A suppressor analysis of residues involved in cation transport in the lactose permease: identification of a coupling sensor.
Four amino acids critical for lactose permease function were altered using site-directed mutagenesis. The resulting Quad mutant (E269Q/R302L/H322Q/E325Q) was expressed at 60% of wild-type levels but found to have negligible transport activity. The Quad mutant was used as a parental strain to isolate suppressors that regained the ability to ferment the alpha-galactoside melibiose. Six different suppressors were identified involving five discrete amino acid changes and one amino acid deletion (Q60L, V229G, Y236D, S306L, K319N and DeltaI298). All of the suppressors transported alpha-galactosides at substantial rates. In addition, the Q60L, DeltaI298 and K319N suppressors regained a small but detectable amount of lactose transport. Assays of sugar-driven cation transport showed that both the Q60L and K319N suppressors couple the influx of melibiose with cations (H(+) or H(3)O(+)). Taken together, the data show that the cation-binding domain in the lactose permease is not a fixed structure as proposed in previous models. Rather, the data are consistent with a model in which several ionizable residues form a dynamic coupling sensor that also may interact directly with the cation and lactose.
Franco
Peter J
PJ
Department of Genetics, Cell Biology and Development and the Biotechnology Institute, University of Minnesota, Minneapolis, MN 55455, USA.
Matzke
Elizabeth A
EA
Johnson
Jerry L
JL
Wiczer
Brian M
BM
Brooker
Robert J
RJ
D019943:Amino Acid Substitution; D001692:Biological Transport; D015153:Blotting, Western; D002412:Cations / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D013862:Thiogalactosides / Q000378:metabolism
D002412:Cations; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013862:Thiogalactosides; C021499:lactose permease; D008553:Melibiose; D007785:Lactose