The SecYEG preprotein translocation channel is a conformationally dynamic and dimeric structure

Abstract

Escherichia coli preprotein translocase comprises a membrane-embedded trimeric complex of SecY, SecE and SecG. Previous studies have shown that this complex forms ring-like assemblies, which are thought to represent the preprotein translocation channel across the membrane. We have analyzed the functional state and the quaternary structure of the SecYEG translocase by employing cross-linking and blue native gel electrophoresis. The results show that the SecYEG monomer is a highly dynamic structure, spontaneously and reversibly associating into dimers. SecG-dependent tetramers and higher order SecYEG multimers can also exist in the membrane, but these structures form at high SecYEG concentration or upon overproduction of the complex only. The translocation process does not affect the oligomeric state of the translocase and arrested preproteins can be trapped with SecYEG or SecYE dimers. Dissociation of the dimer into a monomer by detergent induces release of the trapped preprotein. These results provide direct evidence that preproteins cross the bacterial membrane, associated with a translocation channel formed by a dimer of SecYEG.

Fig. 1. BN– and SDS–PAGE analysis of the purified SecYEG complex. (A) Coomassie Blue staining of the purified SecYEG complex and (B) autoradiography of the ¹²⁵I-labelled SecYEG complex (∼30 000 c.p.m., ∼20 ng) separated by 17% SDS–PAGE. (C) Autoradiography of the [¹²⁵I]SecYEG complex analyzed by linear gradient BN–PAGE (6–15%). The radiolabelled SecYEG complex was diluted in TSG buffer containing the indicated amount of detergent and incubated on ice or at 37°C for 5 min before loading on to the gel. The high molecular weight markers are ¹²⁵I-labelled ferritin (440 and 880 kDa), catalase (232 kDa) and BSA (66 and 132 kDa). The aggregates of SecYEG proteins remain in the loading area of the gel (top).

Fig. 2. Cross-linking analysis of the purified SecYEG complex. (A) Separation of the DSS-linked SecYEG proteins by 10% SDS–PAGE. The [¹²⁵I]SecYEG complex was prepared in CL buffer (50 mM HEPES–KOH pH 8.0, 50 mM KCl) with 0.02 or 0.2% DDM. The DSS reagent was dissolved in dimethylsulfoxide (DMSO) and incubated with the SecYEG complex at the indicated final concentration. After 30 min at room temperature, the cross-linker was quenched by Tris–HCl pH 8.0 (50 mM final) and the cross-linked products were dissolved in Laemmli sample buffer, followed by gel electrophoresis and autoradiography. (B) Separation of the DSS-linked SecYEG complex by 6–15% BN–PAGE. The [¹²⁵I]SecYEG complex was prepared in CL buffer with 0.02% DDM and incubated with the indicated concentration of DSS, as described above. The cross-linked products were dissolved in BN-sample buffer with or without 0.05% SDS. For reference, the native and heat-treated SecYEG complexes were loaded on the same gel (lanes 1 and 2). Incubation with DMSO does not induce the dissociation of the SecYEG dimers (lane 12).

Fig. 3. Dynamic dimeric association of the SecYEG complex. (A) Reversible dissociation of the native SecYEG dimers. To vary the amount of detergent while keeping the SecYEG concentration constant, a stock solution of [¹²⁵I]SecYEG complex (∼1.5 × 10⁶ c.p.m./µg) was first prepared in TSG buffer with 0.02 or 0.2% DDM. Aliquots (∼30 000 c.p.m.) were then diluted on ice to the desired DDM concentration and analyzed by BN–PAGE and autoradiography. (B) Irreversible dissociation of the heat-treated SecYEG complex. The [¹²⁵I]SecYEG complex was prepared in TSG buffer with 0.2% DDM and incubated for 5 min at 37°C (lane 2). The heat-treated SecYEG complex was then diluted on ice to the desired DDM concentration and analyzed by BN–PAGE and autoradiography.

