Multiple SecA Molecules Drive Protein Translocation across a Single Translocon with SecG Inversion

Kazuhiro Morita,Hajime Tokuda,Ken-Ichi Nishiyama

doi:10.1074/jbc.m111.301754

Kazuhiro Morita, Hajime Tokuda + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m111.301754

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Abstract

SecA is a translocation ATPase that drives protein translocation. D209N SecA, a dominant-negative mutant, binds ATP but is unable to hydrolyze it. This mutant was inactive to proOmpA translocation. However, it generated a translocation intermediate of 18 kDa. Further addition of wild-type SecA caused its translocation into either mature OmpA or another intermediate of 28 kDa that can be translocated into mature by a proton motive force. The addition of excess D209N SecA during translocation caused a topology inversion of SecG. Moreover, an intermediate of SecG inversion was identified when wild-type and D209N SecA were used in the same amounts. These results indicate that multiple SecA molecules drive translocation across a single translocon with SecG inversion. Here, we propose a revised model of proOmpA translocation in which a single catalytic cycle of SecA causes translocation of 10-13 kDa with ATP binding and hydrolysis, and SecG inversion is required when the next SecA cycle begins with additional ATP hydrolysis.

Highlights

Preproteins are translocated across membranes by SecA ATPase through SecYEG with dynamic structure changes, including SecG inversion
We found that D209N SecA generated a translocation intermediate of proOmpA that can be translocated into mature by wildtype SecA and proton motive force (PMF), indicating that multiple SecA molecules function on a single translocon
Because SecG inversion is controlled by the ATPase activity of SecA, we examined the effects of the SecA mutants on SecG inversion

Summary

Introduction

Preproteins are translocated across membranes by SecA ATPase through SecYEG with dynamic structure changes, including SecG inversion. Results: A SecA mutant, together with wild-type SecA, caused SecG inversion with accumulation of translocation intermediates. Conclusion: Multiple SecA molecules drive protein translocation across a translocon with SecG inversion. D209N SecA, a dominant-negative mutant, binds ATP but is unable to hydrolyze it This mutant was inactive to proOmpA translocation. An intermediate of SecG inversion was identified when wild-type and D209N SecA were used in the same amounts These results indicate that multiple SecA molecules drive translocation across a single translocon with SecG inversion. We propose a revised model of proOmpA translocation in which a single catalytic cycle of SecA causes translocation of 10 –13 kDa with ATP binding and hydrolysis, and SecG inversion is required when the SecA cycle begins with additional ATP hydrolysis

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Results

Conclusion