PHOSPHOLIPID-ASSISTED PROTEIN-FOLDING - PHOSPHATIDYLETHANOLAMINE IS REQUIRED AT A LATE STEP OF THE CONFORMATIONAL MATURATION OF THE POLYTOPIC MEMBRANE-PROTEIN LACTOSE PERMEASE

Previously we presented evidence that phosphatidylethanolamine (PE) ac ts as a molecular chaperone in the folding of the polytopic membrane p rotein lactose permease (LacY) of Escherichia coil, Here we provide mo re definitive evidence supporting the chaperone properties of PE. Memb rane insertion of LacY prevents its irreversible aggregation, and PE p articipates in a late step of conformational maturation. The temporal requirement for PE was demonstrated in vitro using a coupled translati on-membrane insertion assay that allowed the separation of membrane in sertion from phospholipid-assisted folding. LacY was folded properly, as assessed by recognition with conformation-specific monoclonal antib odies, when synthesized in the presence of PE-containing inside-out me mbrane vesicles (IOVs) or in the presence of IOVs initially lacking PE but supplemented with PE synthesized in vitro either co- or post-tran slationally. The presence of IOVs lacking PE and containing anionic ph ospholipids or no addition of IOVs resulted in misfolded or aggregated LacY, respectively. Therefore, critical folding steps occur after mem brane insertion dependent on the interaction of LacY with PE to preven t illicit interactions which lead to misfolding of LacY.