E. Bochkareva et al., CHAPERONIN-PROMOTED POSTTRANSLATIONAL MEMBRANE INSERTION OF A MULTISPANNING MEMBRANE-PROTEIN LACTOSE PERMEASE, The Journal of biological chemistry, 271(36), 1996, pp. 22256-22261
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.