is dedicated to the transport of folded proteins. In most prokaryotes, it plays only a minor role, with most proteins being secreted through the Sec system. The main difference between the two transport systems lies in the nature of the substrates: Sec-dependent proteins fold after translocation, whereas Tat-dependent proteins fold before. As a result of this, the two systems are mechanistically completely different (reviewed in Robinson & Bolhuis, 2004; Pohlschröder et al., 2005; Natale et al., 2008). Usually, two or three components with distinct functions are involved in the translocation of Tat substrates. These are denoted TatA, TatB, and TatC. TatA and TatB are small proteins with similar topologies, both having one membrane-spanning domain at the N-terminus. The third component, TatC, is a larger protein with six membrane-spanning domains. Organisms such as Gram-positive bacteria and archaea often lack the TatB protein (Robinson & Bolhuis, 2004). In these organisms, the TatA protein is probably bifunctional, fulfilling the role of both TatA and TatB (Barnett et al., 2008).

The signals directing Sec and Tat substrates to their respective translocases are, at first glance, fairly similar. Substrates for both pathways contain a transient amino-terminal stretch of amino acids of about 15–35 residues comprising three basic domains (von Heijne, 1990): a positively charged region at the N-terminus (N-domain), a hydrophobic core (H-domain), and a more polar region that contains the cleavage site for a signal peptidase (C-domain). There are three features that set signal peptides of prokaryotic Sec and Tat substrates apart. Firstly, Tat substrates contain a characteristic twin-arginine motif at the border of the N- and H-domains; secondly, the hydrophobicity of the H-domain in Tat substrates is lower than that of Sec-dependent proteins; and thirdly, Tat signal peptides are, on average, longer than Sec signal peptides (Chaddock et al., 1995; Berks, 1996; Cristobal et al., 1999). The Tat motif contains a pair of arginines (hence the name twin-arginine translocase) that are surrounded by a number of other conserved residues. In Escherichia coli, the motif is S/TRRxFLK (Berks, 1996). The twin-arginine residues are nearly always present, although there appear to be a few exceptions. For instance, the TtrB subunit of Salmonella enterica tetrathionate reductase contains a KR motif instead, but it is still directed to the Tat pathway (Hinsley et al., 2001). In general, however, changes in the two arginines, even if these are conservative, block or drastically reduce protein translocation (see e.g. Chaddock et al., 1995; Stanley et al., 2000; Buchanan et al., 2001; Rose et al., 2002).

The residues surrounding the two arginine residues are present at a high frequency, but can nevertheless still vary. However, only the phenylalanine (the second residue after the arginines) appears to be critical; the functionality of the E. coli Tat substrate SufI was only retained when Phe was replaced with another strongly hydrophobic residue such as Leu (Stanley et al., 2000). Surprisingly, replacing the other residues surrounding the two arginines in SufI or YacK (a SufI homologue) only led to minor effects, if at all (Stanley et al., 2000).

As mentioned before, in most prokaryotes, the Sec system is the dominant export route. In contrast, however, in halophilic archaea (haloarchaea), it is the Tat system that is predicted to be the dominant export route (Bolhuis, 2002; Rose et al., 2002). It has been speculated that this is an adaptation to the highly saline conditions in which these organisms thrive (Bolhuis, 2002; Rose et al., 2002). Haloarchaea contain high concentrations of KCl intracellularly, and it may be that secretory proteins fold very rapidly, which in turn leads to a necessity of the Tat system. As a consequence, the haloarchaeal Tat system is essential for viability (Dilks et al., 2005; Thomas & Bolhuis, 2006), corroborating the dominant role of this transport route.

The haloarchaeal Tat system is different from the Tat system of nonhalophilic organisms in a number of ways. Firstly, as mentioned before, most proteins in haloarchaea are secreted in a Tat-dependent manner. Secondly, the composition and topology of Tat translocase components in haloarchaea are different. There are one or two TatA proteins, and always two TatC proteins, with one of these TatC proteins being a translational fusion between two TatC domains (Bolhuis, 2002); the latter seems unique to haloarchaea. Thirdly, we have shown that transport of the Tat-dependent substrate AmyH, an amylase from the haloarchaeon Haloarcula hispanica, depends on the sodium motive force (Kwan et al., 2008). This is in contrast to bacterial or chloroplast Tat systems, which depend on the proton motive force. For all of those reasons, it is also conceivable that the nature of signal peptides of haloarchaeal Tat substrates is different from those of nonhalophilic Tat substrates. Thus, it was important to investigate the Tat motif of Tat substrates, as any major differences would have an impact on for instance the prediction of the transport routes used by proteins found through genomic sequencing projects. Here, in this study, we analysed the importance of residues in the Tat motif of the aforementioned AmyH to provide.

