Re-examination of the Role of the Amino-Terminus of SecA in Promoting Its Dimerization and Functional State

Abstract

The SecA nanomotor promotes protein translocation in Eubacteria by binding both protein cargo and the protein-conducting channel and undergoing ATP-driven conformation cycles that drive this process. Conflicting reports exist as to whether SecA functions as a monomer or dimer during this dynamic process.Here we re-examine the role of amino and carboxyl termini of SecA in promoting its dimerization and functional state by examining three secA mutants and their respective proteins: SecAD8 lacking residues 2-8, SecAD11 lacking residues 2-11, and SecAD11/N95 lacking both residues 2-11 and its carboxyl-terminal 70 residues. We demonstrate that whether or not SecAD11 or SecAD11/N95 was functional for promoting cell growth depends solely on their vivo levels that appear to govern residual dimerization. Cell fractionation revealed that SecAD11 and SecAD11/N95 were still proficient in membrane association, although they were reduced in the formation of integral membrane SecA. The presence of a modestly higher level of SecAD11/N95 in the membrane and its ability to form dimers as detected by chemical crosslinking were consistent with the higher secA expression level and better growth property of this mutant compared to secAD11. Biochemical studies showed that SecAD11 and SecAD11/N95 displayed identical dimerization defects, while SecAD8 was intermediate between these proteins and wildtype SecA. Furthermore, both SecAD11 and SecAD11/N95 were equally defective in their translocation ATPase specific activity. Our studies show that the non-essential carboxyl-terminal 70 residues of SecA play no role in its dimerization, while increasing truncation of the amino-terminal region of SecA from 8 to 11 residues results in an increasing defect in SecA dimerization and poor in vivo function unless highly overexpressed and also clarify a number of conflicting reports in the literature and support the essential nature of the SecA dimer.