Characterization and sequencing of the lac Y54-41 "uncoupled" mutant of the lactose permease.
The Escherichia coli strain carrying the lac Y54-41 gene encodes a mutant lactose permease which carries out normal downhill transport of galactosides but is defective in uphill accumulation. In this study, the mutant lac Y54-41 gene was cloned onto the multicopy vector pUR270. As expected, the cloned gene was shown to express normal downhill transport activity but was markedly defective in the uphill transport of methyl-beta-D-thiogalactopyranoside. Direct measurements of H+ transport revealed that the mutant permease can transport H+ with methyl-beta-D-thiogalactopyranoside but at a significantly reduced capacity compared to the wild-type strain. However, under conditions where the mutant and wild-type strains both transport lactose at similar rates, no detectable H+ transport was observed in the mutant strain. The entire cloned lac Y54-41 gene was subjected to DNA sequencing, and a single base substitution was found which replaces glycine 262 in the protein with a cysteine residue. Inhibition experiments showed that the mutant permease is dramatically more sensitive to three different sulfhydryl reagents: N-ethylmaleimide, p-hydroxymericuribenzoate, and p-hydroxymercuriphenylsulfonic acid. However, the lactose analogue, thiodigalactoside, was only marginally effective at protecting against inhibition in the mutant strain. The results are consistent with the idea that the sulfhydryl reagents are inhibiting the mutant permease activity by reacting with cysteine 262.
Brooker
R J
RJ
Institute for Advanced Studies in Biological Process Technology, University of Minnesota, St. Paul 55108.
Myster
S H
SH
Wilson
T H
TH
D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D015246:Deoxyribonuclease EcoRI; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D005822:Genetic Vectors; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007785:Lactose / Q000378:metabolism; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D008756:Methylgalactosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010663:Phenylmercury Compounds / Q000494:pharmacology; D010957:Plasmids; D011522:Protons; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism; D014169:Transformation, Bacterial
D029968:Escherichia coli Proteins; D006902:Hydroxymercuribenzoates; C050221:LacY protein, E coli; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D009004:Monosaccharide Transport Proteins; D010663:Phenylmercury Compounds; D011522:Protons; D027981:Symporters; D013862:Thiogalactosides; C030601:4-hydroxymercuribenzoate; C009468:thiomethylgalactoside; C002910:4-hydroxymercuribenzenesulfonate; C021499:lactose permease; D015246:Deoxyribonuclease EcoRI; D007785:Lactose; D005033:Ethylmaleimide