The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.
Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ.
Frillingos
S
S
Howard Hughes Medical Institute, University of California Los Angeles 90095-1662, USA.
Ujwal
M L
ML
Sun
J
J
Kaback
H R
HR
D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D003545:Cysteine / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D027981:Symporters
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D003545:Cysteine; D005033:Ethylmaleimide