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Originally published In Press as doi:10.1074/jbc.M306516200 on July 16, 2003

J. Biol. Chem., Vol. 278, Issue 40, 38428-38436, October 3, 2003
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Sequence-specific Binding of prePhoD to Soluble TatAd Indicates Protein-mediated Targeting of the Tat Export in Bacillus subtilis*,

Ovidiu I. Pop {ddagger}, Martin Westermann §, Rudolf Volkmer-Engert ¶, Daniela Schulz ¶, Cornelius Lemke ||, Sandra Schreiber {ddagger}, Roman Gerlach {ddagger} **, Reinhard Wetzker {ddagger}{ddagger} and Jörg P. Müller {ddagger} §§

From the {ddagger}Institut für Molekularbiologie, Friedrich-Schiller-Universität Jena, Hans-Knöll-Strasse 2, Jena D-07745, Germany, §Institut für Ultrastrukturforschung, Klinikum der Friedrich-Schiller-Universität Jena, Ziegelmühlenweg 1, Jena D-07743, Germany, Abt. f. Molekulare Bibliotheken, Charite, Ziegelstr. 5–9, Berlin 10117, Germany, ||Institut für Anatomie 1, Friedrich-Schiller-Universität Jena, Teichgraben 7, Jena 07743, Germany, and {ddagger}{ddagger}Arbeitsgruppe Molekulare Zellbiologie, Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Strasse 1, Jena D-07747, Germany

Received for publication, June 19, 2003 , and in revised form, July 15, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Tat (twin-arginine protein translocation) system initially discovered in the thylakoid membrane of chloroplasts has been described recently for a variety of eubacterial organisms. Although in Escherichia coli four Tat proteins with calculated membrane spanning domains have been demonstrated to mediate Tat-dependent transport, a specific transport system for twin-arginine signal peptide containing phosphodiesterase PhoD of Bacillus subtilis consists of one TatA/TatC (TatAd/TatCd) pair of proteins. Here, we show that TatAd was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatAd. Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatAd demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatAd was interfered by peptides containing the twin-arginine motif but remained active when the arginine residues were substituted. The selective binding of TatAd to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatAd interaction. Substitution of the binding motif demonstrated the pivotal role of basic amino acid residues for TatA binding. These features suggest that TatA interacts prior to membrane integration with its pre-protein substrate and could therefore assist targeting of twin-arginine pre-proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria have two distinct pathways for the export of proteins across the cytoplasmic membrane. The majority of the periplasmic proteins are exported in an unfolded conformation via the Sec pathway, which is promoted by ATP hydrolysis (1–4). Proteins translocated by this pathway are targeted to the membrane-embedded proteinaceous Sec pore by soluble targeting factors (1). The transport is mediated by N-terminal signal peptides that are similar structurally but do not show sequence conservation (5).

Several proteins use an alternate translocation path. Because of its highly conserved twin-arginine sequence motif present in the signal peptides of proteins using this way, it is called Tat1 (twin-arginine translocation) pathway (6–8). It was originally identified in chloroplasts (9, 10) and has been described recently (9, 11) for Escherichia coli. The currently only known driving force of the translocation is the pH gradient at the membrane (9, 12). Because most of the Tat substrates require the incorporation of cofactors or subunit association (13–15), the Tat pathway appears to be responsible for proteins incompatible with the Sec pathway (16). By a not yet identified control mechanism the Tat system exports only Tat substrates that attained a native conformation (15–17).

The currently best characterized Tat system of E. coli consists of four proteins with calculated membrane spanning domains (10, 18, 19). Sequence analysis predicts that TatA, TatB, and TatE are proteins that comprise a transmembrane N-terminal {alpha}-helix followed by an amphiphatic {alpha}-helix at the cytoplasmic side of the membrane (20, 21). TatC, the fourth protein known to be involved in the Tat system of E. coli, has six predicted transmembrane helices (20). Topology determination revealed that TatC contains four transmembrane helices (18). Although tatB and tatC appear to play a pivotal role in the Tat-dependent protein translocation in E. coli (11, 19, 20), TatA and TatE can, at least partially, functionally substitute each other (20). By analyzing the presence of Tat substrates and components of the Tat machinery surveys of prokaryotic genomes indicate that the Tat pathway is wide spread among bacteria and archaea (8, 22). The standard Tat systems (such as in E. coli) consist of one TatC, at least one copy of TatA/E, and TatB (23). In certain prokaryotes a TatB homolog appears to be absent.

Currently there exist only weak ideas about the targeting of the twin-arginine precursors to the translocase unit and the structure of the Tat transport system. In vitro work with the plant thylakoid Tat system demonstrated that no soluble factors are required of Tat-dependent export (24) and that TatA is required for the transport steps following precursor recognition (25, 26). The TatA/B/E proteins have been predicted to act as membrane receptors for Tat substrates (22) or to form the export channel itself (27–31). The targeting of Tat-dependent iron-sulfur protein HiPIP to the membrane appears to be independent of the Tat components (15). The use of the Tat system by redox proteins in many bacteria or preferably non-redox proteins in other species, as well as the different composition of the Tat proteins, indicates that the Tat system is of heterogeneous nature (8, 22, 23).

Analysis of Tat-like proteins in Bacillus subtilis revealed that the genome encodes three TatA and two TatC-like proteins (31). TatB-like proteins appear to be absent of B. subtilis and of other sequenced bacilli (22, 32). Translocation of the B. subtilis protein PhoD containing a twin-arginine signal peptide was shown to be dependent on the expression of a tatA tatC pair (33). These genes (designated tatAd and tatCd) are co-localized with phoD in one operon resulting in co-regulated expression of phoD and tatAd/Cd. A second copy of tatC (tatCy) was not required for PhoD export (31). The PhoD-specific transport system was functionally active in E. coli. Although PhoD or a fusion consisting of the signal peptide of PhoD and LacZ was not recognized by the E. coli Tat components, co-expression of B. subtilis tatAd/tatCd resulted in translocation of SPPhoD-LacZ. This transport was shown to be {Delta} pH-dependent. These studies revealed that the minimal requirement of a Tat transport system consists of a TatA/TatC pair, a twin-arginine signal peptide, and the pH gradient at the bacterial cytoplasmic membrane (33). PhoD is the only known substrate of the TatAd/TatCd system (31, 34).

