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Journal of Bacteriology, November 2008, p. 7302-7307, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00593-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Reexamination of the Role of the Amino Terminus of SecA in Promoting Its Dimerization and Functional State

Sanchaita Das,¹ Elizabeth Stivison,¹ Ewa Folta-Stogniew,² and Donald Oliver^1*

Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459,¹ W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University School of Medicine, New Haven, Connecticut 06520²

Received 29 April 2008/ Accepted 12 August 2008

The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a monomer or dimer during this dynamic process. Here we reexamined the roles of the amino and carboxyl termini of SecA in promoting its dimerization and functional state by examining three secA mutants and the corresponding proteins: SecA8 lacking residues 2 to 8, SecA11 lacking residues 2 to 11, and SecA11/N95 lacking both residues 2 to 11 and the carboxyl-terminal 70 residues. We demonstrated that whether SecA11 or SecA11/N95 was functional for promoting cell growth depended solely on the vivo level of the protein, which appeared to govern residual dimerization. All three SecA mutant proteins were defective for promoting cell growth unless they were highly overproduced. Cell fractionation revealed that SecA11 and SecA11/N95 were proficient in membrane association, although the formation of integral membrane SecA was reduced. The presence of a modestly higher level of SecA11/N95 in the membrane and the ability of this protein to form dimers, as detected by chemical cross-linking, were consistent with the higher level of secA expression and better growth of the SecA11/N95 mutant than of the SecA11 mutant. Biochemical studies showed that SecA11 and SecA11/N95 had identical dimerization defects, while SecA8 was intermediate between these proteins and wild-type SecA in terms of dimer formation. Furthermore, both SecA11 and SecA11/N95 were equally defective in translocation ATPase specific activity. Our studies showed that the nonessential carboxyl-terminal 70 residues of SecA play no role in its dimerization, while increasing the truncation of the amino-terminal region of SecA from 8 to 11 residues results in increased defects in SecA dimerization and poor in vivo function unless the protein is highly overexpressed. They also clarified a number of conflicting previous reports and support the essential nature of the SecA dimer.

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* Corresponding author. Mailing address: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459. Phone: (860) 685-3556. Fax: (860) 685-2141. E-mail: doliver{at}wesleyan.edu

Published ahead of print on 22 August 2008.

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Journal of Bacteriology, November 2008, p. 7302-7307, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00593-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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