Materials and methods


Unless noted, all chemicals were from Sigma-Aldrich (Dorset, UK) or Fisher Scientific (Loughborough, UK).

Strains and growth conditions

Haloferax volcanii H26 has been described before (Allers et al., 2004) and was routinely grown at 45 °C in a rich medium (YPC) containing 0.5% yeast extract (Difco, Becton Dickinson, Oxford, UK), 0.1% peptone (Oxoid, Basingstoke, UK), 0.1% casamino acids (Difco), and 18% salt water (14.4% NaCl, 2.1% MgSO4·7H2O, 1.8% MgCl2·6H2O, 0.42% KCl, 0.056% CaCl2, and 12 mM Tris-HCl, pH 7.5). Solid media were prepared by the addition of 1.5% agar (Difco). If required, novobiocin was added at 0.3 μg mL−1.

Escherichia coli was routinely grown in Luria–Bertani medium (0.5% yeast extract, 1% peptone, 1% NaCl); if required, 100 μg mL−1 ampicillin was added. For the construction of plasmids, E. coli JM109 (F′traD36 proA+B+ lacIq Δ(lacZ)M15/ Δ(lac-proAB) glnV44 e14 gyrA96 recA1 relA1 endA1 thi hsdR17) was used. To prepare unmethylated DNA for efficient transformation of H. volcanii, E. coli ER2925 (New England Biolabs, Hitchin, UK) was used.

DNA techniques

Transformation of E. coli (Sambrook & Russel, 2001) and H. volcanii (Cline et al., 1989) was performed as described. General DNA techniques were performed as described (Sambrook & Russel, 2001).

AmyH was produced in H. volcanii by transforming this strain with the plasmid pSY-AmyH, which has been described before (Kwan et al., 2008). All mutations in the signal-peptide encoding region of the amyH gene were carried out using the Quickchange mutagenesis system (Stratagene, La Jolla, CA).

Amylase secretion assays

To visualize AmyH secretion on plates, 0.5% starch was added to YPC-agar. After 2 days of growth, starch-YPC plates were stained for 30 s with iodine solution (2% KI, 0.2% I2).

Western blotting

Proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted onto polyvinylidene difluoride membranes (Millipore, Watford, UK) using a semi-dry system. Amylase was visualized with specific antibodies and horseradish peroxidase anti-rabbit IgG conjugates (Promega, Southampton, UK), using the Pico West detection system (Perbio Science, Cramlington, UK).


Proteomes from E. coli K-12 MG1655, Haloarcula marismortui ATCC 43049, Natromonas pharaonis DSM2160, and Halobacterium salinarum NRC1 were obtained through the European Bioinformatics Institute (http://www.ebi.ac.uk/genomes). Proteomes were analysed firstly with tatfind 1.4 at http://signalfind.org/tatfind.html (Dilks et al., 2003). To avoid false-positives, two additional steps were adopted. Firstly, very few (if any) Tat substrates are polytopic integral membrane proteins, and proteins showing one or more additional membrane-spanning domains (using TMHMM at http://www.cbs.dtu.dk/services/TMHMM/) were therefore removed from the dataset. Secondly, proteins in the dataset were analysed for signal peptides using the Hidden Markov model of signalp 3.0 (Bendtsen et al., 2004; http://www.cbs.dtu.dk/services/SignalP/). Any proteins below the threshold score of 0.5 were also removed. For archaea, it is not clear whether the Gram-negative or the Gram-positive model is better; for this reason, both were tested and proteins scoring below the threshold in either model were removed. The final datasets contained 24 Tat substrates for E. coli, 94 for H. marismortui, 41 for H. salinarum, and 74 for N. pharaonis.

The datasets were used to generate sequence logos that show the information content of the different positions. For this the application weblogo 3 (Crooks et al., 2004; http://weblogo.threeplusone.com) was used.

