The lateral gate of SecYEG opens during protein translocation

Abstract

The SecYEG translocon of Escherichia coli mediates the translocation of preproteins across the cytoplasmic membrane. Here, we have examined the role of the proposed lateral gate of the translocon in translocation. A dual cysteine cross-linking approach allowed the introduction of cross-linker arms of various lengths between adjoining aminoacyl positions of transmembrane segments 2b and 7 of the lateral gate. Oxidation and short spacer linkers that fix the gate in the closed state abolished preprotein translocation, whereas long spacer linkers support translocation. The cross-linking data further suggests that SecYEG lateral gate opening and activation of the SecA ATPase are coupled processes. It is concluded that lateral gate opening is a critical step during SecA-dependent protein translocation.

FIGURE 1.

Introduced cysteine mutations in TM2b and TM7 of the lateral gate of SecY. A, topology model of E. coli SecY based on a sequence alignment with the M. jannaschii SecY crystal structure (17). Highlighted are TM segments 2b and 7 and the amino acids residues selected for cysteine mutagenesis (red, Ser-87 and Phe-286; yellow, Met-83, Ile-86, and Phe-279). Also indicated is the OmpT cleavage site in cytoplasmic region C4. B, side view of the M. jannaschii SecY crystal structure showing the putative gate region (Protein Data Bank code 1RHZ (17)). Indicated are amino acids Ser-87 and Phe-286 in TM2b and TM7, respectively. The figure was created using MOLMOL (38) and POV-Ray.

FIGURE 2.

Disulfide cross-linking of cysteines in the lateral gate formed by TM2b and TM7. A, IMVs containing different SecYEG derivatives were treated with the oxidizer sodium tetrathionate (NaTT, 1 mm). To access the cross-linking efficiency of the cysteine residues in SecY, the IMVs were incubated with the protease OmpT and analyzed by Coomassie Brilliant Blue-stained SDS-PAGE. B, immunostaining of the SDS-PAGE using an antibody against the N-terminal His₆ tag in SecY. Note, to prevent the reduction of the disulfide bond during electrophoresis, samples that contained DTT were analyzed on a different gel than samples without DTT. For this reason there is a slight difference in gel mobility of full-length SecY and N-SecY. On the same gel, the N-SecY fragments of all SecY mutants migrate at the same position.

FIGURE 3.

An intramolecular disulfide bond in SecY F286C/S87C inhibits proOmpA translocation. A, translocation of FL-proOmpA into wild-type IMVs (lane 2) or IMVs containing overexpressed levels of Cys-less (lanes 3 and 4), F286C/S87C (lanes 5 and 6), Cys-286 (lanes 7 and 8), or Cys-87 (lanes 9 and 10) SecYEG. Prior to translocation, IMVs were oxidized or reduced with NaTT (lanes 4, 6, 8, and 10) and DTT (lanes 2, 3, 5, 7, and 9), respectively. Lane 1 shows a 20% FL-proOmpA standard. B, SecA translocation ATPase activity of IMVs containing Cys-less, F286C/S87C, Cys-286, or Cys-87 SecYEG and that had been preincubated with (black bars) or without (white bars) NaTT. Error bars represent S.D.

FIGURE 4.

Cross-linking of SecY F286C/S87C with thiol reactive reagents of different spacer lengths. A, IMVs containing Cys-less or F286C/S87C SecYEG were incubated with the reducing agent TCEP, the oxidizer NaTT (2 Å), or the cross-linkers bBBr (5 Å), BMOE (∼8 Å), and BMH (∼13 Å). After 30 min at 37 °C, IMVs were treated with OmpT and cross-linking of the cysteine residues in SecY was analyzed by Coomassie Brilliant Blue-stained SDS-PAGE. Cross-linking efficiencies were calculated from a minimum of three independent gels using the full-length SecY band in lane 2 as 100% control, and lane 3 as background. B, direct fluorescent monitoring of the cross-linking of Cys-87 and Cys-287 of SecY by bBBr. IMVs containing Cys-less, Cys-87, Cys-286, or F286C/S87C SecYEG were incubated with bBBr. After 30 min at 37 °C, IMVs were treated with OmpT and cysteine cross-linking was analyzed by SDS-PAGE and in gel UV fluorescence using a cut-off filter of 520 nm. C, translocation of Texas Red-proOmpA into IMVs containing Cys-less (lanes 1–5) or F286C/S87C (lanes 8–12) SecYEG that had been incubated with different cross-linkers. Lanes 6 and 7 show a 20% Texas Red-proOmpA standard. D, quantification of the Texas Red-proOmpA translocation into IMVs containing F286C/S87C SecYEG treated with different cross-linkers. After cross-linking, the IMVs were incubated with (gray bars) or without (black bars) the reductant DTT, and used for translocation assays as described under “Experimental Procedures.” E, SecA translocation ATPase activity in the presence of IMVs containing F286C/S87C SecYEG treated with different cross-linkers. Values were corrected for the ATP hydrolysis in the absence of preprotein. Error bars represent S.D.

FIGURE 5.

Cys-286 and Cys-87 are in close proximity when a translocation intermediate is arrested in the SecYEG pore. A, a proOmpA-DHFR translocation intermediate blocks the translocation sites of IMVs containing F286C/S87C SecYEG. Unlabeled proOmpA-DHFR (800 μg/ml; suprastoichiometric to the number of translocation sites) and 10 mm NADPH + 10 μm methotexrate (lane 2) or proOmpA (lane 3) was incubated with F286C/S87C SecYEG IMVs (250 μg/ml) and SecA (20 μg/ml) for 10 min at 37 °C in the presence of 2 mm ATP. Membranes were re-isolated by ultracentrifugation, resuspended in translocation buffer with SecA (20 μg/ml), 2 mm ATP (and 10 mm NADPH and 10 μm methotexrate for proOmpA-DHFR), and assayed for a second round of translocation using Texas Red-proOmpA. Lane 1 shows a 10% Texas Red-proOmpA standard. B, F286C/S87C SecYEG IMVs charged with the proOmpA-DHFR translocation intermediate were incubated with the indicated cross-linkers. After 30 min at 37 °C, the protease OmpT was added to determine the amount of cross-linked SecY. Gels were stained with SYPRO Ruby. The positions of proOmpA-DHFR, SecY, OmpT, and the N-terminal fragment of SecY are indicated by arrows. Lanes 7 and 8 are controls showing the NaTT-oxidized F286C/S87C SecYEG IMVs treated with OmpT, and DTT-reduced F286C/S87C SecYEG IMVs, respectively. The cross-linking efficiency was calculated from three independent gels using the full-length SecY band in lane 8 as 100% control, and lane 2 as background.