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Originally published In Press as doi:10.1074/jbc.M513900200 on May 12, 2006

J. Biol. Chem., Vol. 281, Issue 29, 19977-19984, July 21, 2006
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Affinity of TatCd for TatAd Elucidates Its Receptor Function in the Bacillus subtilis Twin Arginine Translocation (Tat) Translocase System*Formula

Sandra Schreiber{ddagger}, Rayk Stengel{ddagger}, Martin Westermann§, Rudolph Volkmer-Engert, Ovidiu I. Pop{ddagger}, and Jörg P. Müller{ddagger}1

From the {ddagger}Institut für Molekulare Zellbiologie, Friedrich-Schiller-Universität Jena, Drackendorfer Strasse 1, D-07747 Jena, Germany, §Elektronenmikroskopisches Zentrum, Klinikum der Friedrich-Schiller-Universität Jena, Ziegelmühlenweg 1, D-07743 Jena, Germany, and Abteilung für Molekulare Bibliotheken, Charite, Ziegelstrasse 5-9, 10117 Berlin, Germany

Received for publication, December 30, 2005 , and in revised form, May 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Twin arginine translocation (Tat) systems catalyze the transport of folded proteins across the bacterial cytosolic membrane or the chloroplast thylakoid membrane. In the Tat systems of Escherichia coli and many other species TatA-, TatB-, and TatC-like proteins have been identified as essential translocase components. In contrast, the Bacillus subtilis phosphodiesterase PhoD-specific system consists only of a pair of TatAd/TatCd proteins and involves a TatAd protein engaged in a cytosolic and a membrane-embedded localization. Because soluble TatAd was able to bind the twin arginine signal peptide of prePhoD prior to membrane integration it could serve to recruit its substrate to the membrane via the interaction with TatCd. By analyzing the distribution of TatAd and studying the mutual affinity with TatCd we have shown here that TatCd assists the membrane localization of TatAd. Besides detergent-solubilized TatCd, membrane-integrated TatCd showed affinity for soluble TatAd. By using a peptide library-specific binding of TatAd to cytosolic loops of membrane protein TatCd was demonstrated. Depletion of TatCd in B. subtilis resulted in a drastic reduction of TatAd, indicating a stabilizing effect of TatCd for TatAd. In addition, the presence of the substrate prePhoD was the prerequisite for appropriate localization in the cytosolic membrane of B. subtilis as demonstrated by freeze-fracture experiments.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In bacteria, most of the exported proteins cross the cytosolic membrane through the Sec-dependent translocase in an unfolded conformation (1–3). A subset of proteins is transported via the twin arginine translocation (Tat)2 pathway. This alternative route accepts folded proteins or protein domains as substrates (4–6). Initially, the Tat pathway was discovered in chloroplasts as a pathway that operates independently of soluble factors and nucleoside triphosphates and is exclusively energized by the proton gradient across the membrane (7–9). In vitro translocation systems using Escherichia coli components demonstrated that also in bacteria this transport system is energized exclusively by the transmembrane proton electrochemical gradient (10, 11). Substrates destined for export by the Tat system are synthesized as preproteins with a signal peptide containing an almost invariant twin arginine sequence motif in the N-region of the otherwise canonically structured signal sequence (12).

In E. coli TatA, TatB, and TatC proteins have been demonstrated to be essential for Tat-dependent protein transport (13–16). The TatA homologous protein TatE has been proven to be functionally redundant (17). In chloroplasts structural and functional counterparts Tha4 (homologous to TatA; Ref. 18), Hcf106 (homologous to TatB; Refs. 16, 19), and cpTatC (homologous to TatC; Ref. 20) have been identified. The sequence-related proteins TatA and TatB are anchored in the cytoplasmic membrane via an amino-proximal {alpha}-helical domain (13). TatCs are a family of proteins with six calculated transmembrane-spanning domains with N and C termini exposed to the cytoplasmic or stromal side of the membrane (4). Gouffi et al. (21) recently proposed function-linked changes of TatA and TatC topologies for the mechanism of folded protein translocation in E. coli. These changes involve a TatA, the C terminus of which shuttles between the cytosol and the periplasmic space, and a TatC, the predicted fourth and fifth transmembrane helices of which shuttle in the periplasmic space. They speculated that this topology of TatC might reflect an operational state of TatC that changes during the protein translocation process (21). The current proposal for the action of Tat transport system of E. coli and plant thylakoids involves the initial binding of substrates to the TatB-TatC high molecular weight complex mediated via a direct contact of the double arginine signal peptide with TatC (20, 22). The signal peptide binding triggers association with TatA to form the active translocation channel driven by the transmembrane proton electrochemical gradient. The folded substrate protein is translocated across the membrane through a channel formed by multiple TatA protomers (recently reviewed in Refs. 23 and 24).

