Cysteine scanning mutagenesis of helix V in the lactose permease of Escherichia coli.
Using a functional lactose permease mutant devoid of Cys (C-less permease), each amino acid residue in putative transmembrane helix V was replaced individually with Cys (from Met145 to Thr163). Of the 19 mutants, 13 are highly functional (60-125% of C-less permease activity), and 4 exhibit lower but significant lactose accumulation (15-45% of C-less permease). Cys replacement of Gly147 or Trp151 essentially inactivates the permease (< 10% of C-less); however, previous studies [Menezes, M. E., Roepe, P. D., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1638; Jung, K., Jung, H., et al. (1995) Biochemistry 34, 1030] demonstrate that neither of these residues is important for activity. Immunoblots reveal that all of the mutant proteins are present in the membrane in amounts comparable to C-less permease with the exception of Trp151-->Cys and single Cys154 permeases which are present in reduced amounts. Finally, only three of the single-Cys mutants are inactivated significantly by N-ethylmaleimide (Met145-->Cys, native Cys148, and Gly159-->Cys), and the positions of the three mutants fall on the same face of helix V.
Weitzman
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Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Kaback
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D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D027981:Symporters; C021499:lactose permease; D003545:Cysteine