CYSTEINE-SCANNING MUTAGENESIS OF PUTATIVE HELIX-VII IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Using a functional lactose permease mutant devoid of Cys residues (C-l ess permease), each amino acid residue in putative transmembrane helix VII and the flanking cytoplasmic and periplasmic regions (from Leu212 to Glu255) was replaced individually with Cys. Of the 44 single-Cys m utants, 40 exhibit high transport activity, accumulating lactose to >5 0% of the steady-state observed with C-less permease. In contrast, per mease with Cys in place of Ala213 or Tyr236 exhibits low but significa nt activity, and Cys substitution for Asp237 or Asp240 yields permease molecules with little or no activity due to disruption of charge-neut ralizing interactions between Asp237 and Lys358 or Asp240 and Lys319, respectively. Immunological analysis reveals that membrane levels of t he mutant proteins are comparable to that of C-less permease with the exception of Tyr228-->Cys, which exhibits reduced but significant leve ls of permease. Finally, the effect of N-ethylmaleimide (NEM) was test ed on each mutant, and the results indicate that the transport activit y of the great majority of the mutants is not affected by the alkylati ng agent. Remarkably, the six positions where Cys replacements render the permease highly sensitive to inactivation by NEM are confined to t he C-terminal half of helix VII, a region that is strongly conserved a mong transport proteins homologous to lactose permease. The results de monstrate that although no residue per se in the region scanned is ess ential, structural features of the C terminus of helix VII may be impo rtant for transport activity.