Although most bacterial and plant Tat systems contain three Tat proteins (TatA, TatB, and TatC), several bacterial and archaeal species miss a TatB-like protein (4, 25). Thus, at least one copy of a TatA homologue and one copy of a TatC homologue are required for a functional Tat pathway (6, 13, 16). The Bacillus subtilis genome encodes three TatA and two TatC-like proteins (4, 26). Despite the frequent presence of the twin arginine motif in the N-domain of signal peptides, the first identified substrate strictly transported Tat dependent was PhoD, a secretory phosphodiesterase (27, 28). We have demonstrated that the tatAd and tatCd genes, co-localized with phoD in one operon, were essential and sufficient to export PhoD (29). The second copy of tatC (tatCy) was not required for PhoD export (26). TatCy obviously forms together with TatAy a second TatAC translocase mediating the export of YwbN (30). TatAd is engaged in a dual localization. Despite the fact that the protein has a calculated N-terminal membrane-spanning {alpha}-helical region, TatAd was also found in the cytosol where it specifically interacted with the twin arginine motif of the prePhoD signal peptide (31). A similar observation has been recently reported for TatA and TatB proteins of the Streptomyces lividans Tat system (32, 33). The ability of soluble Tat proteins to posttranslationally bind Tat-dependent preproteins resulted in speculation that a soluble population of these Tat proteins could serve to recruit the substrates to the translocase. The prerequisite to confirm this thesis would be a definite cross-talk of the substrate-Tat protein complex with the membrane-integrated part of the translocase.

To prove a possible targeting function of TatAd for its substrate, we studied its affinity for TatCd. Because soluble TatAd was able to bind the twin arginine signal peptide of prePhoD prior to membrane integration, it could mediate the recruitment of the substrates to the membrane via the interaction with TatCd. By analyzing the distribution of TatAd and studying the mutual affinity with TatCd in different cellular systems we have shown here that TatCd assists the membrane localization of TatAd. Detergent-solubilized TatCd as well as membrane-integrated TatCd showed specific affinity for soluble TatAd. By analyzing the affinity of TatAd for a peptide library of TatCd, a specific binding to intermembrane cytosolic loops of TatCd was demonstrated. In addition, depletion of TatCd in B. subtilis resulted in a reduction of TatAd, indicating a stabilizing effect of TatCd for TatAd. Further, the presence of the substrate prePhoD affected distribution of TatAd and was the prerequisite for appropriate membrane insertion.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Plasmids, and Media—E. coli strain TG1(pREP4) (Qiagen, Hilden, Germany) was used to overexpress proteins. Plasmids pQE9tatAd, pQE60tatCd, and pQE9phoDp have been described elsewhere (29, 31). E. coli was grown aerobically at 37 °C in TY medium (34). As required, medium was supplemented with ampicillin (100 µg/ml), kanamycin (40 µg/ml), and isopropyl-beta-D-thiogalactopyranoside (IPTG, 1 mM). The shuttle plasmids pREP9tatAd/tatCd or pREP9tatAd mediate the IPTG-inducible expression of tatAd and tatCd or tatAd in B. subtilis and E. coli (31). B. subtilis strains were grown in TY medium to mid-exponential phase, and expression of tat genes was induced with 1 mM IPTG. To induce the phoD-tatAd-tatCd operon, B. subtilis strains were grown in low phosphate defined medium (35). Induction of phosphate starvation response was monitored by determining alkaline phosphatase activity as described previously (36). Membrane-free cell extracts of B. subtilis and E. coli were prepared as described (31).

Purification of His6-tagged Proteins—His6-TatAd, TatCd-His6, and His6-prePhoD were purified using E. coli strains TG1(pREP4, pQE9tatAd), TG1(pREP4, pQE60tatCd), and TG1(pREP4, pQE9phoDp) as described (31). Membrane-embedded TatAd was purified in the presence of 8 M urea under denaturing conditions according to standard protocols when indicated.

In Vivo Labeling of His-TatAd—[35S]Met-labeled His6-TatAd was obtained by pulse labeling of E. coli TG1(pREP4, pQE9tatAd) cultures. The strain was grown in M9 minimal medium, expression of tatAd was induced for 15 min, and cultures were labeled with 50 µCi of [35S]methionine for 5 min. Subsequent purification using Ni2+-NTA affinity chromatography was carried out essentially as described above.

