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.