Cysteine-scanning mutagenesis of helix VI and the flanking hydrophilic domains on the lactose permease of Escherichia coli.
Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in putative transmembrane helix VI and the flanking hydrophilic loops (residues 164- 211) was replaced individually with Cys. Of the 48 mutants, 43 accumulate lactose at highly significant rates to > 80% of the steady state observed with C-less permease. Three mutants (Phe185--> Cys, Ala187--> Cys, and Phe208--> Cys) exhibit lower but significant levels of accumulation (30-60% of C-less). Cys replacement for Ala177 or Leu184 results in low transport activity (ca. 20%) in the C-less background but much higher activity (60-70%) in the wild type. Immunoblot analysis reveals that all of the mutants are inserted into the membrane at concentrations comparable to that of C-less perrmease. The transport activity of the great majority of the mutants is unaffected by treatment with N-ethylmaleimide (NEM). Relatively modest but significant inactivation (ca. 50%) is observed with mutants Phe170--> Cys, Gly173--> Cys, and Ala187--> Cys, and these positions cluster on the same face of the helix VI. Moreover, the two positions where single Cys replacements result in low activity (Ala177 and Lcu184) are on the same face of helix VI. The results demonstrate the following. (i) Permease function is not disrupted by replacement of most residues with Cys, but function is disrupted when some of the residues are further altered by addition of the NeM moiety. (ii) The latter residues lie on a stripe down one face of an alpha-helix, and within the same stripe are residues where Cys substitution itself leads ti inhibition of function.
Frillingos
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Howard Hughes Medical Institute, Department of Physiology and Microbiology, University of California at Los Angeles 90024-1570, USA.
Kaback
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics; D002451:Cell Compartmentation / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D007785:Lactose / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D017433:Protein Structure, Secondary; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D027981:Symporters
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; C021499:lactose permease; D007785:Lactose; D003545:Cysteine; D005033:Ethylmaleimide