TatB functions as an oligomeric binding site for folded Tat precursor proteins

Abstract

Twin-arginine-containing signal sequences mediate the transmembrane transport of folded proteins. The cognate twin-arginine translocation (Tat) machinery of Escherichia coli consists of the membrane proteins TatA, TatB, and TatC. Whereas Tat signal peptides are recognized by TatB and TatC, little is known about molecular contacts of the mature, folded part of Tat precursor proteins. We have placed a photo-cross-linker into Tat substrates at sites predicted to be either surface-exposed or hidden in the core of the folded proteins. On targeting of these variants to the Tat machinery of membrane vesicles, all surface-exposed sites were found in close proximity to TatB. Correspondingly, incorporation of the cross-linker into TatB revealed multiple precursor-binding sites in the predicted transmembrane and amphipathic helices of TatB. Large adducts indicative of TatB oligomers contacting one precursor molecule were also obtained. Cross-linking of Tat substrates to TatB required an intact twin-arginine signal peptide and disappeared upon transmembrane translocation. Our collective data are consistent with TatB forming an oligomeric binding site that transiently accommodates folded Tat precursors.

Figure 1.

Surface-exposed and internal amino acid residues of SufI that were exchanged by the photo-cross-linker benzoyl-phenylalanine. (A) Surface-exposed residues of SufI. Structural data were taken from Tarry et al. (2009a). (B) Internal residues W140, W161, L233, and F261 of SufI.

Figure 2.

On membrane-targeting, the surface of pSufI is found predominantly in the vicinity of TatB. (A) Codons of the sufI DNA that specify the indicated amino acids were mutated to amber stop codons. The resulting DNAs were transcribed and translated in vitro by an E. coli cell-free extract prepared from a strain that expresses an amber suppressor tRNA and the cognate Bpa-specific tRNA-synthetase. Radiolabeled translation products were separated by SDS-PAGE and are visualized by Phosphorimaging. The electrophoretic mobilities of molecular mass standards are indicated on the left. In contrast to the approx. 50-kDa wild-type (wt) precursor of SufI (pSuf), smaller translation products were predominantly expressed from the amber mutant DNAs when Bpa was missing. These smaller translation products match the molecular masses expected for premature termination at the individual amber codons. The shortest fragment of 48 amino acids was not retained by the polyacrylamide gel used here. In addition to each premature termination product, some full-size pSufI was synthesized in the absence of Bpa, obviously resulting from read-through of the mutant mRNAs. Addition of Bpa led to a clear increase in full-size pSufI of each variant, indicating suppression of the amber codons by the incorporation of the cross-linker Bpa. (B) Wild-type pSufI and its indicated Bpa variants were synthesized in vitro in the presence of inside-out inner membrane vesicles, and the transport efficiency of each mutant was analyzed as described in Materials and Methods. Transport efficiency of wt-pSufI was set 100%. (C) After synthesis of each indicated Bpa variant of pSufI in the presence or absence of membrane vesicles (INV), samples were irradiated with UV light or mock-incubated before SDS-PAGE and Phosphorimaging. UV irradiation led to numerous radiolabeled bands larger in size than pSufI, only few of which were specifically obtained in the presence of INV (lanes marked with downward pointing arrows). The most prominent of those photo-adducts are labeled with asterisks and dots. Of notice, no membrane-specific adducts were obtained for SufI variants W140, W161, L233, F261 carrying Bpa in the interior of the folded structure. (D) The indicated Bpa variants of pSufI were synthesized in vitro in the presence of INVs. Samples irradiated with UV light were either directly prepared for SDS-PAGE or only after coimmunoprecipitation with antibodies directed against TatA, TatB, and TatC (αTat). Asterisk-labeled adducts are recognized by anti-TatB antibodies and dot-labeled ones by anti-TatA antibodies. No Tat-specific adduct was observed for the internal W140 variant of pSufI, nor were any specific cross-links obtained with the nonfunctional KK-precursor of SufI.

Figure 3.

A Tat precursor interacts transiently with TatB before its translocation. (A) The two indicated Bpa variants of pSufI were synthesized in the presence of INV, one-half of each reaction containing the uncoupler CCCP at 0.1 mM, the other half only the solvent DMSO. Samples were either digested with Proteinase K (PK) to visualize protease-resistant (i.e., translocated precursor and mature forms of SufI) or otherwise irradiated with UV light. The labeled photo-adducts are those identified in Figure 2. Whereas CCCP completely blocks translocation of pSufI into INVs and also abolishes cross-linking to TatA, the TatB adducts of pSufI were not affected by the uncoupler. (B) After synthesis of pSufI in the presence of INV, vesicles were collected by centrifugation, resuspended, and incubated in the absence or presence of an ATP regenerating system (cf. Materials and Methods). The ATP-dependent increase in translocation is paralleled by a loss of cross-linking to TatB.

Figure 4.