Freeze-Fracture Electron Microscopy—Liposomes were concentrated by centrifugation and resuspended in phosphate-buffered saline (PBS) 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 of platinum/carbon at an angle of 35°. The evaporation of platinum/carbon with electron guns was controlled by a thin layer quartz crystal monitor.

Fracture Labeling of TatAd—For freeze-fracture immunogold labeling and subsequent electron microscopy, the freeze-fracture replicas were transferred to a digesting solution (10 mM Tris-HCl, pH 8.3, containing 30 mM sucrose and 2.5% SDS) and incubated overnight according to Fujimoto (37). The replicas were washed four times in PBS buffer and treated with PBS with 1% bovine serum albumin for 30 min. Next, they were placed in PBS containing bovine serum albumin (0.5%) and monospecific antibodies against TatAd (dilution 1:20) for 1 h. Subsequently the replicas were washed four times with PBS and placed on a 1:50 diluted solution of the secondary gold-conjugated antibody (goat anti-rabbit IgG with 10 nm gold particles; British Biocell International, Cardiff, UK) in PBS containing 0.5% bovine serum albumin for 1 h. After immunogold labeling, the replicas 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-filmed copper 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.

SDS-PAGE and Western Blot Analysis—Protein SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was prepared as described by Laemmli (38). After separation by SDS-PAGE, proteins were transferred to a nitrocellulose membrane (Schleicher and Schüll) as described by Towbin et al. (39). Proteins were visualized using monospecific antibodies against TatAd, TatCd, and alkaline phosphatase-conjugated goat anti-rabbit antibodies (Sigma) according to the manufacturer's instructions.

Isolation of Inverted Membrane Vesicles—E. coli cells producing the TatCd, TatAd, and TatCd, B. subtilis SecYEG (40) or no Tat proteins were harvested by centrifugation, washed, and resuspended in 50 mM Tris-HCl, pH 8.0. The cell suspension was passed three times through a French press at 16,000 lb/in2 to obtain inside-out inner membrane vesicles (IMVs), and the cell debris was removed by low spin centrifugation (10,000 x g for 5 min). Membranes were collected by high spin centrifugation (200,000 x g, 1 h) and resuspended in buffer containing 1 mM dithiothreitol; the inner membranes were subsequently separated from the outer membranes by sucrose density gradient centrifugation (41). IMVs were frozen in liquid nitrogen and stored at –80 °C. Protein content was determined by the DC protein assay (Bio-Rad).

TatAd Binding to IMVs—For TatAd binding reactions, IMVs were resuspended in 100 µl of binding buffer (50 mM HEPES-KOH, pH 7.6, 30 mM KCl, 0.5 mg/ml of bovine serum albumin, 10 mM dithiothreitol, 2 mM magnesium acetate) and incubated for 15 min on ice with various amounts of [35S]Met-labeled His6-TatAd and nonlabeled His6-TatAd as indicated in the figures. Samples were subsequently loaded on a sucrose cushion (0.25 mM sucrose in binding buffer) and fractionated by centrifugation (10 min, 30 lb/in2 in a Beckman Airfuge at room temperature). The amounts of [35S]Met-labeled His6-TatAd in the supernatant and in the pellet were quantified with a {gamma} counter.

Stability Determination of TatAd—B. subtilis strains were grown to mid-exponential growth phase in TY medium. Plasmid-mediated expression of tatAd was induced with IPTG (final concentration 1 mM). After 1 h further protein synthesis was stopped by addition of tetracycline (12.5 µg/ml) and chloramphenicol (10 µg/ml). Cells were withdrawn, lysed, and equalized for identical protein content. TatAd in total cell extracts was detected immunologically using SDS-PAGE and subsequent immunoblotting. Relative TatAd level was estimated by quantification of chemiluminescence-labeled protein.

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) (42, 43). Before screening, the membranes were washed in methanol for 10 min and 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, the peptide arrays were incubated with [35S]Met-labeled His6-TatAd (50 ng/ml; 10,000 cpm/µl) in blocking buffer for 16 h at room temperature with gentle shaking. Unbound protein was washed out with TBST buffer. Relative amounts of radioactivity were estimated by using phosphorimaging (Fuji) and associated image analytical software PC-BAS.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mutual Interaction of TatAd and TatCd—Affinity of the particular Tat proteins has been extensively investigated for E. coli as well as in plant Tat systems (44–48). Because functionality of the B. subtilis TatAd/TatCd transport system was demonstrated in E. coli (29), we assumed the formation of a functional transport unit in this host. To study the interaction of B. subtilis TatAd/TatCd proteins, we analyzed their mutual affinity in E. coli. His6-TatAd was amplified simultaneously with the untagged translocase proteins TatAd/TatCd using E. coli TG1(pQE9tatAd, pREP9tatAd/tatCd). Cells were harvested, and release of membrane-integrated proteins was obtained by lysing cells in phosphate buffer containing 0.5% octyl glucoside. His6-TatAd was immobilized on Ni2+-NTA superflow-agarose from membrane-free cell extracts. After removal of unbound material His6-TatAd was eluted with imidazole (250 mM) and assayed for retained TatCd. As demonstrated in Fig. 1A, lane 1, His6-TatAd eluate contained untagged TatCd.


