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-less 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 mutants, 40 exhibit high transport activity, accumulating lactose to > 50% of the steady-state observed with C-less permease. In contrast, permease with Cys in place of Ala213 or Tyr236 exhibits low but significant activity, and Cys substitution for Asp237 or Asp240 yields permease molecules with little or no activity due to disruption of charge-neutralizing interactions between Asp237 and Lys358 or Asp240 and Lys319, respectively. Immunological analysis reveals that membrane levels of the mutant proteins are comparable to that of C-less permease with the exception of Tyr228-->Cys, which exhibits reduced but significant levels of permease. Finally, the effect of N-ethylmaleimide (NEM) was tested on each mutant, and the results indicate that the transport activity of the great majority of the mutants is not affected by the alkylating agent. Remarkably, the six positions where Cys replacements render the permease highly sensitive to inactivation by NEM are confined to the C-terminal half of helix VII, a region that is strongly conserved among transport proteins homologous to lactose permease. The results demonstrate that although no residue per se in the region scanned is essential, structural features of the C terminus of helix VII may be important for transport activity.
Frillingos
S
S
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1662.
Sahin-Tóth
M
M
Persson
B
B
Kaback
H R
HR
D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007785:Lactose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; 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