Cysteine scanning mutagenesis of the N-terminal 32 amino acid residues in the lactose permease of Escherichia coli.
Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in the hydrophilic N-terminus and the first putative transmembrane helix was systematically replaced with Cys (from Tyr-2 to Trp-33). Twenty-three of 32 mutants exhibit high lactose accumulation (70-100% or more of C-less), and an additional 8 mutants accumulate to lower but highly significant levels. Surprisingly, Cys replacement for Gly-24 or Tyr-26 yields fully active permease molecules, and permease with Cys in place of Pro-28 also exhibits significant transport activity, although previous mutagenesis studies on these residues suggested that they may be required for lactose transport. As expected, Cys replacement for Pro-31 completely inactivates, in agreement with previous findings indicating that "helix-breaking" propensity at this position is necessary for full activity (Consler TG, Tsolas O, Kaback HR, 1991, Biochemistry 30:1291-1297). Twenty-nine mutants are present in the membrane in amounts comparable to C-less permease, whereas membrane levels of mutants Tyr-3-->Cys and Phe-12-->Cys are slightly reduced, as judged by immunological techniques. Dramatically, mutant Phe-9-->Cys is hardly detectable when expressed from the lac promoter/operator at a relatively low rate, but is present in the membrane in a stable form when expressed at a high rate from the T7 promoter. Finally, studies with N-ethylmalemide show that 6 Cys-replacement mutants that cluster at the C-terminal end of putative helix I are inactivated significantly.(ABSTRACT TRUNCATED AT 250 WORDS)
Sahin-Tóth
M
M
Howard Hughes Medical Institute, Department of Physiology, University of California at Los Angeles 90024-1662.
Persson
B
B
Schwieger
J
J
Cohan
P
P
Kaback
H R
HR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active / Q000187:drug effects; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose; D003545:Cysteine; D005033:Ethylmaleimide