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Volume 271, Number 36, Issue of September 6, 1996 pp. 22256-22261
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Chaperonin-promoted Post-translational Membrane Insertion of a Multispanning Membrane Protein Lactose Permease

(Received for publication, March 28, 1996, and in revised form, July 8, 1996)

From the Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel

Using an in vitro membrane-free translation system from Escherichia coli, it is shown that chaperonin GroEL added cotranslationally interacts with newly synthesized lactose permease (LacY), a polytopic membrane protein, thereby preventing aggregation. Subsequently, when the isolated GroEL-LacY complex is incubated with inverted membrane vesicles, the permease is inserted into the membrane in a MgATP-dependent manner. Post-translational membrane insertion is also observed when aggregation of newly synthesized LacY is prevented by addition of the nonionic detergent n-dodecyl-,D-maltoside during translation in place of GroEL. No membrane integration occurs with right-side-out vesicles, indicating that LacY interacts specifically only with the cytosolic face of the membrane. Ligand thiodigalactoside protection against alkylation of the Cys-148 residue in the permease shows proper post-translational insertion. Moreover, limited proteolysis of soluble LacY either complexed with GroEL or in detergent indicates that the newly synthesized protein assumes a conformation that is comparable to that of native, membrane-embedded permease prior to insertion into the membrane.

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