R E S E A R C H L E T T E R

Analysis ofthe twin-argininemotifofa haloarchaeal Tat substrateDaniel Kwan & Albert Bolhuis

Department of Pharmacy and Pharmacology, University of Bath, Bath, UK

Correspondence: Albert Bolhuis,

Department of Pharmacy and Pharmacology,

University of Bath, Bath BA2 7AY, UK. Tel.:

144 1225 383 813; fax: 144 1125 386 114;

e-mail: a.bolhuis@bath.ac.uk

Present address: Syntaxin Ltd, Abingdon

OX14 3YS, UK.

Received 1 March 2010; revised 20 April 2010;

accepted 22 April 2010.

Final version published online 17 May 2010.

DOI:10.1111/j.1574-6968.2010.02001.x

Editor: Marco Moracci

Keywords

twin-arginine translocation; protein secretion;

halophilic archaea; Tat motif; signal peptide.

Abstract

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. There-

fore, it was important to test which features in haloarchaeal Tat substrates were

important for transport, as these might also be different from those of nonhalo-

philic 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.

Introduction

The twin-arginine translocase (Tat) is a protein transloca-

tion 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 mechanisti-

cally completely different (reviewed in Robinson & Bolhuis,

2004; Pohlschroder et al., 2005; Natale et al., 2008). Usually,

two or three components with distinct functions are in-

volved 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 mem-

brane-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 hydro-

phobicity 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,

FEMS Microbiol Lett 308 (2010) 138–143c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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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 intra-

cellularly, 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 ha-

loarchaeon 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 haloarch-

aeal 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

Chemicals

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 1C in a rich

medium (YPC) containing 0.5% yeast extract (Difco, Bec-

ton Dickinson, Oxford, UK), 0.1% peptone (Oxoid, Basing-

stoke, 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 mg mL�1.

Escherichia coli was routinely grown in Luria–Bertani

medium (0.5% yeast extract, 1% peptone, 1% NaCl); if

required, 100 mg mL�1 ampicillin was added. For the con-

struction of plasmids, E. coli JM109 (F0 traD36 proA1B1

lacIqD(lacZ)M15/D(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 de-

scribed 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 im-

munoblotted onto polyvinylidene difluoride mem-

branes (Millipore, Watford, UK) using a semi-dry system.

Amylase was visualized with specific antibodies and horse-

radish peroxidase anti-rabbit IgG conjugates (Promega,

FEMS Microbiol Lett 308 (2010) 138–143 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

139Twin-arginine motif of haloarchaea

Southampton, UK), using the Pico West detection system

(Perbio Science, Cramlington, UK).

Bioinformatics

Proteomes from E. coli K-12 MG1655, Haloarcula maris-

mortui 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 there-

fore 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

Bioinformatics

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-con-

taining 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 transmem-

brane 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

Fig. 1. Tat motifs of Escherichia coli, Haloarcula marismortui, Natromo-

nas 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).

FEMS Microbiol Lett 308 (2010) 138–143c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

140 D. Kwan & A. Bolhuis

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 differ-

ences 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.

Replacement of the arginine residues in the Tatmotif 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 (pre-

AmyH-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, in-

dicating 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).

Importance of the other residues in the Tatmotif 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).

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

(a)

(b)

(c)

Fig. 2. 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.

(a)

(b)

Fig. 3. 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.

FEMS Microbiol Lett 308 (2010) 138–143 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

141Twin-arginine motif of haloarchaea

(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 obser-

vation that none of the haloarchaeal proteins in our datasets

contained an Ala in that position.

Conclusion

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 con-

sensus 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.

Acknowledgements

D.K. was sponsored by a studentship from the Biotechnol-

ogy and Biological Sciences Research Council, and A.B. was

supported by a University Research Fellowship from the

Royal Society.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Table S1. Uniprot accession numbers and their Tat motifs.

Please note: Wiley-Blackwell is not responsible for the

content or functionality of any supporting materials sup-

plied by the authors. Any queries (other than missing

material) should be directed to the corresponding author

for the article.

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143Twin-arginine motif of haloarchaea