title: Cysteine-scanning mutagenesis of helix II and flanking hydrophilic domains in the lactose permease of Escherichia coli.
abstract: Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in putative transmembrane helix II and flanking hydrophilic loops (from Leu34 to Lys74) was replaced individually with Cys. Of the 41 single-Cys mutants, 28 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 10 mutants exhibit lower but significant levels of accumulation (25-60% of C-less). His35-->Cys permease exhibits very low activity (ca. 20% of C-less), while Gly64-->Cys or Asp68-->Cys permease is unable to accumulate lactose. However, His35 can be replaced with Arg without effect on transport activity [Padan, E., Sarkar, H.K., et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6765-6768]. In addition, even though mutant Gly64-->Cys or Glu68-->Cys is inactive both in the C-less background and in the wild-type, neither Gly64 [Jung, K., Jung, H., et al. (1995) Biochemistry 34, 1030-1039] nor Glu68 [Jessen-Marshall, A.E., & Brooker, R.J. (1996) J. Biol. Chem. 271, 1400-1404] is essential for active lactose transport. Immunoloblot analysis reveals that all of the mutants except His35-->Cys permease are inserted into the membrane at concentrations comparable to that of C-less permease. The transport activity of the single-Cys mutants is altered by N-ethylmaleimide (NEM) treatment in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to NEM inactivation at positions that cluster in a manner indicating that they are on one face of an alpha-helix (Thr45-->Cys, Gly46-->Cys, Phe49-->Cys, Ser53-->Cys, Ser56-->Cys, Gln60-->Cys, and Ser67-->Cys). Interestingly, the same face contains positions where Cys substitution itself leads to low transport activity (Ile52-->Cys, Leu57-->Cys, Gln60-->Cys, and Gly64-->Cys). The results demonstrate that although no residue per se in this region of the permease is irreplaceable, the surface of one face of helix II is important for active lactose transport.
authors.affiliation: Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1570, USA.
authors.forename: S
authors.initials: S
authors.lastname: Frillingos
authors.forename: J
authors.initials: J
authors.lastname: Sun
authors.forename: A
authors.initials: A
authors.lastname: Gonzalez
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D007785:Lactose / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D027981:Symporters
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose; D003545:Cysteine; D005033:Ethylmaleimide