Results and discussion


Sequence logos have been useful in visualizing patterns in aligned sequence motifs (Schneider & Stephens, 1990) and have indeed been used to analyse Tat motifs (see e.g. Bendtsen et al., 2005). We used this to compare the Tat motifs of haloarchaeal Tat substrates with that of the consensus E. coli motif (S/TRRxFLK). Signal peptide-containing sequences were extracted from genomes of E. coli and three fully sequenced haloarchaea: H. marismortui, N. pharaonis, and H. salinarum. The datasets obtained (see Supporting Information, Table S1) were filtered as outlined in Materials and methods to minimize the number of false-positive hits. Current information of prokaryotic signal peptides in general and the Tat system more specifically is mostly derived from bacterial systems, and as such, our searches may have been biased towards bacterial-like signal peptides. This, and our additional filtering, has most likely led to the absence of some genuine Tat signal peptides. Indeed, some proteins that are known to be Tat substrates in E. coli are missing from our dataset, including FdnH, HyaA, and HybO, all of which have been shown experimentally to be Tat substrates (Hatzixanthis et al., 2003; Berks et al., 2005). However, these three contain C-terminal transmembrane helices, which is the reason why our filtering steps rejected them. Nevertheless, only a fairly small proportion of Tat substrates have such additional membrane-spanning domains, and we think that this approach has also resulted in datasets with very few or no false-positive proteins.

The twin-arginine motifs obtained were aligned manually and used to generate sequence logos (Fig. 1). As can be observed from the top panel, our method used indeed led to a motif with the consensus SRRxFLK as observed before (Berks, 1996). The twin-arginine motifs in haloarchaea were similar, but with a number of notable differences. Firstly, the dominance of Phe in position 5 is less pronounced than in E. coli; Val is found in that position in a very similar frequency. Secondly, Leu in position 6 appears to be far more frequent in haloarchaeal Tat motifs as compared with the E. coli Tat motif. Finally, the Lys in position 7 is less common in haloarchaea as compared with E. coli. Some of these differences may be attributable to the overall differences in the amino acid composition between halophilic and nonhalophilic proteins. For instance, haloarchaea contain, on average, fewer large hydrophobic residues such as Phe, as well as a relatively low percentage of lysine residues as compared with bacteria such as E. coli or Bacillus subtilis (Bolhuis et al., 2007). In this respect, the prominence of Leu in position 6 is actually interesting as this residue is, like Phe, less frequent in haloarchaeal proteins.


Tat motifs of Escherichia coli, Haloarcula marismortui, Natromonas pharaonis, and Halobacterium salinarum were obtained as outlined in Materials and methods. The final datasets contained 24 Tat substrates for E. coli, 94 for H. marismortui, 41 for H. salinarum, and 74 for N. pharaonis. Sequence logos were generated with weblogo 3, with positively charged residues in blue, negatively charged residues in red, hydrophobic residues in green, and polar residues in black. The x-axis shows the position in the twin-arginine motif, with the two arginines as positions 2 and 3. The y-axis shows the information content expressed in bits (Crooks et al., 2004).

Replacement of the arginine residues in the Tat motif of AmyH with lysines

We and others have shown that the substitution of the two arginines in the signal peptide by two lysines (a conservative change as the positive charges are retained) leads to a block in secretion in a number of haloarchaeal Tat substrates (Rose et al., 2002; Shi et al., 2006; Gimenez et al., 2007; Kwan et al., 2008). To test whether single R to K substitutions affected translocation, we individually replaced the arginine residues at positions 14 and 15 of the AmyH signal peptide (Fig. 2a) with lysine residues. The secretion of these variants (preAmyH-KR and preAmyH-RK) was compared with wild-type AmyH (preAmyH-RR) and the earlier constructed mutant containing two lysines (preAmyH-KK). As shown in Fig. 2 with starch-plate assays and Western blotting, neither preAmyH-KR nor preAmyH-RK was secreted, indicating that both arginine residues of the Tat motif are critical to translocation. Western blotting indicated a small amount of AmyH in the supernatant fractions of the KK and KR mutants, but, as the precursor and mature forms of AmyH run very close together on SDS-PAGE, we could not determine whether those corresponded to precursor (the result of cellular lysis) or mature AmyH (the result of secretion).


