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 α-galactoside melibiose. Six different suppressors were identified involving five discrete amino acid changes and one amino acid deletion (Q60L, V229G, Y236D, S306L, K319N and ΔI298). All of the suppressors transported α-galactosides at substantial rates. In addition, the Q60L, ΔI298 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 H3O+). 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.
The Journal of Membrane Biology – Springer Journals
Published: Sep 18, 2006
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