The B. subtilis phoD gene encodes an secretory enzyme with alkaline phosphatase and phosphodiesterase activities (36). Slow processing maintains the protein in a cell wall-associated localization before release (37). It has been demonstrated that PhoD is a member of the so-called Pho regulon of B. subtilis (38). The Pho regulon comprises a group of genes that are induced in response to the depletion of inorganic phosphate in the growth medium and is regulated by the two component signal transduction system PhoR/PhoP (39). A phoR12 mutation in B. subtilis strain GCH871 was shown to be functionally active under phosphate replete conditions resulting in the induction of Pho regulon genes (40).

To investigate the selectivity and specificity of the PhoD transport system further, we analyzed the localization and affinity of TatAd by combining genetic and in vitro approaches in B. subtilis and E. coli. Unexpectedly, we found TatAd in the cytoplasmic membrane, as well as in the cytosol. By using purified TatAd and prePhoD we demonstrated the interaction of both peptides. Inefficient binding of TatAd to mature PhoD or Sec-dependent or cytosolic proteins demonstrated the particular role of the twin-arginine-containing signal peptide of PhoD in the recognition process. The selective affinity of TatAd to peptides derived from the signal peptide of PhoD showed that the twin-arginine motif acts as a binding site of TatAd. Substitution of amino acid residues of the binding motif elucidated the role of particular amino acids of this motif for TatAd recognition.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids, Bacterial Strains, and Media—Table I lists the plasmids and bacterial strains used. TY medium (tryptone/yeast extract) contained Bacto tryptone (1%), Bacto yeast extract (0.5%), and NaCl (1%). For pulse labeling of proteins M9 minimal medium was used (41). To induce or repress the phoD operon in B. subtilis 168 cultures were grown in low or high phosphate defined medium, as described (40). B. subtilis phoR12 was cultivated in absence of isopropyl-{beta}-D-thiogalactopyranoside (IPTG). Induction of phosphate starvation response was monitored by determining alkaline phosphatase activity as described previously (39). When required, media were supplemented with ampicillin (80 µg/ml), kanamycin (20 µg/ml), chloramphenicol (20 µg/ml or 5 µg/ml), tetracycline (12.5 µg/ml), erythromycin (5 µg/ml), arabinose (0.2%), or IPTG (1 mM).


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  		TABLE I
Plasmids and strains

 

DNA Techniques—Procedures for DNA purification, restriction, ligation, agarose gel electrophoresis, and transformation of E. coli were carried out as described by Sambrook et al. (42). Restriction enzymes were from MBI Fermentas. PCR was carried out with the VENT DNA polymerase (New England Biolabs) using chromosomal DNA of B. subtilis 168 as template.

To construct pQE9phoDm, the phoD gene lagging the 5'-terminal region encoding the signal sequence was amplified from the chromosome of B. subtilis strain 168 by PCR using the primers P1 (5'-GTA GGA TCC GCG CCT AAC TTC TCA AGC-3') containing a BamHI site and P2 (5'-CGA TCC TGC AGG ACC TCA TCG GAT TGC-3') containing a PstI site. The amplified fragment was cleaved with BamHI and PstI and cloned in the corresponding sites of pQE9. The resulting plasmid pQE9phoD allowed the IPTG-inducible synthesis of N-terminally His6-tagged mature PhoD in E. coli.

To overexpress tatAd and tatCd genes in E. coli, genes were amplified using primers Ad1 (5'-GTG GGA TCC ATG TTT TCA AAC ATT GG-3') containing a BamHI site and primer Ad2 (5'-CCT CTG CAG CAT TCA GCC CGC G-3') containing a PstI site and Cd1 (5'-TTT CCA TGG ATA AAA AAG AAA CCC-3') containing a NcoI restriction site and Cd2 (5'-GCG GGA TCC GGC CGC CGC TGT TTC TTC-3') containing a BamHI restriction site, respectively. The tatAd fragment was cleaved with BamHI and PstI and cloned in the corresponding sites of pQE9. The resulting plasmid pQE9tatAd allowed the IPTG-inducible synthesis of His6-TatAd. The tatCd fragment was cleaved with NcoI and BamHI and subsequently cloned into pQE60, cleaved with NcoI and BglII. The resulting plasmid pQE60tatCd allowed the synthesis of TatC-His6.

To amplify the DNA region encoding preYvaY primer Y1 (5'-ATG GAT CCA TGA AAA GTA AAT TAC TTA GGC T-3'), incorporating a BamHI restriction site, and primer Y2 (5'-TAA AAG CTT ATT GAT GAA TCA ATT TT-3'), incorporating a HindIII restriction site, were used. For amplification of the DNA fragment encoding the mature part of YvaY primer Y3 (5'-CAG GAT CCA AAG AAA ACC ATA CAT TT-3'), incorporating a BamHI restriction site, and primer Y2 were used. The PCR fragments were digested with BamHI and HindIII and inserted 3'-terminal of the His coding region of pQE9 digested with the same enzymes. The resulting plasmids pQE9yvaYp and pQE9yvaYm were transformed into E. coli TG1(pREP4).

