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J. Biol. Chem., Vol. 283, Issue 23, 15709-15715, June 6, 2008

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Analysis of Polypeptide Movement in the SecY Channel during SecA-mediated Protein Translocation^*

From the Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

In bacteria most secretory proteins are transported across the plasma membrane by the interplay of the ATPase SecA with the translocation channel formed by the SecY complex; SecA uses cycles of ATP hydrolysis to "push" consecutive segments of a polypeptide substrate through the channel. Here we have addressed the mechanism of this process by following the fate of stalled translocation intermediates. These were generated by using a polypeptide substrate containing a bulky disulfide-bonded loop, thus preventing the final residues from passing through the channel. Protease protection experiments showed that the intermediates were stable in the presence of ATP and could complete translocation once the block was removed. The translocation intermediate was also stable when SecA associated with ATPS, a poorly hydrolyzable ATP analog, or ADP plus AlF₄, which mimics the transition state during ATP hydrolysis. In contrast, when SecA was in its ADP-bound state, the translocating polypeptide moved back into the cytosol, as indicated by the disappearance of the protected fragment. Backsliding was not significantly altered by deletion of the plug domain, a short helix in the center of the SecY channel, but it was slowed down when changes were introduced into the pore ring, the constriction of the hourglass-shaped channel. In all cases, backsliding was significantly slower than forward translocation. Together, these data suggest that SecA binds the polypeptide chain in its ATP state and releases it in the ADP state. The channel itself does not bind the polypeptide chain but provides "friction" that minimizes backsliding when ADP-bound SecA resets to "grab" the next segment of the substrate.

Received for publication, December 19, 2007 , and in revised form, March 21, 2008.

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^* This work was supported, in whole or in part, by National Institutes of Health Grant GM052586 (to T. A. R.). 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 and S2.

¹ Present address: Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115.

² Present address: Dept. of Pathology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611.

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³ To whom correspondence should be addressed: Howard Hughes Medical Inst. and Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0676; E-mail: tom_rapoport{at}hms.harvard.edu.

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This article has been cited by other articles:

				J. Gumbart and K. Schulten The Roles of Pore Ring and Plug in the SecY Protein-conducting Channel J. Gen. Physiol., December 1, 2008; 132(6): 709 - 719. [Abstract] [Full Text] [PDF]