Abstract
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.
Highlights
Synthesized in vitro [4, 5] and inserted cotranslationally into INV [5]
There is no evidence for post-translational insertion of LacY or other polytopic membrane proteins in E. coli, and, generally, little is known about biogenesis of polytopic membrane proteins
In order to determine whether or not the LacY bound to GroEL is able to interact with membranes, sucrose gradient fractions containing the GroEL-LacY complex were incubated with E. coli INV at 37 °C
Summary
LacY, lactose permease; LacY(Cys148), the permease mutant containing a single native Cys residue at position 148; INV, inverted plasma membrane vesicle(s); DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; DM, n-dodecyl-,D-maltoside; TEA, triethanolamine; TDG, thiodigalactoside (-D-galactopyranosyl 1-thio--D-galactopyranoside); NEM, N-ethylmaleimide; NEM-biotin, 3-(N-maleimidopropionyl)-biocytin; avidin-agarose, avidin immobilized on 6% beaded agarose; Tricine, N-tris(hydroxymethyl)methylglycine; CAPS, 3-(cyclohexylamino)propanesulfonic acid. Membrane proteins are very hydrophobic and tend to aggregate in membrane-free translation systems [5]. In this respect, molecular chaperones may act to prevent aggregation. Molecular chaperones may act to prevent aggregation It has been demonstrated by Bochkareva et al [6] that chaperonin GroEL maintains the translocation-competent unfolded state of newly synthesized pre--lactamase and that translocation across the membrane requires MgATP-promoted discharge of the protein from GroEL. The physiological role of molecular chaperones, including GroEL, is to interact with transiently exposed hydrophobic patches and to promote folding, assembly, or secretion of various proteins mainly by preventing aggregation [7,8,9]. The possible role of GroEL or other molecular chaperones in the targeting and insertion of polytopic membrane proteins into the membrane is an open question
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