To synthesize epitope-tagged TatAd the DNA region encoding phoD and tatAd was amplified from the chromosome of B. subtilis 168 by using primer P1 (33) and primer T1 (5'-CG GAA TTC CAT AAT TTC CAC TCC TTA ATT CGT GA-3') containing an EcoRI site. A HindIII-EcoRI DNA fragment encoding the 359 3'-terminal amino acids of phoD and tatAd was fused to the DNA region encoding the TAP (calmodulin-binding-protein and protein A) epitopes (43) resulting in an in-frame fusion of tatAd and TAP. The 'phoD-tatAd-TAP DNA fragment was placed on the B. subtilis nonreplicative vector pORI22 (44) and was transformed into the B. subtilis 168. Campbell-like integration into the chromosome resulted in B. subtilis strain 168::pORI22-tatAd-TAP (Fig. 1). As a control a DNA fragment encoding phoD and the start codon of tatAd was amplified using primer P1 and A2 (5'-CG GAA TTC GCC CGC GTT TTT GTC CTG CTT TAC CGC-3') containing an EcoRI site and fused to the TAP region resulting in the fusion of the start codon of tatAd and TAP. After subcloning of the DNA fragment into pORI22 and transformation into B. subtilis, strain 168::pORI22-tatAd'-TAP was obtained (Fig. 1). Appropriate mode of integration of pORI22-derived plasmids into the chromosome of B. subtilis 168 was validated by PCR amplification of junction fragments. For inducible expression of tatAd/tatCd genes in B. subtilis a 1046-bp BamHI-PstI DNA fragment from pQE9tatAd/Cd (33) was inserted into pREP9 (45) resulting in pREP9tatAd/Cd.



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  		FIG. 1.
Construction of mutant strains of B. subtilis tatAd-TAP. Schematic presentation of the phoD operon of B. subtilis 168 (A), 168::pORI22-tatAd-TAP (B), and 168::pORI22-tatAd'-TAP (C). By a Campbell-type integration of the pORI22-derivative plasmids were integrated into the chromosome of B. subtilis 168. B. subtilis 168::pORI22-tatAd-TAP or 168::pORI22-tatAd'-TAP allowed the phosphate starvation-inducible synthesis of fusion proteins consisting of TatAd and TAP epitopes or the ATG start codon of TatAd and TAP, respectively. The tatCd gene in the latter strains was placed under the control of the in B. subtilis constitutive repC promoter.

 

Freeze-fracture Electron Microscopy—Cells were concentrated by centrifugation, washed two times with growth medium, and resuspended in 10% (v/v) of the initial volume of growth medium containing 15% (w/v) glycerol. Aliquots were enclosed between two 0.1-mm copper profiles as used for the sandwich double-replica technique. The sandwiches were rapidly frozen by plunging them into liquid propane, cooled by liquid nitrogen. Freeze-fracturing was performed in a BAF400T (BAL-TEC, Liechtenstein) freeze-fracture unit at –150 °C using a double-replica stage. The fractured samples were shadowed without etching with 2.0–2.5 nm platinum/carbon at an angle of 35°. The evaporation of platinum/carbon with electron guns was controlled by a thin-layer quartz crystal monitor.

Freeze-fracture Labeling—For freeze-fracture immunogold labeling and subsequent electron microscopy the freeze-fracture replica were transferred to a digesting solution (2.5% SDS in 10 mM Tris buffer, pH 8.3, and 30 mM sucrose) and incubated overnight (46). The replica were washed four times in PBS buffer and treated with PBS + 1% bovine serum albumin for 30 min. Next they were placed in PBS containing bovine serum albumin (0.5%) and monospecific antibodies against TatAd, SecY, or DnaK (dilution 1:20) for 1 h. Subsequently the replica were washed four times with PBS and placed on a 1:50 diluted solution of the second gold-conjugated antibody (goat anti-rabbit IgG with 10 nm of gold; British Biocell International, Cardiff, UK) in PBS containing 0.5% bovine serum albumin for 1 h. After immunogold labeling, the replica were immediately rinsed several times in PBS, fixed with 0.5% glutaraldehyde in PBS for 10 min at room temperature, washed four times in distilled water, and finally picked onto Formvar-coated grids for viewing in an EM 902 electron microscope (Zeiss, Oberkochen, Germany). Freeze-fracture micrographs were mounted with direction of shadowing from bottom to top at a magnification as indicated in the figures.

Post-embedding Labeling of B. subtilis—Phosphate-starved B. subtilis cells were embedded in Lowicryl K4M as described (37). Ultrathin sections of Lowicryl-embedded cells were mounted on nickel grids with carbon-coated Formvar films and subsequently labeled with specific rabbit antibodies and goat anti-rabbit IgG conjugated to 10 nm of gold (British Biocell International, Cardiff, UK) as a secondary antibody (37). Control experiments were performed by staining sections under similar conditions by omitting the specific antibodies.

Cell Fractionation of B. subtilis and E. coli—B. subtilis cells were harvested and resuspended in PBS buffer (140 mM NaCl, 2.7 mM KCl, 1.3 mM KH2PO4, 10 mM Na2HPO4, pH 7.3) containing 5 mM phenylmethylsulfonyl fluoride. Cell suspension was passed three times through a French press at 16,000 lb/in2. Unbroken cells were removed by centrifugation at 10,000 x g for 10 min. To obtain membrane-free cytosolic protein cell lysate was centrifuged at 150,000 x g for 2 h at 4 °C, and pellet was used as membrane fraction. Cytosolic and membrane fractions of E. coli were obtained from spheroblasted cells. Spheroblasts were lysed in 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2 and subsequently centrifuged at 100,000 x g for 1 h. Pellets contained membrane fraction and supernatant cytosolic protein.

SDS-PAGE and Western Blot Analysis—SDS-PAGE were prepared as described (47). After separation by SDS-PAGE, proteins were transferred to a nitrocellulose membrane (Schleicher & Schüll) (48). Proteins were visualized using monospecific antibodies against PhoD (37), TatAd, or TatCd and alkaline phosphatase-conjugated goat anti-rabbit antibodies (Sigma) according to the manufacturer's instructions.