Figure 1
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  		FIGURE 1.
Mutual interaction of TatAd and TatCd. His6-TatAd (A) or TatCd-His6 (B) were purified from E. coli strains TG1(pQE9tatAd, pREP9tatAd/tatCd) or TG1(pQE60tatCd, pREP9tatAd/tatCd) in phosphate buffer containing 0.5% octyl glucoside using Ni2+-NTA-agarose and subsequently eluted with 250 mM imidazol. Eluates were assayed for Tat proteins using SDS-PAGE and immunodetection (lanes 1). Strain TG1(pREP9tatAd/tatCd) processed under identical conditions was used as control for unspecific binding (lanes 2). C, TatCd-His6 loaded (lane 1) or unloaded (lane 4) Ni2+-NTA-agarose beads were incubated with membrane-free cell extracts derived from B. subtilis 168(pREP9tatAd/tatCd) overexpressing TatAd (lanes 2 and 5). After washing, eluates were assayed for Tat proteins using SDS-PAGE and immunodetection (lanes 3 and 6).

 
A reciprocal experiment was carried out by using TatCd-His6 as bait protein. E. coli strain TG1(pQE60tatCd, pREP9tatAd/tatCd) produces TatCd-His6 in addition to the untagged translocase proteins TatAd/TatCd. TatCd-His6 was purified and immobilized on Ni2+-NTA superflow-agarose as described above and analyzed for retained TatAd using SDS-PAGE and immunoblotting. As shown in Fig. 1B, lane 1, TatCd-His6 retained untagged TatAd co-expressed in the same cells. As control, strain TG1(pREP9tatAd/tatCd) was processed under essentially identical conditions. In this case neither TatAd nor TatCd was retained at the column material (Fig. 1, A and B, lanes 2). To optimize release of Tat proteins from the bacterial membrane, octyl glucoside was replaced by dodecyl maltoside or Triton X-100. Essentially similar co-purification of the untagged protein could be observed by its His6-tagged translocase partner protein (data not shown).

TatCd Has Affinity for Soluble TatAd—Based on our previous data TatAd is found, in addition to its expected membrane localization, soluble in the cytosol both in B. subtilis and in E. coli, where it specifically interacted with its substrate prePhoD (31). To assist substrate transport, TatAd should interact with specific receptor sites at the membrane surface. A possible candidate is the membrane-embedded TatCd. To investigate this thesis, affinity of TatAd to TatCd was analyzed using immobilized purified TatCd. Therefore, TatCd-His6 was overexpressed in and subsequently purified from E. coli TG1(pREP4, pQE60tatCd) using Ni2+-NTA superflow-agarose in the presence of 0.5% octyl glucoside. TatCd-His6-loaded agarose beads were incubated with membrane-free cell extracts derived from B. subtilis 168(pREP9tatAd/tatCd) cells producing soluble TatAd (Fig. 1C, lane 2). After removal of unbound material, TatAd was co-eluted with TatCd-His6 from the column material (Fig. 1C, lane 3). As a control experiment Ni2+-NTA superflow-agarose was incubated with cell lysate of E. coli TG1, washed, and incubated with membrane-free cell extracts of B. subtilis 168(pREP9tatAd/tatCd) (Fig. 1C, lanes 4 and 5). Because here no TatAd remained bound to the beads (Fig. 1C, lane 6), it can be concluded that soluble TatAd was retained via its interaction with immobilized TatCd-His6.


Figure 2
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  		FIGURE 2.
Membrane-localized TatCd has affinity for soluble TatAd. A, binding of [35S]Met-labeled His6-TatAd to IMVs derived from E. coli TG1 control cells (lane 3) and cells that overproduce TatCd (lanes 1 and 4) or TatAd/TatCd (lane 2) in the absence (lanes 1–3) or presence (lane 4) of a 50-fold excess of nonlabeled TatAd. B, binding of [35S]Met-labeled His6-TatAd to IMVs derived from E. coli cells that overproduce TatCd (black bars) or TatAd/TatCd (gray bars) in the presence of a 1-, 2.5-, 5-, or 7.5-fold excess of labeled TatAd (lanes 1–4).

