A phospholipid acts as a chaperone in assembly of a membrane transport protein.
A mutant of Escherichia coli lacking phosphatidylethanolamine (PE) and a monoclonal antibody (mAb 4B1) directed against a conformationally sensitive epitope (4B1) of lactose permease were used to establish a novel role for a phospholipid in the assembly of a membrane protein. Epitope 4B1 is readily detectable in spheroplasts and right-side-out membrane vesicles from PE-containing but not from PE-deficient cells expressing lactose permease. Lactose permease from PE-containing membranes, but not from PE-deficient membranes, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis is also recognized by mAb 4B1. If total E. coli phospholipids or PE (but not phosphatidylcholine, phosphatidylglycerol, or cardiolipin) are blotted on nitrocellulose sheets (Eastern blot) prior to transfer of proteins from SDS-polyacrylamide gels, the permease from PE-deficient cells regains its recognition by mAb 4B1. Therefore, PE is required during assembly to form epitope 4B1, but, once formed, sufficient "conformational memory" is retained in the permease to either retain or reform this epitope in the absence of PE. Lactose permease lacking epitope 4B1 can be induced to form the epitope if partially denatured and then renatured in the presence of PE specifically. These results establish for the first time a role for PE as a molecular chaperone in the assembly of the lactose permease.
Bogdanov
M
M
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225, USA.
Sun
J
J
Kaback
H R
HR
Dowhan
W
W
D000911:Antibodies, Monoclonal / Q000276:immunology; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D018832:Molecular Chaperones / Q000502:physiology*; D009004:Monosaccharide Transport Proteins; D010714:Phosphatidylethanolamines / Q000502:physiology*; D011487:Protein Conformation; D027981:Symporters
D000911:Antibodies, Monoclonal; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D009004:Monosaccharide Transport Proteins; D010714:Phosphatidylethanolamines; D027981:Symporters; C021499:lactose permease