Purification of His-tagged Proteins—His-tagged proteins were prepared from IPTG-induced E. coli TG1(pREP4, pQE9tatAd), TG1(pREP4, pQE60tatCd), TG1(pREP4, pQE9phoDp), TG1(pREP4, pQE9phoDm), TG1(pREP4, pQEC), TG1(pREP4, pQE9yvaYp), and TG1(pREP4, pQE9yvaYm) cultures as abundant proteins and purified by nickel-nitrilotriacetic acid affinity chromatography. The purification of TatCd, CopR, PhoD, and YvaY proteins were carried out under denaturating conditions. Soluble His6-TatAd was purified under native conditions with sodium phosphate buffer under standard conditions (Qiagen).

Preparation and Purification of Antibodies against TatAd—Purified His6-TatAd or TatCd-His6 emulsified in MPL + TDM + CWS adjuvant (catalog number M6661; Sigma) at 100 µg/ml were used to immunize New Zealand White rabbits (Charles River, Hilden, Germany). Specific antibodies were affinity-purified from the sera by adsorption to and elution (with 0.1 M glycine at pH 2.5) from nitrocellulose (37). As proved by Western blotting, monospecific antibodies specifically cross-reacted with TatAd or TatCd.

In Vivo Labeling of His-tagged Proteins—[35S]-Labeled His6-tagged proteins were obtained by pulse labeling of E. coli cultures. Strains were grown in M9 minimal medium, expression of genes of interest was induced for 15 min, and cultures were labeled in 50 µCi of [35S]methionine for 5 min. Subsequent purification using nickel-nitrilotriacetic acid affinity chromatography was carried out essentially as described above.

Co-immunopurification—Purification of TAP proteins was carried out following standard procedures (43). Cytosolic fractions of B. subtilis cell lysates were prepared in purification buffer IPP150 (43) as described above.

Co-immunoprecipitation—Binding of His6-tagged [35S]-labeled proteins to unlabeled TatAd was measured as follows: [35S]-labeled proteins were incubated with 10-M excess of His6-TatAd (total 0.4 µg) at room temperature in 50 µl of PBS buffer supplemented with 0.5% n-octyl-{beta}-D-glucothiopyranoside if indicated. For competition experiments synthetic peptides QNNTFDRRKFIQGAGKIAG or QNNTFDAAAFIQGAGKIAG (0.4 M) were added to the reaction mixture. After 60 min, 50 µl of PBS buffer containing 1 µl of monospecific antibodies against the unlabeled protein, pre-complexed with 10 µl of Dynabeads (Dynal, Oslo, Norway), were added, and the mixture was further incubated for 60 min while shaking. Subsequently, the protein A Dynabeads were washed five times with 500 µl of PBS buffer. [35S]-Labeled proteins bound to protein A beads were counted in scintillation liquid. Immunoprecipitation experiments were carried out at least three times.

Synthesis and Screening of the Cellulose-bound Peptide Arrays— Peptide arrays were prepared by automated spot synthesis using the AMS SPOT-robot (Abimed, Langenfeld, Germany) (49, 50). Before screening, the membranes were washed in methanol for 10 min, three times in TBS buffer (50 mM Tris, 137 mM NaCl, 27 mM KCl, pH 8.0), and subsequently incubated in blocking buffer (10% GENOSYS SU-07–250; 5% sucrose, TBST buffer (TBS with 0.05% Tween)) for 3 h. After washing with TBST buffer peptide arrays were incubated with [35S]-labeled His6-TatAd (50 ng/ml; 10,000 cpm/ml) in blocking buffer for 16 h at room temperature with gentle shaking. Unbound protein was washed out with TBST buffer. Amount of retained [35S]-labeled protein was quantified using phosphorimage analysis. Relative amounts of radioactivity were estimated by using a phosphorimager (Fuji) and associated image analytical software PC-BAS.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Immunogold Labeling of TatAd—The E. coli Tat system has the ability to transport folded proteins and enzyme complexes across the cytoplasmic membrane (22, 27). To transport these folded peptides would result in a translocation pore with a minimum diameter of 5 nm (51). We have shown that the TatAd/Cd transport system of B. subtilis is able to transport PhoD with a molecular mass of 62.7 kDa, as well as a hybrid protein consisting of the signal peptide of PhoD and LacZ, resulting in a molecular mass of 120 kDa (31, 33), resulting in a necessarily similar sized translocation pore. Because freeze-fracture cytochemistry is known to be a powerful technique to study macromolecular architecture of biomembranes (46, 52) we used this method to investigate the cytoplasmic membrane of B. subtilis and E. coli containing TatAd/Cd proteins that could form detectable structures. Immunogold labeling of TatAd with monospecific antibodies was carried out to localize TatAd and to identify visible ultrastructures and TatAd-containing translocation particles. Immunogold labeling of TatAd in freeze-fractured membranes of B. subtilis 168 cells grown under phosphate starvation revealed an even distribution of TatAd in the cell envelope. Interesting, most immunogold labels appeared to be clustered both at the protoplasmic (PF) and the exoplasmic side (EF) of the cytoplasmic membrane (Fig. 2, A and B). Absence of immunogold labeling of freeze fractures of B. subtilis 168 wild type cells grown under phosphate replete conditions demonstrated specificity of labeling (Supplemental Fig. 1C). Because no labeling of gold-conjugated anti rabbit antibody could be observed in absence of primary antibodies, unspecific binding of the secondary antibody could be excluded (data not shown). To rule out that the monospecific TatAd antibodies cross-reacted with another phosphate starvation-induced cytosolic protein, we compared the immunodetection of soluble B. subtilis proteins between cell extracts obtained from cells grown under phosphate replete and phosphate depleted conditions. No other proteins, except for TatAd, could be detected in the Western blot (data not shown). Unexpectedly, a substantial amount of gold particles was localized at the cytosol indicating that B. subtilis TatAd is not an exclusively membrane integrated protein (Fig. 2A). Artificial induction of the phosphate starvation response by using B. subtilis 168 phoR12 resulted in similar distribution of TatAd except for a higher expression level of TatAd (Supplemental Fig. 1, A, B, and D). Again clusters of TatAd-labeled protein could be observed but were not linked to vesicle-like structures in the membrane (Supplemental Fig. 1A). To validate immunogold labeling for protein localization, freeze-fractured cells were immunogold-labeled with antibodies against the chaperone DnaK and integral membrane protein SecY, a part of the Sec-translocase unit (3). As expected, the chaperone DnaK could be detected in the cytosol only (Supplemental Fig. 2B), and immunogold labeling of SecY indicated that SecY was predominantly localized in the cytoplasmic membrane (Supplemental Fig. 2A).