 
Membrane-localized TatCd Has Affinity for Soluble TatAd—To circumvent experimental artifacts due to the detergent-mediated extraction of TatCd from the membrane, we studied the ability of membrane-localized TatCd to bind TatAd. IMVs were prepared from E. coli TG1(pREP4, pQE60tatCd) and then incubated with [35S]Met-labeled and purified soluble His6-TatAd. IMVs bearing overexpressed TatCd supported significantly higher binding of [35S]Met-labeled soluble His6-TatAd compared with that of the control IMVs (Fig. 2A, lane 1). [35S]Met-labeled TatAd binding was effectively reduced nearly to the background level by the addition of a 50-fold excess of nonlabeled TatAd (Fig. 2A, lane 4). Relative high background binding of TatAd to the IMVs was due to unspecific binding as shown by TatAd binding to liposomes (data not shown). In contrast, it was not possible to discern specific binding to any of the SecYEG components of the B. subtilis Sec translocase unit (data not shown). If IMVs were produced from E. coli TG1(pREP4, pQE9tatAd/tatCd) simultaneously producing TatAd and TatCd, lower levels of specific binding of [35S]Met-labeled TatAd were observed compared with IMVs containing TatCd only (Fig. 2A, lane 2). Increasing amounts of [35S]Met-labeled TatAd reduced the rate of binding for TatCd, the same as for TatAd/TatCd-containing IMVs (Fig. 2B). It was concluded that TatAd specifically binds to TatCd, which provides a limited number of receptor sites at the surface of the membrane.


Figure 3
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  		FIGURE 3.
Distribution of TatAd depends on the presence of TatCd. A, as indicated, synthesis of Tat proteins or no additional proteins was induced for 1 h using E. coli strains TG1(pREP4, pQE9), TG1(pREP4, pQE9tatAd) or TG1 (pREP4, pQE9tatAd /Cd). Identical amounts of cytosolic (C) and membrane fraction (M) were assayed for TatAd using SDS-PAGE and immunodetection. B, relative distribution of TatAd (mean and standard deviation) of two independent experiments was quantified using imaging software.

 
Distribution of TatAd Depends on the Presence of TatCd—Having established that TatCd provides receptor sites for TatAd at the membrane, we next investigated its influence on the distribution of TatAd. Cytosolic and membrane localization of TatAd was analyzed in the presence or absence of TatCd in E. coli. Whereas in strain TG1(pREP4, pQE9tatAd) the majority of TatAd remained in the cytosol, more pronounced membrane localization was observed using strain TG1(pREP4, pQE9tatAd/tatCd) co-expressing tatAd and tatCd (Fig. 3). This result suggests that TatCd stimulates the membrane localization of TatAd.

Allocation of TatAd in B. subtilis—To study the distribution of TatAd in the natural host, synthesis of the TatAd and TatCd translocase components in B. subtilis 168 was induced upon growing the strains to phosphate starvation. Induction of the starvation response was monitored by measuring the enzymatic activity of alkaline phosphatases, which are induced immediately after depletion of inorganic phosphate from the growth medium (35) (data not shown). In parallel, cells were withdrawn at the time points indicated, separated in cytosolic and membrane fraction, and the presence of TatAd was analyzed immunologically (Fig. 4A). 15 min after phosphate starvation, a significant amount of TatAd was already detected in both fractions, reaching its highest level 30 min after induction. Up to 60 min after the onset of the induction the majority of TatAd was found in the cytosolic fraction. Upon prolonged incubation the protein quickly disappeared in both the cytosolic and membrane fractions, indicating a short half-life of TatAd. A similar distribution of TatAd was observed using strain B. subtilis 168 {Delta}tatCy (Fig. 4B). A raised level of TatAd could reproducibly be observed in B. subtilis 168 {Delta}tatCy strains.


Figure 4
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  		FIGURE 4.
Allocation of TatAd in B. subtilis. B. subtilis strains 168 (A), 168 {Delta}tatCy (B), 168 {Delta}tatCd (C), and 168 {Delta}tatCd/y (D) were grown in low phosphate defined medium. Cells were withdrawn at the indicated time points after induction of phosphate starvation and fractionated in cytosolic and membrane fractions. Identical amounts of fractions were assayed for TatAd using SDS-PAGE and immunodetection.

 


Figure 5
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  		FIGURE 5.
TatCd stabilizes TatAd. B. subtilis 168 containing plasmids pREP9tatAd (A, and filled circles in panel C) or pREP9tatAd /tatCd (B, and open circles in panel C) were grown to mid-exponential growth. Plasmid-mediated expression was induced by additon of IPTG (1 mM final concentration). After 1 h protein synthesis was stopped by addition of tetracycline and chloramphenicol, and samples were withdrawn after the indicated time points. Cells were lysed, and total cell extracts were equalized for their protein content. The presence of TatAd in cell extracts was detected using Western blotting with monospecific antibodies against TatAd. C, the amount of TatAd was quantified using chemiluminescense detection and imaging software.