Secretion of AmyH with changes in positions 14 and 15 of the AmyH signal peptide. (a) The signal peptide of AmyH, with the residues that were substituted in this study indicated in bold. The arrow indicates the position where the signal peptide is cleaved. (b) AmyH secretion assays on starch-agar plates; (c) Western blot showing AmyH in cells (C) and medium (M). Replacements in Arg14 and/or Arg15 of the signal peptide are indicated. P, precursor of AmyH; M, mature AmyH.

Importance of the other residues in the Tat motif of AmyH

To investigate the importance of the other residues in the twin-arginine motif, the residues at positions 13, 16, 17, 18, and 19 in preAmyH were all changed to alanine residues. As used in many other studies, alanine was chosen as it removes most of the side chain without affecting the backbone of the peptide chain. As shown in Fig. 3, the secretion was again tested using the starch-plate assays and Western blotting. Ser13, Thr16, and Lys19 were not critical, as Ala residues on those positions did not affect translocation. This was not entirely surprising because residues in the same positions in the E. coli Tat substrate SufI also had no significant effect on translocation (Stanley et al., 2000).


Secretion of AmyH with replacements in the signal peptide of residues surrounding the two arginines. (a) AmyH secretion assays on starch-agar plates; (b) Western blot showing AmyH in cells (C); and medium (M). Replacements in the signal peptide are indicated. P, precursor of AmyH; M, mature AmyH.

Two residues that were shown to be important were Val17 and Leu18. When Val17 was substituted by Ala, no amylase activity was detected in the supernatant (Fig. 3a). This was confirmed by Western blotting (Fig. 3b), which showed a complete absence of AmyH in the medium fraction of the V17A substitution. Our finding that this residue is critical to translocation is similar to what was found for E. coli SufI (which has a Phe in this position; Stanley et al., 2000). At this position, a strongly hydrophobic residue is important and the most common residues found here are Phe, Val, and Leu. It is interesting to note that a number of haloarchaeal Tat substrates, nine out of a total of 209 proteins in our datasets (see Table S1), do contain an Ala in that position. None of these nine proteins have been characterized, but homology searches indicate that at least some of them appear to be genuinely extracytoplasmic proteins (data not shown). This suggests that signal peptides with an Ala in the position equivalent to Val17 in AmyH can still be secreted, possibly through compensation by other features in their signal peptides. An alternative explanation is that these proteins are not Tat substrates, but are translocated through another route, such as for example the Sec pathway.

The next residue (Leu18 in AmyH) is also commonly a strongly hydrophobic residue, usually Leu, Ile, or Val, but changing this residue to Ala in SufI does not lead to a block in its translocation (Stanley et al., 2000). In contrast, it is critical in AmyH, as the L18A mutant is not translocated at all, shown both by the starch-plate assays and Western blotting (Fig. 3). This finding is corroborated by the observation that none of the haloarchaeal proteins in our datasets contained an Ala in that position.


As outlined in the introduction, the haloarchaeal Tat system differs on several aspects from those of nonhalophilic Tat systems. Therefore, we could not exclude the possibility that, for instance, proteins with RK or KR motifs would also be Tat-dependent substrates. However, we found that residues that are critical to the translocation of an E. coli Tat substrate are also critical to the export of AmyH, including both arginine residues and the first of the pair of hydrophobic residues that follow the arginines. In addition, the second hydrophobic residue in the Tat motif is also essential for AmyH secretion, while this residue seems to be of less importance in the E. coli Tat substrate SufI. The sequence logos indicate that this residue can also be another strongly hydrophobic amino acid such as Val or Ile, but further mutational analysis has to be performed to confirm this. It is interesting to note that the importance of this residue was already indicated by our bioinformatics analysis. The consensus motif for haloarchaeal Tat substrates can be denoted as (S/T)RRx(F/L)L, even though the first residue (Ser or Thr) does not appear to be essential for translocation. This information is useful in the prediction of Tat substrates encoded by genes found in haloarchaeal genomes. We do need to note, though, that our conclusions are based on the analysis of only one haloarchaeal Tat substrate, and it is clear that the characterization of other signal peptides is needed to understand the requirements for Tat-dependent export fully.

Supporting Information

Table S1. Uniprot accession numbers and their Tat motifs.


D.K. was sponsored by a studentship from the Biotechnology and Biological Sciences Research Council, and A.B. was supported by a University Research Fellowship from the Royal Society.


  • Editor: Marco Moracci


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