Intramolecular dislocation of the COOH terminus of the lac carrier protein in reconstituted proteoliposomes.
A dodecapeptide corresponding to the carboxyl terminus of the lac carrier of Escherichia coli was synthesized, coupled to thyroglobulin, and the conjugate was used to generate site-directed polyclonal antibodies. The antibodies react with the carboxyl-terminal peptide and with the lac carrier protein, while monoclonal antibody 4B1 reacts with intact lac carrier protein, but not with the carboxyl-terminal peptide. Antibody 4B1 binds preferentially to right-side-out membrane vesicles relative to inside-out vesicles, confirming the presence of the 4B1 epitope on the periplasmic surface of the membrane. Alternatively, anti-carboxyl-terminal antibody binds preferentially to inside-out vesicles, demonstrating that the carboxyl terminus of the lac carrier protein is on the cytoplasmic surface. Surprisingly, both antibodies bind to proteoliposomes reconstituted with purified lac carrier protein, and quantitative binding assays indicate that the epitopes are equally accessible. When proteoliposomes containing purified lac carrier protein are digested with carboxypeptidases A and B, binding of anti-carboxyl-terminal antibodies decreases by greater than 80%, while binding of antibody 4B1 and various transport activities remain essentially unchanged. It is suggested that during reconstitution, the lac carrier protein undergoes intramolecular dislocation of the carboxyl terminus with no significant effect on its catalytic activity.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001693:Biological Transport, Active; D002268:Carboxypeptidases; D002462:Cell Membrane / Q000648:ultrastructure; D004926:Escherichia coli / Q000648:ultrastructure*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000276:immunology; D009004:Monosaccharide Transport Proteins; D011487:Protein Conformation; D011510:Proteolipids; D013329:Structure-Activity Relationship; D027981:Symporters
D000911:Antibodies, Monoclonal; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C021499:lactose permease; D002268:Carboxypeptidases