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  		FIG. 2.
Localization of TatAd in B. subtilis. Cells of B. subtilis 168 was grown in low phosphate defined medium to phosphate starvation, freeze-fractured, and subsequently labeled with TatAd-specific antibodies and 10 nm of gold-conjugated secondary antibody. Electron micrographs demonstrate the protoplasmic face (PF; A) and the fracture through the cytosol (Cy; A) and the exoplasmic face (EF; B) of the cytoplasmic membrane. Scale bar represents 0.2 µm.

 

Localization of TatAd was further elucidated by immunogold labeling of ultrathin sections of B. subtilis 168 cells grown under phosphate starvation. Again beside the expected membrane associated localization of the gold particles, about 50% could be detected in the cytosol of the cell (Fig. 3). As a control DnaK and SecY protein were immunogold-labeled in B. subtilis 168. Although DnaK was found in the cytosol, SecY was membrane-associated (Supplemental Fig. 3).



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  		FIG. 3.
Localization of TatAd in B. subtilis 168. Ultrathin sections of phosphate-starved cells were incubated with TatAd-specific anti-serum and subsequently incubated with anti-rabbit IgG-gold. Scale bar represents 0.2 µm.

 

To elucidate whether the localization of TatAd was depending on the presence of prePhoD, we analyzed its localization in a B. subtilis phoD strain. Strain MH5444 deleted for phoD was transformed with plasmid pREP9tatAd/Cd allowing the IPTG-inducible synthesis of the TatAd/TatCd proteins. MH5444-(pREP9tatAd/Cd) was grown to phosphate starvation, expression of Tat proteins was induced, and localization of TatAd was detected using immunogold labeling of freeze-fracture cells essentially as described above. No TatAd could be detected in the cytosol of the cell (data not shown).

Because the PhoD-specific TatAd/TatCd translocation system has been demonstrated to be functionally active in E. coli (33), we analyzed the localization of TatAd in E. coli. Cells of E. coli TG1(pREP4, pQE9tatAd/Cd)-expressing B. subtilis TatAd/TatCd proteins were analyzed by freeze-fracture technique with subsequent immunogold labeling of TatAd. Like in B. subtilis, immunologically detected TatAd was localized at the cytoplasmic membrane, as well as in the cytosol (Supplemental Fig. 4).

TatAd Is Localized in the Cytoplasmic Membrane and the Cytosol—The above results indicated that TatAd is not exclusively localized in the membrane. Immunological detection of TatAd in cytosolic cell fractions of B. subtilis strains 168, GCH871, and TG1(pREP4, pQE9tatAd/Cd) confirmed that a substantial amount of the protein was localized in the cytosol (Fig. 4). Absence of membrane proteins in the cytosolic fraction was monitored by detecting TatCd. No TatCd could be detected in the cytosolic fractions either of B. subtilis strains or of E. coli TG1(pREP4, pQE9tatAd/Cd) (Fig. 4).



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  		FIG. 4.
Subcellular localization of TatAd and TatCd in B. subtilis and E. coli. The presence of TatAd in cytoplasmic and membrane fractions of B. subtilis (lanes 1–3) and E. coli strains (lanes 4 and 5) was detected by using Western blotting with monospecific antibodies against TatAd and TatCd. B. subtilis strain 168 was grown in low phosphate defined medium to phosphate starvation (lane 2); B. subtilis strains 168 (lane 1) and GCH871 (lane 3) were grown in high phosphate defined medium, E. coli strains TG1(pREP4, pQE9tatAd/Cd) (lane 4) and TG1(pREP4) (lane 5) were grown in TY medium to exponential growth, and expression of tat genes was induced for 1 h with IPTG (1 mM). Bands representing TatAd and TatCd are indicated.

 

A kinetic study revealed that cytosolic localization of TatAd in phosphate-starved B. subtilis 168 was variable. At the onset of phosphate starvation substantial amounts of TatAd was detected in the cytosol. Upon ongoing starvation cytosolic TatAd decreased (data not shown). The fraction of TatAd that was co-purified with membranes, resisted carbonate extraction, indicating that this protein is membrane-integrated (data not shown).

Co-purification of TatAd with prePhoD—In vivo synthesized TAP epitope-tagged proteins have been successfully used to demonstrate the interaction between proteins in yeast, plants, and mammalian cells (53–55). To elucidate the affinity of TatAd for its substrate prePhoD, TAP-tagged TatAd was synthesized in B. subtilis. Strains 168::pORI22-tatAd-TAP and 168::pORI22-tatAd'-TAP were grown to phosphate starvation, and TAP peptides were subsequently purified from the cytosolic fraction using IgG-Sepharose beads (43). Both TatAd'-TAP and TatAd-TAP peptides could be detected in the cytosolic fraction. Strain 168::pORI22-tatAd'-TAP mediating the synthesis of the N-terminal methionine of TatAd with the TAP epitope (TatAd'-TAP) showed inducible synthesis of a protein with the molecular weight of the TAP epitope (Fig. 5, lane 2). Strain 168::pORI22-tatAd-TAP produced a protein according to the molecular weight of TatAd-TAP. TatAd-TAP was detected by protein A (Fig. 5, lane 1), as well as TatAd antibodies (data not shown). IgG-purified TAP peptides were assayed for presence of PhoD. Although prePhoD was co-purified with TatAd-TAP, no PhoD could be co-purified with TatAd'-TAP (Fig. 5). This result demonstrated the in situ interaction of TatAd and prePhoD in the cytosol of the cell.



