A Suppressor Analysis of Residues Involved in Cation Transport in the Lactose Permease: Identification of a Coupling Sensor |
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Authors: | Peter J Franco Elizabeth A Matzke Jerry L Johnson Brian M Wiczer Robert J Brooker |
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Institution: | (1) Department of Genetics, Cell Biology and Development and the Biotechnology Institute, University of Minnesota, Minneapolis, Minnesota 55455, USA |
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Abstract: | 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. |
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Keywords: | Suppressor analysis Cation Transport Lactose permease Coupling sensor |
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