Control of H+/lactose coupling by ionic interactions in the lactose permease of Escherichia coli.
A combinatorial approach was used to study putative interactions among six ionizable residues (Asp-240, Glu-269, Arg-302, Lys-319, His-322, and Glu-325) in the lactose permease. Neutral mutations were made involving five ion pairs that had not been previously studied. Double mutants, R302L/E325Q and D240N/H322Q, had moderate levels of downhill [(14)C]-lactose transport. Mutants in which only one of these six residues was left unchanged (pentuple mutants) were also made. A Pent269(-) mutant (in which only Glu-269 remains) catalyzed a moderate level of downhill lactose transport. Pent240(-) and Pent 322(+) also showed low levels of downhill lactose transport. Additionally, a Pent240(-) mutant exhibited proton transport upon addition of melibiose, but not lactose. This striking result demonstrates that neutralization of up to five residues of the lactose permease does not abolish proton transport. A mutant with neutral replacements at six ionic residues (hextuple mutant) had low levels of downhill lactose transport, but no uphill accumulation or proton transport. Since none of the mutants in this study catalyzes active accumulation of lactose, this is consistent with other reports that have shown that each residue is essential for proper coupling. Nevertheless, none of the six ionizable residues is individually required for substrate-induced proton cotransport. These results suggest that the H(+) binding domain may be elsewhere in the permease or that cation binding may involve a flexible network of charged residues.
Johnson
J L
JL
Department of Genetics, Cell Biology and Development, and the Biotechnology Institute, University of Minnesota, Twin Cities, MN, USA.
Brooker
R J
RJ
D001693:Biological Transport, Active / Q000502:physiology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007477:Ions; D007700:Kinetics; D007785:Lactose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008657:Metabolic Clearance Rate; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D007477:Ions; D026901:Membrane Transport Proteins; D017494:Proton Pumps; D011522:Protons; D011994:Recombinant Proteins; C021499:lactose permease; D007785:Lactose