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  		FIG. 5.
Co-purification of TatAd and prePhoD. B. subtilis strains 168::pORI22-tatAd-TAP (lane 1) and 168::pORI22-tatAd'-TAP (lane 2) were grown to phosphate starvation for 2 h. Cells were lysed, and TAP-containing peptides were purified from the cytosolic fraction by using IgG beads. Purified TAP proteins were separated via SDS-PAGE and subsequently detected by using Western blotting with monospecific antibodies against protein A and PhoD. Bands representing TatAd-TAP, TatAd'-TAP (TAP), IgG, and prePhoD are indicated.

 

TatAd Has Affinity for prePhoD—After demonstrating interaction of TatAd with prePhoD in the cytosol, co-immunoprecipitation experiments were carried out to further investigate specificity of this interaction in vitro. Complex formation of purified [35S]-labeled His6-prePhoD (Table II) with purified His6-TatAd was assessed by immunoprecipitation with TatAd-specific antibodies. Taking into account that soluble TatAd forms high molecular weight homomultimers,2 10-M excess of TatAd was used to co-immunoprecipitate peptides. His6-prePhoD could be co-immunoprecipitated with His6-TatAd, whereas only low levels of His6-prePhoD were immunoprecipitated when either the TatAd or the TatAd antibodies were omitted from the mixture (Table II).


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  		TABLE II
Co-immunoprecipitation of TatAd with purified [35S]-His6 proteins

Purified [35S]-labeled proteins were immunoprecipitated with TatA-specific antibodies pre-complexed with protein A Dynabeads. In parallel, TatAd or TatAd antibodies were omitted from the reaction as indicated. Immunoprecipitates were collected, and the amount of bound [35S]-labeled proteins was quantified by counting in scintillation liquid. Amount of bound prePhoD was quantified by counting in scintillation liquid. Bound prePhoD is indicated in percent of input. Data were obtained from a representative experiment.

 

To analyze the specificity of TatAd we investigated affinity to radiolabeled mature PhoD, to the precursor and mature Sec-dependent B. subtilis protein YvaY (56), as well as CopR, a cytosolic protein involved in replication control of plasmids (57). His6-tagged proteins were purified as abundant proteins (Supplemental Fig. 5). As shown in Table II only 25% of mature His6-PhoD could be co-immunoprecipitated by His6-TatAd compared with 58% of His6-prePhoD (Table II). Because the amount of bound mature [35S]-His6-PhoD was hardly higher when antibodies or TatAd were omitted, only weak interaction can be concluded. Amounts of co-immunoprecipitated [35S]-labeled His6-preYvaY, His6-YvaY, and His6-CopR fairly protruded the level of bound protein when TatAd or TatAd antibody was omitted, demonstrating no interaction with Sec-dependent or cytosolic proteins. These data indicate that the signal peptide of PhoD contains specific information mediating the affinity to TatAd. Essentially similar results were obtained when octylglucoside was omitted from the reactions.

TatAd Specifically Binds to Twin-arginine Peptides of the Signal Peptide of PhoD—Cellulose-bound peptide arrays have been used successfully to characterize substrate binding motifs of proteins (58). To determine the sequence-specific information necessary for binding of TatAd to prePhoD, we screened a cellulose-bound peptide scan of the N-terminal region of pre-PhoD for TatAd binding. The peptide scan was composed of 20-mer peptides that overlap by 19 residues over the sequence of 60 N-terminal localized amino acids and covering the 56-amino acid residue-long signal peptide of PhoD. The cellulose-bound peptides were incubated with [35S]-labeled His6-TatAd. TatAd showed selective affinity to peptides containing the twin-arginine motif (Fig. 6A). Binding was most pronounced when these residues were localized at the N-terminal flexible end of the peptide and gradually decreased when they moved to the C-terminal end of the peptides. Optimal affinity was observed for the peptide 26 containing R26R27 at its N-terminal end. Interesting, peptide starting with amino acids R27K28 also showed pronounced binding (Fig. 6B). In addition, affinity could be observed to peptides containing amino acid residues K13L14K15 (numbers indicate amino acid position in prePhoD). Binding of [35S]-labeled SecB, the targeting factor of the Sec translocation system of E. coli, to the PhoD signal peptide library was different from TatAd peptide recognition (data not shown) and followed rules identified by Knoblauch et al. (59).



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  		FIG. 6.
TatAd binding to cellulose-bound peptide scan of prePhoD. A scan composed of 20-mer peptides derived from the prePhoD sequence that overlap by 19 residues was screened for TatAd binding. The position of arginine and lysine residues are bold and in italics. The peptide scan (A) was incubated with [35S]-His6-TatAd, and the amount of TatAd bound to peptides was visualized by phosphorimaging. The numbers on the left indicate the first residue of the first spot of each row. In B, amount of bound TatAd of the 10 best binding peptides of the scan is shown. The numbers on the left indicate the position in the scan, and the horizontal bars demonstrate amount of activity detected.

 

To study the role of particular amino acids of the proposed binding motif, the 10 N-terminal amino acid residues of the twin-arginine containing peptide DRRKFIQGAGKIAGLSLGLT25–44 were substituted each in turn by all gene-encoded amino acids (Fig. 7). Substitution of the basic amino acid RRK cluster decreased binding of TatAd most seriously. In this region amino acid substitution except of arginine, histidine, or lysine interfered with the TatAd peptide interaction. Replacement by acidic amino acids almost abolished affinity of TatAd. Substitution of N-terminal aspartic acid by none acidic residues stimulated binding than compared with the wild type peptide. Replacements of amino acids localized C-terminal of the RRK motif hardly altered binding. Substitutions by arginine, lysine, or histidine stimulated TatAd binding in the N-terminal half of the peptide. Despite its low affinity, peptide SFQNNTFDRRKFIQGAGK18–35 was selected to study the role of amino acid residues localized N-terminal of the twin-arginine motif for TatAd recognition. Substitution of residues Ser-18 to Phe-24 each in turn by all gene-encoded amino acids hardly changed binding to TatAd (data not shown). Again, charge alteration had most pronounced effects. Although substitution by aspartic acid or glutamic acid reduced binding, introduction of basic amino acids stimulated binding above the TatAd affinity observed for the wild type-derived peptide (data not shown).