Incorporation of the cross-linker Bpa into TatB reveals multiple contacts between an extended part of the molecule and precursor proteins. (A) Model of TatB depicting its predicted transmembrane and amphipathic helices and the approximate positions of Bpa. (B) In vitro synthesis of radiolabeled pSufI in the presence of INVs containing the indicated Bpa variants of TatB. UV-irradiation yielded cross-links of varying intensities (asterisks) that were immunoprecipitated by anti-TatB antibodies. Note the different intensities by which the three positions on a helix turn of TatB cross-link to pSufI. (C) In vitro synthesis of radiolabeled pSufI in the presence of INVs containing the I36Bpa variant of TatB. Cross-linking was completely abolished if the signal sequence of pSufI carried a KK-mutation of the consensus RR-motif (lanes 1–6). Cross-linking was unimpaired (lanes 7–12), if INVs were added posttranslationally after depleting all energy sources to prevent translocation into INV (cf. Materials and Methods). Protease-protected SufI species shown in lane 7 demonstrate the translocation activity of the TatB(I36Bpa) variant and the lack of PK protection in lane 10 confirms the successful depletion of energy. (D) In vitro synthesis of radiolabeled pSufI in the presence of INVs containing the indicated Bpa variants of TatB. Note the adducts to TatB (L54Bpa) larger than 100 kDa that likely contain more than one TatB monomer. The origin of the TatB-adduct running at 90 kDa is not clear. This and the 75-kDa adduct were also obtained with a noncleavable mutant of pSufI (pSufI-ΔSP) indicating that all cross-links were between TatB and the precursor of SufI (lanes 15–19). Cross-linking of TatB to pSufI occurred independently of translocation (lanes 20–25). (E) In vitro synthesis of radiolabeled pSufI in the presence of INVs containing the indicated Bpa variants of TatB reveals a position-dependent cross-linking of the transmembrane helix of TatB to pSufI (lanes 1–12). Note the high molecular mass adduct to TatB(L9Bpa) indicative of a TatB oligomer. Cross-linking of TatB to pSufI occurred independently of translocation (lanes 13–18).

Figure 5.

TorAPhoA shows the same interaction with TatB as pSufI. (A) Surface-exposed residues I174, W270, I329, and L467 of PhoA used for the incorporation of the cross-linker Bpa. Structural data were taken from Wang et al., (2005). (B) The 55 kDa Tat precursor protein TorA-PhoA carrying Bpa at the surface position W270 (arrow) was synthesized in vitro with INVs added as indicated. When irradiated with UV-light, a major photo-cross-link (asterisk) was obtained that was recognized by anti-TatB antibodies (αTatB) and was absent from reactions devoid of membranes (lane 6). It was completely digested by Proteinase K (PK) (lane 3) in contrast to the PK-resistant translocated TorA-PhoA species (arrowheads) and therefore not membrane-protected. The three translocated species of TorA-PhoA from top to bottom are precursor, mature form and a translocation-arrested species (Panahandeh et al., 2008). Solubilization of the INVs' Tat machinery by 1% Triton X-100 prevented formation of the TatB adduct of TorA-PhoA (lane 5), whereas dissipation of the PMF by CCCP did not (lane 8). (C) TorA-PhoA (arrow) carrying Bpa at the surface position I329 was synthesized in vitro in the presence of INVs, irradiated with UV-light, and immunoprecipitated with antisera against TatA, TatB, and TatC (lanes 1–5). The only Tat-specific photo-adduct obtained was the one recognized by anti-TatB antibodies (asterisk). Cross-linking was completely abolished if the signal sequence of TorA-PhoA carried a KK-mutation of the consensus RR-motif (lanes 6–10). (D) Internal residues I99 and V196 of PhoA used for the incorporation of Bpa. (E) Comparison between the surface-exposed Bpa variant I329 of TorA-PhoA and the internal variant I99. Lanes 1–4 illustrate that transport of TorA-PhoA into INV proceeded only when TorA-PhoA was synthesized under oxidizing conditions, which were achieved by the addition of oxidized glutathione (GSSG). Transport is indicated in lane 4 by the appearance of the PK-resistant TorA-PhoA species specified in B (arrowheads). In contrast to the internal Bpa variant I99, the surface-exposed variant I329 formed adducts with TatB (asterisk) both under reducing and oxidizing conditions. (F) Cross-linking of the transmembrane and amphipathic helix of TatB to TorA-PhoA. The two TatB variants L9Bpa and L54Bpa again yielded higher molecular mass adducts (arrows). Cross-linking was observed both under reducing and oxidizing conditions.

Figure 6.

Models of possible TatB-precursor interactions. (A) The gray bar represents the lipid bilayer. Each TatC molecule is depicted by six stippled transmembrane helices. Four TatB monomers are shown (diagonally hatched cylinders) with their transmembrane helices contacting one TatC protomer each (the front TatC subunit has been omitted for clarity) and with their cytosolic amphipathic helices encapsulating a folded Tat precursor (black ellipse). The twin-arginine signal sequence (++) is represented by a black line. Its contacts to TatB and TatC are speculative. The tetrameric nature of the TatBC complex is based on previous findings (Lee et al., 2006), but the model would easily accommodate higher order oligomeric structures. (B) Two Tat precursor molecules simultaneously binding to TatB as suggested by recent data (Tarry et al., 2009b; Ma and Cline, 2010).