Cysteine mutagenesis of the amino acid residues of transmembrane helix I in the melibiose carrier of Escherichia coli.
The melibiose carrier of Escherichia coli is a sugar-cation cotransport system that utilizes Na(+), Li(+), or H(+). This membrane transport protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 17-37, which includes all of the residues in membrane helix I. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloro- mercuribenzenesulfonic acid (PCMBS). Cysteine substitution caused loss of transport activity in six of the mutants (G17C, K18C, D19C, Y32C, T34C, and D35C). PCMBS caused greater than 50% inhibition in eleven mutants (F20C, A21C, I22C, G23C, I24C, V25C, Y26C, M27C, Y28C, M30C, and Y31C). We suggest that the residues whose cysteine derivatives were inhibited by PCMBS face the aqueous channel and that helix I is completely surrounded by aqueous environment. Second site revertants were isolated from K18C and Y31C. The revertants were found to have mutations in helices I, IV, and VII.
Ding
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PZ
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Wilson
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TH
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects / Q000235:genetics*; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D002414:Cations, Monovalent / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000037:antagonists & inhibitors / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D007106:Immune Sera / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism*; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000187:drug effects; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000187:drug effects / Q000235:genetics; D027981:Symporters
D002414:Cations, Monovalent; D007106:Immune Sera; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D027981:Symporters; D002731:4-Chloromercuribenzenesulfonate; C021548:melibiose permease; D008553:Melibiose; D003545:Cysteine