MPIase depletion causes the accumulation of TAT substrate precursors.

(A) Detection of SufI and TorA precursors upon MPIase depletion. SufI (top) and TorA (bottom) were expressed in the indicated strains. IPTG (1.5 mM) was added at early log phase, and cultivation was continued for 2 h. The cells were subjected to TCA precipitation, followed by SDS-PAGE/immunoblotting. The precursor and mature forms were then detected as indicated. MPIase depletion was confirmed in KS23 (middle left). Defects in the processing of SufI (middle right) and TorA (bottom right) with the KK signal were also confirmed. The symbol ‘★’ denotes degradation products of the SufI precursor. (B) Pulse-chase experiments of SufI processing after MPIase depletion. EK413/pTac-TatABC harboring pTet-SufI(RR) (top left) or pTet-SufI(KK) (top right), and KS23/pAra-CdsA/pTac-TatABC harboring pTet-SufI(RR) (+MPIase; bottom left, ΔMPIase; bottom right) were pulse-labeled for 30 s and then chased for the indicated periods. SufI(RR) and SufI(KK) were isolated using the attached His tags, followed by SDS-PAGE/autoradiography. (C) Subcellular localization of SufI and TorA-GFP after MPIase depletion. KS23/pTet-TatABC/pTac-SufI(RR) (left) and KS23/pTet-TatABC/pTac-TorA-GFP(RR) (right) were cultivated in the presence (+MPIase) or absence (ΔMPIase) of arabinose. The TAT substrates were leaky expressed from the tac promoter. The cells were then fractionated into periplasmic (peri) and spheroplast (sph) fractions. The indicated proteins were detected by immunoblotting. (D) Subcellular localization of TorA-GFP after MPIase depletion as revealed by fluorescence microscopy. The indicated strains were cultivated, TorA-GFP was induced, and then it was observed by fluorescence microscopy. The scale bars represent 10 μm. A magnified view of part of the ‘+MPIase’ sample is shown on the left.

TAT substrates are translocated into INV in vitro when TatABC and MPIase are both overproduced.

(A) Translocation of SufI and TorA-GFP into TatABC-overproducing INV. SufI (top) and TorA-GFP (bottom) were synthesized in vitro in the presence of INV prepared from the indicated strains. INV was omitted in the ‘-’ samples. The signal sequences on the left are wild-type (RR), while the sequences on the right are mutants (KK). Translocated materials were generated after PK digestion (+). One-fifth of the reaction mixture was analyzed as the translation standard (-). Translocation activities, expressed as a percentage of the amount of the synthesized proteins, are shown at the bottom of each autoradiogram. (B) Depletion of MPIase resulted in the failure of TAT translocation. INV were prepared from KS46/pAra-CdsA harboring pT7-TatABC, which was cultivated in the presence (+MPIase) or absence (ΔMPIase) of arabinose. After washing arabinose, IPTG was added at 1.5 mM to induce TatABC, and cultivation was continued for 2 h prior to INV preparation. SufI translocation was then performed as described in (A). (C) The level of TatC in INV used in (A) and (B). Each INV (10 μg) was analyzed by SDS-PAGE, followed by immunoblotting using anti-RpmJ-His antibodies. The position of TatC-His was shown. (D) MPIase is upregulated upon TatABC overproduction. The expression levels of MPIase in the strains used in (B) were determined by immunoblotting. CdsA and TatABC were induced as indicated. SecB levels were determined as a loading control. MPIase is co-purified with TatABC. INV prepared from BL21/pT7-TatABC-His induced with 1.5 mM IPTG (+++) or not induced (-), and BL21/pT7-Pm-F0b-His induced with 1 mM IPTG, solubilized with DDM, were applied to a TALON column, then eluted. The eluates were analyzed by TLC, followed by immunodetection of MPIase. The positions of the origin/front and MPIase are shown. Purified MPIase was also analyzed at right.

Reconstitution of the TAT system.

(A) Membrane fusion of TatABC proteoliposomes with MPIase liposomes enabled the detection of the SufI translocation. INV, prepared from BL21/pT7-TatABC, were solubilized with OG, and then mixed with purified F0F1-ATPase. The proteoliposomes were then formed by removing the detergent. PL/DAG liposomes or PL/DAG/MPIase liposomes were fused to the reconstituted proteoliposomes. SufI translocation was performed co-translationally (left) or post-translationally (right) in the presence of these proteoliposomes. SufI(RR) (top left) or SufI(KK) (bottom left) was used as the substrate. One-ninth of the reaction mixture was analyzed as the translation standard (-) for the co-translational reactions. For the post-translational reactions, ATP was added as indicated. Translocation activities are shown at the bottom of the left panels. (B) Purification of F0F1-ATPase and the Tat components. F0F1-ATPase from a thermophilic Bacillus PS3 (left), and E. coli TatA-His (lane 1), TatB-His (lane 2), and TatC-His (lane 3) (right) were subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining. (C, D) Complete reconstitution of the TAT system using the purified components. (C) Purified TatA, TatB, and TatC (in a 1:1:1 ratio) and F0F1-ATPase were mixed with phospholipids. Then, detergent was removed to form proteoliposomes. The reconstituted proteoliposomes were then fused with PL/DAG liposomes or PL/DAG/MPIase liposomes. Translocation activity was assayed with SufI(RR) (left) or SufI(KK) (right). (D) Proteoliposomes were reconstituted with the indicated components, followed by TorA-GFP translocation. The TatA:TatB:TatC ratio was 10:1:1. CCCP (20 μM), a protonophore, was added to the sample on the left. TorA-GFP(RR) (top) or TorA-GFP(KK) (bottom) was used as the substrate. Translocation activities are shown at the bottom of each gel.

MPIase is a membrane receptor for TAT substrates.

(A-C) TorA-GFP is targeted to the membrane in an MPIase-dependent manner. TorA-GFP(RR) (A, B; left half) or TorA-GFP(KK) (A, B; right half) was expressed in the specified strains. The cells were then disrupted to obtain the supernatant (sup; cytosol and periplasm) and the precipitate (ppt; membranes) fractions. The fluorescence intensity of each fraction was measured. The ratio to the whole cell was determined and shown as percentages. TatABC was overexpressed in (B), but not in (A). The means and standard deviations were calculated from at least three independent experiments. *, P<0.05; **, P<0.01. In (C), the indicated strains were cultivated, TorA-GFP was induced, and then it was observed by fluorescence microscopy. The scale bars represent 10 μm. Part of each picture was magnified on each side. Scale bars represent 10 μm. (D) TorA-GFP interacts with INV expressing MPIase. Purified TorA-GFP was mixed with the indicated INV, followed by fluorescence detection (top). As a control, each INV was mixed with NBD-PE, a fluorescent phospholipid, and observed (bottom). Scale bars represent 10 μm.

Working model of mechanisms underlying MPIase-dependent TAT translocation.

MPIase recognizes the signal sequence of the TAT substrates and insert them into the membrane. The TatBC complex on the membrane then determines whether the signal sequence of the membrane-inserted TAT substrate is valid. The TatABC translocon then forms a pore the same size as the substrate. This allows the substrate protein to be translocated across the membrane. Finally, Lep cleaves the signal sequence, and the mature protein is released into the periplasm. See the text for details.