The Bacterial ATPase SecA Functions as a Monomer in Protein Translocation

Eran Or,Dana Boyd,Stéphanie Gon,Jonathan Beckwith,Tom Rapoport

doi:10.1074/jbc.m413947200

Eran Or, Dana Boyd + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m413947200

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Abstract

The ATPase SecA drives the post-translational translocation of proteins through the SecY channel in the bacterial inner membrane. SecA is a dimer that can dissociate into monomers under certain conditions. To address the functional importance of the monomeric state, we generated an Escherichia coli SecA mutant that is almost completely monomeric (>99%), consistent with predictions from the crystal structure of Bacillus subtilis SecA. In vitro, the monomeric derivative retained significant activity in various assays, and in vivo, it sustained 85% of the growth rate of wild type cells and reduced the accumulation of precursor proteins in the cytoplasm. Disulfide cross-linking in intact cells showed that mutant SecA is monomeric and that even its parental dimeric form is dissociated. Our results suggest that SecA functions as a monomer during protein translocation in vivo.

Highlights

Many bacterial proteins are transported post-translationally across the inner membrane by the Sec machinery, which consists of two essential components (1– 4)
Our results suggest that SecA functions as a monomer during protein translocation in vivo
The x-ray structure of B. subtilis SecA (7) later showed that four of the six residues were close to the interface between the monomers

Summary

Introduction

Many bacterial proteins are transported post-translationally across the inner membrane by the Sec machinery, which consists of two essential components (1– 4). SecA interacts with the SecY channel (8) and with acidic phospholipids (9 –11) and with both the signal sequence and the mature part of a substrate protein (12). It binds the chaperone SecB, which ushers some precursor proteins to SecA (8, 13, 14). The equilibrium favors dimers, it is shifted almost completely toward monomers in the presence of membranes containing acidic phospholipids or upon binding to the SecY complex (21). The previous results do not exclude models in which SecA cycles between monomeric and oligomeric states during the translocation of a polypeptide chain (22, 23). Our results suggest that SecA functions as a monomer

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