 
In parallel B. subtilis strains 168 {Delta}tatCd (Fig. 4C) and 168 {Delta}tatCd/tatCy (Fig. 4D) were cultivated and processed under essentially identical conditions. Surprisingly, almost no TatAd could be monitored in the particular cellular fractions of both strains. The presence of TatCd was obviously the prerequisite for stabile TatAd production.

TatCd Stabilizes TatAd—To analyze whether TatCd has a stabilizing effect on TatAd, we monitored the stability of TatAd in B. subtilis. Half-life determination of TatAd using [35S]methionine protein pulse labeling of B. subtilis cultures starved for inorganic phosphate was inefficient. In addition, TatAd could not be quantitatively immunoprecipitated from pulse-labeled cell lysates. Therefore, plasmids pREP9tatAd and pREP9tatAd/tatCd mediating the IPTG-inducible synthesis of TatAd or TatAd and TatCd were transformed into B. subtilis 168. To study the stability of TatAd, plasmid-mediated expression of tatAd was induced. After 1 h protein synthesis was stopped by addition of antibiotics. Samples were taken at indicated time points, equalized for their protein content, and immunologically characterized for their TatAd level. Whereas in strain 168(pREP9tatAd/tatCd) the majority of the protein remained stable over several hours, TatAd disappeared quickly in strain 168(pREP9tatAd) (Fig. 5). Thus, it can be concluded that co-expression of tatCd is the prerequisite for stable maintenance of TatAd.

TatAd Specifically Binds Cytosolic Loops of TatCd—A cellulose-bound peptide array has been successfully used to decipher the substrate binding motif of TatAd (31). To determine the sequence-specific information necessary for binding of TatAd to TatCd, we screened a cellulose-bound peptide scan of TatCd for TatAd binding. The peptide scan was composed of 13-mer peptides that overlap by 12 residues over the sequence of TatCd. The cellulose-bound peptides were incubated with 35[S]Met-labeled His6-TatAd. TatAd showed selective affinity to peptides (Fig. 6A). Binding was most pronounced to peptides derived from regions of the cytosolic domains of TatCd. TatCd regions preferably bound by His6-TatAd are displayed in Fig. 6B. Particularly, the cytosolic domains between TMS (transmembrane segment) II and TMS III as well as TMS IV and TMS V showed preferable binding by TatAd.

Absence of Substrate prePhoD Results in Localization of TatAd at the Inner Side of the Cytosolic Membrane—To elucidate whether the distribution of TatAd was also dependent on the presence of prePhoD in B. subtilis, we analyzed its localization in a B. subtilis strain depleted for phoD. Strain MH5444 deleted for phoD was transformed with plasmid pREP9tatAd/tatCd, allowing the IPTG-inducible synthesis of the TatAd/TatCd proteins. The strain was grown to phosphate starvation, expression of Tat proteins was induced, and localization of TatAd was detected using immunogold labeling of freeze-fractured cells. Although in B. subtilis tat wild-type cells TatAd was evenly distributed between cytosol and membrane (Fig. 7A), in MH5444 (pREP9tatAd/tatCd) the protein was exclusively localized in the membrane (31). Strikingly, the TatAd protein was almost entirely localized at the cytosolic (PF, protoplasmic face) side of the membrane (Fig. 7, compare panels B and C). Freeze fracture through the B. subtilis cell further elucidated localization of TatAd at the inner side of the cell envelope (Fig. 7C, arrow). Cells grown without IPTG showed no immunogold labeling, demonstrating the specificity of TatAd labeling (supplemental Fig. S1). Immunogold labeling of freeze-fractured cells of B. subtilis tatCd strains failed due to inefficient labeling.3


