Document Type

Article

Publication Date

4-1-1997

Publication Title

Proceedings of the National Academy of Sciences of the United States of America

Abstract

SecA, a 102-kDa hydrophilic protein, couples the energy of ATP binding to the translocation of preprotein across the bacterial inner membrane. SecA function and topology were studied with metabolically labeled [35S]SecA and with inner membrane vesicles from cells that overex- pressed SecYEGDFyajC, the integral domain of preprotein translocase. During translocation in the presence of ATP and preprotein, a 65-kDa N-terminal domain of SecA is protected from proteolytic digestion through insertion into the mem- brane, as previously reported for a 30-kDa C-terminal domain [Economou, A. & Wickner, W. (1994) Cell 78, 835–843]. Insertion of both domains occurs at saturable SecYEGDFyajC sites and is rapidly followed by deinsertion. SecA also asso- ciates nonsaturably and unproductively with lipid. In the presence of ATP, yet without involvement of preprotein or SecYEG, lipid-bound SecA forms domains that are protease- resistant and that remain so even upon subsequent membrane disruption. Unlike the [35S]SecA that inserts into the mem- brane at SecYEGDFyajC as it promotes preprotein translo- cation, lipid-associated [35S]SecA does not chase from its protease-resistant state upon the addition of excess SecA. The finding that two domains of SecA (which together represent most regions of the polypeptide chain) cycle into the mem- brane during preprotein translocation, as well as the distinc- tion between the membrane association of SecA at transloca- tion sites of SecYEGDFyajC and at nonproductive lipid sites, are fundamental to the study of the role of SecA in preprotein movement.

DOI

10.1073/pnas.94.11.5574

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