Investigating the SecY plug movement at the SecYEG translocation channel

Abstract

Protein translocation occurs across the energy-conserving bacterial membrane at the SecYEG channel. The crystal structure of the channel has revealed a possible mechanism for gating and opening. This study evaluates the plug hypothesis using cysteine crosslink experiments in combination with various allelic forms of the Sec complex. The results demonstrate that the SecY plug domain moves away from the center of the channel toward SecE during polypeptide translocation, and further show that the translocation-enhancing prlA3 mutation and SecG subunit change the properties of channel gating. Locking the plug in the open state preactivates the Sec complex, and a super-active translocase can be created when combined with the prlA4 mutation located in the pore of the channel. Dimerization of the Sec complex, which is essential for translocase activity, relocates the plug toward the open position. We propose that oligomerization may result in SecYEG cooperative interactions important to prime the translocon function.

Figure 1

Schematic cross-sectional view of the closed Sec channel depicting the plug, the pore and the amino-acyl positions engineered in this study. The prlA4 (I408N) and prlA3 (F67C) mutations are labeled A4 and A3, respectively. Drawing kindly provided by Ms Kailun Jiang.

Figure 2

Crosslinking between the SecY plug and SecE. (A) About 1 μg of IMVs enriched for the cysteine-mutagenized SecYE_HAG (odd lanes) or SecY_HAEG (even lanes) complexes were solubilized with the Laemmli-sample buffer (without reducing agent), then analyzed by 13% SDS–PAGE and transferred onto polyvinyldene difluoride membrane for immunostaining with anti-HA antibodies. (B) IMVs enriched for the SecYE_HAG complex carrying the mutations SecY–F67C or SecY–S68C in combination with SecE–S120C (labeled F67C or S68C, respectively) were incubated with reducing (DTT) or oxidizing (Cu²⁺(phe)₃) agent for 5 min at RT at the indicated final concentration. Unreacted cysteines were blocked with NEM (8 mM final, 10 min, RT) prior to IMV solubilization and analysis by SDS–PAGE and Western blotting. (C) IMVs enriched for the SecY–F67C/SecE–S120C SecYE_HAG complex were first oxidized with 400 μM Cu²⁺(phe)₃ or reduced with 4 mM DTT, then the cysteines modified by NEM (8 mM final, 5 min, RT). IMVs were solubilized with 0.2% dodecyl-maltoside (DDM) and the oligomeric state of the Sec complex revealed by blue native gel electrophoresis (4–13%) and Western blotting.

Figure 3

SecY-plug movement during preprotein translocation. (A) IMVs enriched for the WT or cysteine-mutagenized SecYE_HAG complexes were tested for their translocase activity using the preprotein substrate [¹²⁵I]proOmpA, as described in Materials and methods. (B) IMVs were incubated in the same conditions as in (A), but using unlabeled proOmpA. Unreacted cysteines were blocked with NEM (8 mM, 5 min, RT) prior to IMV solubilization and analysis by SDS–PAGE and Western blotting with anti-HA antibodies. (C, D) IMVs were incubated in the same conditions as in (B), but using the indicated concentration of proOmpA, or using a preprotein substrate with a deleted (OmpA) or altered (LpK) leader peptide.

Figure 4

Relation between translocase activity and dynamic of the plug. (A) IMVs enriched for the indicated Sec complexes were tested for their translocase activity using [¹²⁵I]proOmpA, as descibed in Materials and methods. In all, 20% of [¹²⁵I]proOmpA added to the reaction was loaded on the gel as standard. (B) The same set of IMVs was analyzed for the SecY–SecE crosslinking efficiency either without (left panel) or with oxidation with 80 μM Cu²⁺(phe)₃ (5 min, RT; right panel), followed by SDS–PAGE and Western blotting with anti-HA antibodies. (C) The translocation-dependent movement of the SecY plug was tested using the IMVs enriched for the SecY–S68C/SecE–S120C complex depleted for SecG. (D) The IMVs were incubated in the presence of the cystein-reactive crosslinker BMOE (0.1 mM final, 5 min, RT), before analysis by SDS–PAGE and Western blotting.

Figure 5

Stabilization of the plug in the open state increases the translocase activity. (A) IMVs enriched for the SecY–S68C/SecE–S120C complex were oxidized with Cu²⁺(phe)₃ at the indicated final concentration (5 min, RT), then re-isolated by ultra-centrifugation. The left lane (−) corresponding to IMVs treated with 1 mM DTT. After IMV resuspension, the amount of SecY–SecE crosslinks obtained was monitored by SDS–PAGE and Western blotting (top panel), while the translocase activity was measured using [¹²⁵I]proOmpA as substrate (8 min, 37°C; bottom panel). (B, C) IMVs enriched for the indicated Sec complexes were treated and analysed as in (A). The percentage of translocated proOmpA (% translo) is indicated.

Figure 6

Sec mutations at R357 relocate the plug toward its closed state. (A) IMVs enriched for the SecY–S68C/SecE–S120C complex (labeled ‘WT'), or carrying the mutations R357E or RPG357EDP, were analyzed for the translocation-dependent plug movement (10 min, 37°C). To increase the detection of the SecY–SecE crosslinks, IMVs were incubated with 80 μM Cu²⁺(phe)₃ (5 min, RT) at the end of the translocation reaction. (B) The same set of IMVs was analyzed for the amount of SecY–SecE crosslinks occurring in the absence of preprotein translocation and using the indicated final concentration of oxidizing agent. (C) The same set of IMVs was incubated with the cysteine-reactive crosslinkers BMOE and BMH (0.1 mM final; 5 min, RT), before analysis by SDS–PAGE and Western blotting.

Figure 7

Sec mutations at R357 monomerize the Sec complex. (A) IMVs enriched for the SecY–S68C/SecE–S120C complex and carrying the mutations R357E or RPG357EDP were solubilized with DDM (0.06%). The membrane extracts were analyzed by BN–PAGE and Western blotting using anti-HA antibodies. The monomeric (M) and dimeric (D) forms of the Sec complex are indicated. (B) The R357-mutagenized Sec complexes were purified and radiolabeled, as described in Materials and methods, then analyzed by BN–PAGE and autoradiography. (C) The reversible dissociation of the ¹²⁵I-labeled and purified Sec complex was analyzed by BN–PAGE. A stock solution of [¹²⁵I]YEG (in 0.2% DDM) was diluted on ice to the indicated detergent concentration, as previously described (Bessonneau et al, 2002). (D) IMVs enriched for the SecYE_HAG complex carrying the mutation SecE–L106C and R357E or RPG357EDP were incubated with Cu²⁺(phe)₃ (80 μM final; 5 min, RT) or with the cysteine-reactive crosslinker BMOE and BMH (0.1 mM final; 5 min, RT). Unreacted cysteines were blocked with NEM (8 mM; 5 min, RT) before analysis of the crosslinked products by SDS–PAGE and Western blotting using anti-HA antibodies.