Figure 6
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  		FIGURE 6.
TatAd specifically binds cytosolic loops of TatCd. A, a scan composed of 13-mer peptides derived from the TatCd sequence that overlap by 12 residues was screened for TatAd binding. The peptide scan was incubated with 35[S]Met-labeled His6-TatAd, and the amount of TatAd bound to peptides was visualized by phosphorimaging. The position of the N-terminal amino acid residue of the peptides in the TatCd protein is indicated on the right. The positions of calculated transmembrane-spanning regions (labeled with Roman numbers) are bold and italic. B, schematic presentation of domains bound by TatAd. Bold lines and shadowed cylinders in the calculated transmembrane segments of TatCd demonstrate regions interacting with TatAd.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
TatAd of the B. subtilis PhoD-specific Tat translocase is engaged in a dual localization. Despite the fact that the protein has a calculated N-terminal membrane spanning the {alpha}-helical region, we demonstrated previously that a substantial fraction of the protein was found to be soluble in the cytosol of the cell in addition to its expected membrane localization (31). A similar observation has been recently reported for TatA and TatB proteins of the Streptomyces lividans Tat system (32, 33). The ability of soluble Tat proteins to posttranslationally bind Tat-dependent preproteins resulted in speculation that a soluble population of these Tat proteins could serve to recruit the substrates to the translocase. The prerequisite to confirm this thesis would be a definite cross-talk of the substrate-Tat protein complex with the membrane-integrated part of the translocase. By analyzing the distribution of TatAd and studying binding to TatCd in different cellular systems, data presented above elucidated a mutual affinity of TatAd and TatCd. Besides detergent-solubilized TatCd, membrane-integrated TatCd showed affinity for soluble TatAd. TatCd promotes membrane localization of TatAd via domain-specific interactions of cytosolic loops of the protein. In addition, depletion of TatCd in B. subtilis resulted in a drastic reduction of TatAd, indicating a stabilizing effect of TatCd for TatAd. The presence of prePhoD was the prerequisite for appropriate membrane insertion as demonstrated by freeze-fracture experiments.

Besides characterization of the affinity of the TatAd and TatCd proteins in the membrane, we focused our studies on the affinity of TatCd for soluble TatAd to get further insight into the function of soluble TatAd. IMVs produced from E. coli strains overexpressing TatCd showed significant elevation of affinity for TatAd compared with the binding to IMVs derived from E. coli wild-type cells. Interference of binding with unlabeled TatAd as well as reduced rates of binding in response to the elevation of the amount of labeled TatAd demonstrated specificity of TatAd binding to TatCd-bearing IMVs. Basal affinity of TatAd to IMVs could be due to lipid interactions of TatAd ranging from an apposition to the membrane surface up to a temporary embedding in the membrane as discussed by Gouffi et al. (21). Currently it cannot be excluded that TatAd interacts with E. coli Tat proteins. Because the depletion of E. coli Tat translocase units did not affect the functionality of the TatAd/TatCd translocase in E. coli as demonstrated previously (29), specific interaction with E. coli seems to be unlikely. IMVs prepared from cells co-expressing tatAd and tatCd reduced binding of labeled TatAd compared with IMVs containing TatCd only, obviously due to a saturation of TatCd binding sites by endogenously expressed TatAd.

Affinity of TatAd to a 13-mer peptide library of TatCd elucidated domain specificity of TatAd recognition. As shown for the sequence-specific affinity of TatAd for double arginine-containing peptides derived from the signal peptide of PhoD, recognition of peptide-specific epitopes of peptide libraries was position dependent (31). Selective binding to peptides derived from cytosolic regions of TatCd demonstrates that the cytosolic loops between the second and the third transmembrane helices as well as the fourth and the fifth transmembrane helices form the basis for this interaction as displayed in Fig. 6B. Complex formation of individual Tat proteins has been extensively investigated in the E. coli (44–48) and the plant (49) Tat systems, but currently there exists no information about the specific interaction sites between TatA and the TatB-TatC-substrate complex. The TatC proteins are highly conserved within various organisms (4). This group of proteins shares a similar orientation in the membrane with N and C termini exposed to the cytoplasmic or stromal side of the membrane (20, 44, 50, 51). Particularly, alignment of TatC proteins revealed that conserved residues are preferably localized in the region exposed to the cytosolic site (52, 53). Mutagenesis of conserved positions of E. coli TatC has revealed that some of these residues are critical for its function, which would be consistent with a role in substrate binding (52, 53). Elucidated site-specific affinity of TatAd for TatCd could be an additional driving force for the assembly step of the Tat translocases of other Tat systems.


Figure 7
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  		FIGURE 7.
The absence of substrate prePhoD results in altered localization of TatAd. Cells of B. subtilis 168 phoR12 (A) and MH5444 (pREP9tatAd / tatCd)(B and C) were grown in low phosphate defined medium to phosphate starvation in the presence of 1 mM IPTG. Cells were freeze fractured and subsequently labeled with TatAd-specific antibodies and 10 nm gold-conjugated secondary antibody. Electron micrographs demonstrate the protoplasmic face (PF) and the exoplasmic face (EF) of the cytoplasmic membrane and fractured cytosol (Cy). Scale bar, 0.5 µm.

