Identification of a residue in the translocation pathway of a membrane carrier.
Preliminary work using directed mutagenesis proved that cysteine is not required for operation of UhpT, the anion exchange protein responsible for glucose 6-phosphate transport by E. coli. We then made a detailed study of C143 and C265, because these cysteines impart sensitivity to p-chloromercuribenzosulfonate (PCMBS), a sulfhydral agent resembling glucose 6-phosphate in size, shape, and charge. We showed that C143 was exposed to the cytoplasm, as expected from hydropathy analysis, but we found no sidedness for C265. Rather, C265 was accessible to PCMBS from both membrane surfaces. And since the attack at C265 was blocked by glucose 6-phosphate, position 265 must lie directly on the pathway taken by the substrate as it moves through this membrane carrier.
Yan
R T
RT
Department of Physiology, Johns Hopkins University Medical School, Baltimore, Maryland 21209.
Maloney
P C
PC
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005958:Glucosephosphates; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; C063498:UhpT protein, E coli; D019298:Glucose-6-Phosphate; D002731:4-Chloromercuribenzenesulfonate; D003545:Cysteine