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J. Biol. Chem., Vol. 282, Issue 7, 4661-4668, February 16, 2007

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In Vivo Membrane Topology of Escherichia coli SecA ATPase Reveals Extensive Periplasmic Exposure of Multiple Functionally Important Domains Clustering on One Face of SecA^*

From the Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459

The Sec-dependent protein translocation pathway promotes the transport of proteins into or across the bacterial plasma membrane. SecA ATPase has been shown to be a nanomotor that associates with its protein cargo as well as the SecYEG channel complex and to undergo ATP-driven cycles of membrane insertion and retraction that promote stepwise protein translocation. Previous studies have shown that both the 65-kDa N-domain and 30-kDa C-domain of SecA appear to undergo such membrane cycling. In the present study we performed in vivo sulfhydryl labeling of an extensive collection of monocysteine secA mutants under topologically specific conditions to identify regions of SecA that are accessible to the trans side of the membrane in its membrane-integrated state. Our results show that distinct regions of five of six SecA domains were labeled under these conditions, and such labeling clusters to a single face of the SecA structure. Our results demarcate an extensive face of SecA that interacts with SecYEG and is in fluid contact with the protein-conducting channel. The observed domain-specific labeling patterns should also provide important constraints on model building efforts in this dynamic system.

Received for publication, November 22, 2006

^* This work was supported by Grant GM42033 from NIGMS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ To whom correspondence should be addressed: Molecular Biology and Biochemistry Dept., Wesleyan University, Middletown, CT 06457. Tel.: 860-685-3556; Fax: 860-685-2141; E-mail: doliver{at}wesleyan.edu.

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