Volume 272, Number 32, Issue of August 8, 1997 pp. 20082-20087
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

In Vitro Analysis of the Stop-transfer Process during Translocation across the Cytoplasmic Membrane of Escherichia coli

(Received for publication, March 14, 1997, and in revised form, May 12, 1997)

Ken Sato ^§ , Hiroyuki Mori ^§ , Masasuke Yoshida , Mitsuo Tagaya ^§ and Shoji Mizushima ^§

From the  Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226 and ^§ School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji, Tokyo 192-03, Japan

In this study, using a derivative of proOmpA containing an artificial stop-transfer sequence (proOmpA2xH1), we analyzed the process of stop-transfer during translocation across the cytoplasmic membrane of Escherichia coli. ProOmpA2xH1 did not interfere with the transit of wild-type proOmpA. When proOmpA2xH1 was anchored in the membrane, membrane-inserted SecA was deinserted with the reversion of the inverted topology of SecG. Cross-linking experiments revealed that the anchored proOmpA2xH1 that does not interact with either SecY or SecA. These results, taken together, suggest that proOmpA2xH1 leaves the translocation pathway by means of a specific interaction between the stop-transfer sequence and the translocational channel.

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