The melibiose carrier of Escherichia coli: cysteine substitutions for individual residues in helix XI.
The melibiose carrier from Escherichia coli is a sugar-cation cotransport system. Previously evidence was obtained that this integral membrane protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 374-396, which includes all of the residues in the proposed helix XI. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloromercuribenzenesulfonic acid (PCMBS). Studies were carried out on both intact cells and inside out vesicles. Cysteine substitution caused loss of transport activity in seven of the mutants (K377C, G379C, A383C, F385C, L391C, G395C and Y396C). PCMBS produced more than 50% inhibition in six of the mutants (S380C, A381C, A384C, F387C, A388C and L391C). Preincubation of the cells with melibiose protected five of these residues from the inhibitory action of PCMBS. It was concluded that the residues whose cysteine derivatives were inhibited by PCMBS probably faced the aqueous channel.
Ding
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PZ
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Wilson
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TH
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D019943:Amino Acid Substitution; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D008094:Lithium / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D012964:Sodium / Q000378:metabolism; D027981:Symporters
D026901:Membrane Transport Proteins; D027981:Symporters; D002731:4-Chloromercuribenzenesulfonate; C021548:melibiose permease; D008553:Melibiose; D008094:Lithium; D012964:Sodium; D003545:Cysteine