Topology of the lac carrier protein in the membrane of Escherichia coli.
Proteolysis of topologically sealed right-side-out and inside-out membrane vesicles from Escherichia coli with chymotrypsin, trypsin, or papain inactivates lac carrier function in a symmetrical manner. Concomitantly, the electrophoretic mobility of lac carrier protein photoaffinity labeled in situ with p-nitro[2-3H]phenyl-alpha-D-galactopyranoside is altered from a relative Mr of 33,000 to 20,000, and the time course of proteolysis is almost identical in vesicles of opposite polarities. In contrast, solubilization of the vesicles in NaDodSO4 followed by proteolysis causes fragmentation of the Mr 33,000 band into material that electrophoreses at the solvent front. Notably, proteolysis has no effect whatsoever on the ability of the lac carrier protein to bind substrate, as judged by photoaffinity-labeling experiments. Furthermore, the electrophoretic patterns of samples proteolyzed prior to photoaffinity labeling are the same as those observed when the procedures are reversed. These results show that the lac carrier protein spans the membrane and indicate that the binding site resides within a segment that is embedded in the bilayer.
D000345:Affinity Labels / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002918:Chymotrypsin / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D009598:Nitrophenylgalactosides / Q000494:pharmacology; D027981:Symporters
D000345:Affinity Labels; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D009598:Nitrophenylgalactosides; C021499:lactose permease; D002918:Chymotrypsin