 
For E. coli TatC a dual topology has been suggested: Its calculated 6 transmembrane-spanning domains were confirmed by Behrendt et al. (54). Alternatively, Gouffi et al. (50) predicted that the fourth and fifth transmembrane helices and the predicted second cytoplasmic loop between the two helices of E. coli TatC protein are located in the periplasm. They speculated that this topology might reflect an operational state of TatC that changes during the protein translocation process. In combination with topology changes of TatA they propose a model in which topology rearrangements of TatC and TatA assist the movement of the substrate into the membrane and the formation of a hydrophilic cavity to provide a protein-conducting channel for substrate transport (see Ref. 21 for details). Direct affinity of TatCd for TatAd combined with topological flexibility of TatCd would provide a possible mechanism for how the TatAd-substrate complex could integrate into the membrane driven by the proton motive force at the membrane.

Monitoring of TatAd in the cytosol and the membrane in B. subtilis showed that at the onset of the induction it is distributed between the cytosol and the membrane fraction. Upon ongoing incubation the protein quickly disappears in both the cytosolic and the membrane fractions, obviously due to proteolytic degradation.

Our attempts to study the influence of TatCd for the distribution of TatAd in B. subtilis failed because TatAd could be hardly detected in B. subtilis {Delta}tatCd strains in both fractions. Because the half-life of TatAd was much shorter when the protein was expressed in the absence of TatCd it can be concluded that TatCd has a stabilizing effect on TatAd. Absence of TatCd obviously results in the TatAd mislocalization and/or misfolding becoming a target for host-specific proteases. After mediating the targeting and the transport of prePhoD, TatAd could be either recycled to the cytosol or could stay in the membrane. Short half-life of TatAd in B. subtilis indicates that it is, once integrated into the membrane, rapidly degraded by host-specific proteases. Thus, we currently favor the thesis that TatAd is faced to a one-way use and is subsequently degraded from the membrane.

Membrane localization of TatAd was dependent on the presence of the substrate prePhoD. Freeze-fracture experiments carried out in a B. subtilis strain deleted for phoD revealed that TatAd was almost exclusively localized in the membrane (31). Striking was the observation that the absence of the substrate resulted also in altered membrane localization of TatAd. Although freeze-fracture experiments using B. subtilis wild-type cells showed a even distribution of the protein in both sites of the membrane irrespective of the expression level of the proteins,3 absence of the substrate resulted in the accumulation of TatAd in the inner side of the membrane. A possible explanation for this observation is that the formation of the heterodimeric substrate-TatAd complex is obviously the prerequisite for the proper structure and/or folding to obtain the appropriate membrane integration. Chaperone-assisted substrate proofreading or quality control activity during assembly of complex endogenous substrates has been suggested for the E. coli Tat system (55).

The current understanding of the mechanism of the protein translocation of TatABC systems involves a cyclical assembly model proposed by Cline and Mori (49) for plant thylakoids. In E. coli it has been demonstrated that a complex consisting of TatB and TatC serves as substrate binding site in which TatC mediates the specific substrate interaction (22). The pH gradient triggers Tha4 (TatAd homologue) recruitment to the precursor-bound TatC-TatB complex and causes a rearrangement of components to form an active translocon. Upon completion of protein translocation, TatA is released while TatB remains assembled with TatC (reviewed in Ref. 24). Transport systems containing soluble Tat proteins might act fundamentally different as evidenced for the PhoD-specific TatAd/TatCd transport system: Soluble Tat proteins have been discussed as mediating the targeting of the Tat substrates to the membrane-integrated part of the translocase (31–33). The formation of the TatAd-prePhoD complex is obviously the prerequisite for the appropriate integration of TatAd into the membrane. At the cis side of the membrane TatCd serves as receptor for the TatAd-prePhoD complex and stabilizes TatAd in the membrane, probably by assisting the formation of the protein-conducting channel to mediate prePhoD transport. Because TatAd in the absence of the substrate directly interacted with TatCd, besides a possible direct interaction of the substrate with TatC, a direct affinity of both proteins can be concluded. In addition, a specific affinity of TatC for the Tat substrates is currently under investigation. After prePhoD translocation, TatAd obviously becomes a target of proteolytic degradation by host-specific cell wall proteases.


   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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed. Tel.: 49-3641-9325671; Fax: 49-3641-9325652; E-mail: joerg.mueller2{at}med.uni-jena.de.

2 The abbreviations used are: Tat, twin arginine translocation; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PBS, phosphate-buffered saline; IMV, inner membrane vesicle. Back

3 M. Westermann, O. I. Pop, and J. P. Muller, unpublished data. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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J. P. Barnett, R. T. Eijlander, O. P. Kuipers, and C. Robinson
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