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  		FIG. 7.
Substitution analysis of the twin-arginine binding motif of TatAd. N-terminal 10 amino acids of peptide DRRKFIQGAGKIAGLSLGLT (indicated on the left) derived from the signal peptide of PhoD were substituted by amino acids indicated on the top. Lane + indicates wild type sequence. Peptide matrix was treated with [35S]-labeled His6-TatAd as described.

 

To elucidate the function of the RRK sequence motif for the interaction of TatAd with prePhoD further, co-immunoprecipitation experiments were carried out in the presence of synthetic peptide containing the twin arginine. Addition of QNNTFDRRKFIQGAGKIAG peptide significantly reduced co-immunoprecipitation of prePhoD by TatAd (Table III). In contrast, peptide QNNTFDAAAFIQGAGKIAG containing a substitution of the RRK sequence motif showed less interference of the prePhoD-TatAd interaction (Table III).


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  		TABLE III
Co-immunoprecipitation of TatAd with [35S]-prePhoD

Purified TatAd and [35S]-prePhoD proteins were mixed in PBS buffer containing 0.5 % n-octyl-{beta}-D-glucothiopyranoside. After 60 min of incubation at room temperature, TatAd-specific antibodies pre-complexed with protein A Dynabeads were added, and the incubation was continued for another 60 min. Amount of bound prePhoD was quantified by counting in scintillation liquid. Bound prePhoD is indicated in percent of input with standard deviation.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several components with one or six calculated membrane spanning domains have been described to mediate the transport of twin-arginine signal peptide-containing proteins (22). Therefore, Tat-dependent protein export is believed to be independent of soluble cytosolic factors (60). In the present study we demonstrate that a substantial fraction of TatAd protein of the PhoD-specific B. subtilis translocation system can be found beside its expected membrane-integrated localization in the cytosol. Soluble TatAd was functional active as demonstrated by its affinity to its substrate prePhoD and sequence-specific interaction with twin-arginine containing peptides. Therefore, we currently favor the thesis that TatAd on its way to the cytoplasmic membrane could fulfill a function as targeting factor for Tat substrate prePhoD.

We have shown previously (33) that the transport of PhoD can be mediated by only two Tat proteins, one similar to TatA and one similar to TatC of E. coli. To investigate the function of TatA proteins further, we studied the distribution of the TatAd protein in B. subtilis and in E. coli by using freeze-fracture technique and subsequent immunogold labeling. In B. subtilis, membrane-bound TatAd was equally distributed at both sides of the freeze-fractured cytoplasmic membrane. Immunogold-labeled protein was found to be aggregated with proteinaceous structures with a size of less than 5 nm. Absence of larger structures stimulates the hypothesis that the transport of proteins is independent of vesicle formation, which would be visible in the freeze-fractured membranes (22). Freeze-fractured membranes of E. coli cells overexpressing TatAd/TatCd demonstrated the uniform distribution of TatAd over the surface of the cell envelope.

In addition to its expected membrane-associated localization, freeze fractures through the cytosol indicated that a substantial amount of TatAd was localized in the cytosol in both bacterial systems. Ultrathin sections of B. subtilis cells confirmed an abundant localization of TatAd in the cytosol. Immunogold labeling of reference proteins demonstrated the reliability of both freeze-fracture analysis and labeling of ultrathin sections. The cytosolic chaperone DnaK could be detected in the cytosol, and SecY was detected predominately in the cytoplasmic membrane. Cytosolic localization of TatAd was first observed in E. coli TG1(pREP4, pQE9tatAd) cells overexpressing His6-TatAd. An abundant amount of His6-TatAd was soluble after sonication of the cells under native buffer conditions (data not shown). This unexpected observation was of crucial interest, because soluble TatAd is functional as it could be demonstrated to bind prePhoD- and prePhoD-derived peptides specifically. Identification of TatAd in membrane-free cell extracts of B. subtilis 168 confirmed the cytosolic localization of TatAd. Absence of TatCd in the cytosolic fractions of B. subtilis, as well as E. coli, demonstrated absence of membrane proteins in the cytosolic fraction. Interestingly, the amount of soluble TatAd varied in dependence of the induction time of the phoD operon. Because the amount of cytosolic TatAd decreased during prolonged phosphate starvation, presence of soluble TatAd might depend on the availability of newly synthesized prePhoD substrate to be targeted to the membrane. Absence of cytosolic TatAd in a strain not producing prePhoD confirmed this thesis. Thus, transient presence of cytosolic homomultimeric TatAd is linked to its substrate prePhoD. In addition, this observation points out that TatAd interacts specifically with prePhoD.

