CHAPERONIN-PROMOTED POSTTRANSLATIONAL MEMBRANE INSERTION OF A MULTISPANNING MEMBRANE-PROTEIN LACTOSE PERMEASE

Using an in vitro membrane-free translation system from Escherichia co li, it is shown that chaperonin GroEL added cotranslationally interact s with newly synthesized lactose permease (LacY), a polytopic membrane protein, thereby preventing aggregation, Subsequently, when the isola ted GroEL-LacY complex is incubated with inverted membrane vesicles, t he permease is inserted into the membrane in a MgATP-dependent manner. Post-translational membrane insertion is also observed when aggregati on of newly synthesized LacY is prevented by addition of the nonionic detergent n-dodecyl-beta,D-maltoside during translation in place of Gr oEL. No membrane integration occurs with right-side-out vesicles, indi cating that LacY interacts specifically only with the cytosolic face o f the membrane. Ligand thiodigalactoside protection against alkylation of the Cys-148 residue in the permease shows proper posttranslational insertion. Moreover, limited proteolysis of soluble LacY either compl exed with GroEL or in detergent indicates that the newly synthesized p rotein assumes a conformation that is comparable to that of native, me mbrane-embedded permease prior to insertion into the membrane.