Cysteine scanning mutagenesis of putative helix XI in the lactose permease of Escherichia coli.
Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid in putative transmembrane helix XI was individually replaced with Cys (from Ala347 to Ser366). Fifteen of the 20 mutants are highly functional and accumulate lactose to > 60% of the level achieved by C-less permease, and an additional three mutants, all located at the cytoplasmic end of the helix, exhibit lower but significant lactose accumulation. Cys replacements for Thr348 or Lys358 result in virtually inactive permease. Lys358, however, is not essential for active lactose transport but plays a role in permease folding or membrane insertion by interacting with Asp237. Immunoblots reveal that all mutant proteins are present in the membrane in amounts comparable to C-less with the exception of Lys358-->Cys which is hardly detectable, as expected. The results highlight Thr348 as a potentially important residue for further analysis. Finally, all active mutants were assayed after treatment with the sulfhydryl reagent N-ethyl-maleimide, and results range from nearly complete inhibition to almost 2-fold stimulation. Remarkably, all of the strongly inhibited positions lie on one face of helix XI. The implications of the findings for packing of transmembrane helices in the C-terminal half of the permease are discussed.
Dunten
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RL
Howard Hughes Medical Institute, Department of Physiology, University of California Los Angeles 90024-1574.
Sahin-Tóth
M
M
Kaback
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HR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active / Q000187:drug effects; D003545:Cysteine / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship; 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; D007785:Lactose; D003545:Cysteine; D005033:Ethylmaleimide