Co-immunoprecipitation demonstrated preferred binding of TatAd to prePhoD. The weak co-purification of mature PhoD indicated that secondary, but obviously less important binding sites, might be present in the mature part of the protein. The Sec-dependent transported B. subtilis protein preYvaY, mature YvaY, or cytosolic CopR were not recognized by TatAd. It can therefore be concluded that the twin-arginine signal peptide is the preferred binding site of TatAd. At the moment we can not quantify the stoichiometric ratio of the TatAd-prePhoD complexes. Gel filtration of purified TatAd indicated that the soluble protein forms complexes bigger than 100 kDa indicating that functional TatAd acts as a homomultimeric protein.2

Affinity of TatAd to a 20-mer peptide library of the N-terminal region of PhoD elucidated sequence specificity of TatAd recognition. Selective binding to peptides containing the twin-arginine motif could be observed. The different binding pattern of SecB to the peptide library demonstrated that binding was motif-specific. Recognition of the sequence motif was position-dependent. Localization of the motif at the flexible N-terminal end resulted in stronger TatAd peptide interaction and gradually decreased when the motif moved to the C-terminal attachment site of the peptide. Accessibility of the recognition motif for TatAd, especially because we observed that TatAd forms large homomultimeric complexes, might be sterically hampered if it is localized close to the inflexible C terminus of the peptide. Therefore, preferred binding of peptides with N-terminal localized RRK can be explained. The use of 20-mer peptides might underestimate the role of the secondary and tertiary structures of the signal peptide of PhoD for TatAd recognition. Still, selective binding to twin-arginine motif-containing peptides indicated that the twin-arginine motif is the target site recognized by TatAd and could play, in general, an essential role for the interaction of the Tat-translocase component TatA with its substrates. This result is consistent with the observation that TatAd binds preferable prePhoD, but less efficiently the mature PhoD as shown by co-immunoprecipitation.

Additional evidence about the role of particular amino acids for TatAd binding was obtained by substitution of 10 N-terminal amino acid residues of peptide DRRKFIQGAGKIAGLSLGLT25–44 localized in the signal peptide of PhoD. Substitution of the twin-arginine motif unambiguously demonstrated the essence of these residues for TatAd recognition. Beside substitution of the arginine residues, replacement of the lysine residue had similar consequences for TatAd binding. Therefore, it can be speculated that this third basic residue belongs to the recognition motif of TatAd. Surprisingly, binding of TatAd to peptides with altered RRK cluster was mainly charge-dependent. Although conservative substitution hardly reduced binding, uncharged amino acids reduced and acidic amino acid residues abolished TatAd binding. Remarkably, variation of amino acids localized C-terminal or N-terminal of the RRK cluster had only inferior effects on TatAd binding indicating that these amino acid residues are not essentially involved in the TatAd recognition. Based on these data we tend to speculate that RRK26–28 is involved in the TatAd recognition motif. This indicates that the proposed conserved (S/T)RRX-FLK sequence motif of the Tat signal peptides (27) might be involved in recognition of other Tat components. Moreover, it cannot be excluded that TatAd, functionally active in the cytosol of the B. subtilis cell, is acting different from TatA proteins of other organisms and therefore shares different recognition specificities. Absence of a TatB-like protein indicates that the Tat export in B. subtilis might be functionally different from E. coli. Most of the bacterial and plant Tat signal peptides studied demonstrated that both arginine residues of the consensus motif were critically important for the Tat transport (7, 13, 61–64). However, it has been demonstrated recently that a single lysine substitutions for arginine either naturally occurring (65) or replaced experimentally (61) were still transported in a Tat-dependent manner. Our data confirm that one of the arginine residues and, in addition, the lysine residue can be substituted by another positively charged amino acid residue and will still be recognized by TatAd. The physiological relevance of this observation is currently being studied in more detail.

Interference of twin-arginine peptides with Tat substrates for interaction with Tat components has been first demonstrated in an in vitro translocation system by Alami and co-workers (66). We demonstrated that co-immunoprecipitation of prePhoD by TatAd was interfered by addition of a peptide containing the RRK motif. Substitution of this motif by alanine residues resulted in a far lesser extent of this interference. These data confirmed that interaction of soluble peptides is sequence-specific, and co-immunoprecipitation experiments reflect in vivo function of TatAd. The discrepancy between the efficient interference of peptide QNNTFDRRKFIQGAGKIAG in the co-purification of prePhoD by TatAd and the inefficient recognition in the cellulose-bound peptide library can be explained by a higher flexibility of soluble peptide.

Despite the fact that TatA has an unusual and not very hydrophobic predicted structure in which only the extreme N terminus has the potential to form a classic hydrophobic transmembrane helix, TatA of E. coli has been found membrane-associated only (67, 68). Tha4, the plant TatA orthologue, was localized entirely in the membrane of thylacoids (51, 69). Other proteinaceous factors having affinity to TatA proteins could possibly mediate the targeting of selected substrates (70). In vitro translocation systems established for the E. coli Tat system demonstrated that no soluble factors are necessary to obtain protein translocation into inverted membrane vesicles (66, 71). Vesicle-based transport systems might underestimate the role of peptide-mediated targeting. In addition, because Tat substrates fold prior to translocation, the necessity for a fast targeting process to maintain export competence might be reduced compared with the Sec translocation system. Cytosolic localization of TatAd could reflect that Tat translocation in B. subtilis acts functionally different from other systems. Although soluble TatAd could mediate targeting of newly synthesized prePhoD to the translocation site, membrane-integrated TatAd could be involved in the translocation process. Structural and functional data of homomultimeric TatAd complexes present in the cytosol, as well as in the membrane,3 will help to uncover the relevance of the dual localization of TatAd.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains supplemental figures. Back

** Present address: Friedrich-Alexander-Universität Erlangen-Nürnberg, Institut für Klinische Mikrobiologie, Immunologie und Hygiene, Wasserturmstrasse 3–5, 91054 Erlangen, Germany. Back

§§ To whom correspondence should be addressed. Tel.: 49-3641-657577; Fax: 49-3641-657520; E-mail: jmueller{at}imb-jena.de.

1 The abbreviations used are: Tat, twin arginine translocation; IPTG, isopropyl-{beta}-D-thiogalactopyranoside; PBS, phosphate-buffered saline. Back

2 O. Pop and J. P. Müller, unpublished data. Back

3 M. Westermann, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Albert Bolhuis, Colin Robinson, and Jan Maarten van Dijl for useful discussions and Eckardt Birch-Hirschfeld for synthesizing oligonucleotides.



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