Characterization and sequencing of the lac Y54-41 'uncoupled' mutant of the lactose permease

Robert J Brooker, S. H. Myster, T. H. Wilson

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Abstract

The Escherichia coli strain carying 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-β-D-thiogalactopyranoside. Direct measurements of H+ transport revealed that the mutant permease can transport H+ with methyl-β-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.

Original languageEnglish (US)
Pages (from-to)8135-8140
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number14
StatePublished - Jan 1 1989

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Membrane Transport Proteins
Genes
Thiogalactosides
Sulfhydryl Reagents
Lactose
Cysteine
Galactosides
Ethylmaleimide
Active Biological Transport
DNA Sequence Analysis
Glycine
Escherichia coli
Substitution reactions
Acids
lactose permease
DNA
Proteins
Experiments

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Characterization and sequencing of the lac Y54-41 'uncoupled' mutant of the lactose permease. / Brooker, Robert J; Myster, S. H.; Wilson, T. H.

In: Journal of Biological Chemistry, Vol. 264, No. 14, 01.01.1989, p. 8135-8140.

Research output: Contribution to journalArticle

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