THE ROLE OF HELIX-VIII IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI .1. CYS-SCANNING MUTAGENESIS

Using a functional lactose permease mutant devoid of Cys residues (C-l ess permease), each amino acid residue in transmembrane domain VIII an d flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced in dividually with Cys. Of the 34 single-Cys mutants, 26 accumulate lacto se to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Cry 262 --> Cys, Gly 268 --> Cys, Asn 272 --> Cy s, 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-T6th M, Persson B, Kaback HR, 199 4, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lacto se transport. Immunoblot analysis reveals that the mutants are inserte d into the membrane at concentrations comparable to C-less permease, w ith the exceptions of mutants Pro 280 --> Cys, Gly 287 --> Cys, and Ly s 289 --> Cys, which are expressed at reduced levels. The transport ac tivity of the mutants is inhibited by N-ethylmaleimide (NEM) in a high ly specific manner. Most of the mutants are insensitive, but Cys repla cements render the permease sensitive to inactivation by NEM at positi ons 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 26 8 --> 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, al though only a single residue in this region of the permease is essenti al for activity (Glu 269), one face of the helix plays an important ro le in the transport mechanism. More direct evidence for the latter con clusion is provided in the companion paper (Frillingos S, Kaback HR, 1 997, Protein Sci 6:438-443) by using site-directed sulfhydryl modifica tion of the Cys-replacement mutants in situ.