Covalently Dimerized SecA Is Functional in Protein Translocation

Jeanine De Keyzer,Arnold J.M Driessen,Ben De Kruijff,Niels Nijstad,Chris Van Der Does,Nico Nouwen,Eli O Van Der Sluis,Robin E.J Spelbrink

doi:10.1074/jbc.m506157200

Abstract

The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.

Highlights

In solution, SecA exist as a homodimer (5) that equilibrates with its monomeric form in a temperature, salt- and protein concentration-dependent manner (6)
SecYEG Overexpression Results in an Increased Level of Dimeric SecA— After solubilization of inner membrane vesicles (IMVs) at room temperature with SDS, membraneassociated SecA migrates on SDS-PAGE as a mixture of monomers and dimers (25)
Overexpression of SecYEG results in a dramatic increase in the level of SecA dimers (Fig. 3, lane YEGϩ), demonstrating that SecYEG stabilizes a dimeric state of SecA that is resistant to room temperature SDS

Summary

Covalently Dimerized SecA Is Functional in Protein Translocation*

Recent studies with a monomeric SecA mutant carrying a deletion of only the first 11 amino-terminal amino acids (SecA(⌬2–11)) show that this protein is entirely inactive for in vitro preprotein translocation (9, 19) and unable to complement the growth defect of a SecA temperature-sensitive strain (19). We have studied the functional requirement for the postulated dissociation of the SecA dimer by making use of covalently linked SecA dimers This covalently linked SecA dimer no longer supports SecB-dependent protein translocation, it still binds the SecYEG complex with high affinity and displays normal preprotein-stimulated ATPase and translocation activity in the absence of SecB. These results demonstrate that SecA can bind SecYEG as a functional dimer

EXPERIMENTAL PROCEDURES

RESULTS

TABLE ONE

DISCUSSION