Chaperonin-promoted post-translational membrane insertion of a multispanning membrane protein lactose permease.
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-beta,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.[1]References
- Chaperonin-promoted post-translational membrane insertion of a multispanning membrane protein lactose permease. Bochkareva, E., Seluanov, A., Bibi, E., Girshovich, A. J. Biol. Chem. (1996) [Pubmed]
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