Fig. 4. The oligomeric form of the SecYEG complex in the membranes. (A) Immunodetection of the Sec complexes in membranes enriched for the SecYE_HAG or SecYE_HA proteins. IMVs (∼10 µg) were solubilized on ice in TSG buffer with the indicated detergent concentration. Protein aliquots (∼0.2 µg for SecYE_HAG and ∼0.1 µg for SecYE_HA IMVs) were analyzed by BN–PAGE and immunostained with anti-HA IgG. For reference, ∼20 ng of the purified SecYE_hisG complex was loaded on the same gel and immunostained with anti-SecG IgG (left panel). (B) Thermal stability of the oligomeric SecYEG complexes. IMVs (∼10 µg) enriched for the SecYE_HAG or SecY₄E_HAG complexes were solubilized with 0.05% DDM and incubated at the indicated temperature for 5 min. Aliquots were then analyzed by BN–PAGE and immunostained with anti-HA IgG. (C) Oligomeric state of SecYEG during translocation. The translocation assays were performed in 100 µl as described in Materials and methods using proteoliposomes (0.35 µg of SecYEG proteins) or IMVs (∼10 µg of proteins), then solubilized using the indicated concentrations of DDM. Aliquots (∼0.2 µg of proteins) were analyzed by BN–PAGE and immunostained with anti-SecG IgG.

Fig. 5. Multimerisation of the SecYEG complex. (A) Nucleation of the purified SecYEG complex. A solution of purified SecYEG (12 mg/ml) was diluted to the desired protein concentration in TSG buffer-0.1% DDM. Aliquots were analyzed by BN–PAGE (gel 4–15% left panel; and 4–12% right panel) and stained with Coomassie Blue. The SecYEG complex was also incubated with 0.05% SDS where indicated on the figure. (B) Immunodetection of the SecYEG complex in wild-type membranes. IMVs (∼10 µg) were solubilized in TSG buffer with 0.2% DDM. Aliquots (1 µg of protein for the wild-type membranes) were diluted four times in TSG buffer containing the indicated concentration of DDM, then analyzed by BN–PAGE and immunostaining with anti-SecG IgG. (C) Oligomeric state of SecYEG during translocation in wild-type IMVs. The translocation assays were performed in 100 µl as described in Materials and methods using wild-type IMVs (∼10 µg), then solubilized on ice using the indicated concentrations of DDM. Aliquots were analyzed by BN–PAGE and immunostained with anti-SecG IgG.

Fig. 6. The active preprotein translocation channel is comprised of a dimeric SecYEG complex. (A) Creation of trapped translocation intermediate. [¹²⁵I]proOmpA–BPTI was incubated with SecYEG- or SecYE-enriched IMVs in 100 µl of TL buffer, as described in Materials and methods. Addition of 2 mM DTT after 10 min of incubation allowed proOmpA to complete translocation across the membrane (lane 4). Translocation reactions were treated with proteinase K (1 mg/ml, 15 min on ice), then TCA-precipitated and analyzed by 12% SDS–PAGE and autoradiography. (B) Immunoprecipitation of a SecYEG–proOmpA complex. The translocation reactions described in (A) were scaled up to 250 µl, then layered over an equal volume of 0.2 M sucrose and centrifuged (100 000 g, 10 min, 4°C). Membranes were resuspended in half-volume TSG buffer (125 µl) and solubilized with 0.1% DDM (30 min, 4°C). The same reaction loaded in lane 5 was also incubated further with either 0.1% DDM for 5 min at 37°C (lane 7), 0.4% DDM for 30 min at 4°C (lane 8) or 0.4% DDM for 30 min at 4°C, then diluted four times to 0.1% DDM (lane 9). After centrifugation (142 000 g, 30 min, 4°C), the SecYEG complex was immunoprecipitated with anti-SecG protein A–Sepharose beads. Co-immunoprecipitated proOmpA was monitored by SDS–PAGE and autoradiography. (C) Molecular weight of the SecYEG–proOmpA complex. Aliquots of the detergent extracts prepared in (B) were directly loaded on BN–PAGE and analyzed by autoradiography. For the M_r reference, the purified [¹²⁵I]SecYEG complex in TSG–0.02% DDM (lane EYG) and [¹²⁵I]proOmpA–BPTI (left lane) were loaded on the same gel.

Fig. 7. The dissociation of the SecYEG dimer promotes the release of the trapped translocation intermediate. Radiochemical [¹²⁵I]proOmpA– BPTI (∼50 000 c.p.m.) was incubated with SecYEG-enriched IMVs (10 µg) in 100 µl of TL buffer in the presence or the absence of ATP, as described in Materials and methods, then layered over an equal volume of 0.2 M sucrose and centrifuged (100 000 g, 10 min, 4°C). Membranes were resuspended in half-volume TSG buffer and solubilized with the indicated concentration of DDM (30 min, 4°C). (A) Aliquots (∼2 µg proteins) were loaded on BN–PAGE and analyzed by autoradiography or (B) by western blotting (∼0.4 µg aliquots), followed by immunostaining with anti-SecG antibodies.