title: Alteration of Na(+)-coupled transport in site-directed mutants of the melibiose carrier of Escherichia coli.
abstract: Asn-58 of the Escherichia coli melibiose carrier was replaced by Ala, Leu, Ser, and Gln. Trp-54 was replaced by Leu and a double mutant Leu-54/Ala-58 was constructed using site-directed mutagenesis. Cation/sugar cotransport and sugar-induced cation uptake were studied for each mutant. The change of Asn-58 to Ala results in a nearly complete loss of Na(+)-stimulated galactoside transport as well as sugar-stimulated Na+ uptake. Substitutions of Leu, Gln, and Ser for Asn-58 were also defective in Na(+)-stimulated sugar transport. The Trp-54 to Leu mutant shows moderate sugar accumulation with cation selectivity similar to wild-type. The double mutant Leu-54/Ala-58 shows elevated H(+)-melibiose cotransport as well as reduced Na(+)-stimulated melibiose cotransport. These results suggest that Asn-58 is important for Na+ recognition.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Franco
authors.forename: T H
authors.initials: TH
authors.lastname: Wilson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D004926:Escherichia coli / Q000737:chemistry*; D006859:Hydrogen / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008756:Methylgalactosides / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D012964:Sodium / Q000378:metabolism*; D013329:Structure-Activity Relationship; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism
chemical_list: D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D027981:Symporters; D013862:Thiogalactosides; C009468:thiomethylgalactoside; D006859:Hydrogen; C021548:melibiose permease; D008553:Melibiose; D012964:Sodium; D007785:Lactose