Functional interactions between putative intramembrane charged residues in the lactose permease of Escherichia coli.
Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys. Individual replacements for Asp-237, Asp-240, Glu-269, Arg-302, Lys-319, His-322, Glu-325, or Lys-358 abolish active lactose transport. When Asp-237 and Lys-358 are simultaneously replaced with Cys and/or Ala, however, high activity is observed. Therefore, when either Asp-237 or Lys-358 is replaced with a neutral residue, leaving an unpaired charge, the permease is inactivated, but neutral replacement of both residues yields active permease [King, S. C., Hansen, C. L. & Wilson, T. H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Remarkably, moreover, when Asp-237 is interchanged with Lys-358, high activity is observed. The observations provide a strong indication that Asp-237 and Lys-358 interact to form a salt bridge. In addition, the data demonstrate that neither residue nor the salt bridge plays an important role in the transport mechanism. Thirteen additional double mutants were constructed in which a negative and a positively charged residue were replaced with Cys. Only Asp-240-->Cys/Lys-319-->Cys exhibits significant activity, accumulating lactose to 25-30% of the steady state observed with C-less permease. Replacing either Asp-240 or Lys-319 individually with Ala also inactivates the permease, but double mutants with neutral substitutions (Cys and/or Ala) at both positions exhibit essentially the same activity as Asp-240-->Cys/Lys-319-->Cys. In marked contrast to Asp-237 and Lys-358, interchanging Asp-240 and Lys-319 abolishes active lactose transport. The results demonstrate that Asp-240 and Lys-319, like Asp-237 and Lys-358, interact functionally and may form a salt bridge. However, the interaction between Asp-240 and Lys-319 is clearly more complex than the interaction between Asp-237 and Lys-358. In any event, the findings suggest that putative transmembrane helix VII lies next to helices X and XI in the tertiary structure of lactose permease.
Sahin-Tóth
M
M
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1574.
Dunten
R L
RL
Gonzalez
A
A
Kaback
H R
HR
D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000201:enzymology; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017433:Protein Structure, Secondary; D027981:Symporters
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease