title: Escherichia coli alpha-ketoglutarate permease is a constitutively expressed proton symporter.
abstract: Escherichia coli kgtP which maps at 56.5 min codes for alpha-ketoglutarate permease (KgtP). This protein, expressed from the cloned gene using the T7 polymerase system and [35S]methionine labeling, fractionated with cell membranes. Right-side-out (RSO) membrane vesicles prepared from a kgtP negative mutant strain did not transport alpha-ketoglutarate, but RSO vesicles from the same strain expressing KgtP from a transforming plasmid transported alpha-ketoglutarate effectively as measured by uptake of the 14C-labeled substrate. E. coli JC7623 strain grown in M9 minimal medium with glucose, glycerol, or alpha-ketoglutarate as carbon source contained a 1.3-kilobase RNA which hybridized to nick-translated kgtP probe. In addition, strain MC1061 cultures grown under these same conditions were all capable of transporting alpha-ketoglutarate, demonstrating that KgtP is constitutively expressed. The Km and Vmax of KgtP assayed in strain MC1061 vesicles were 13-46 microM and 8 nmol/min/mg protein, respectively. Uncouplers that permeabilized the membrane to protons inhibited alpha-ketoglutarate transport into energized vesicles, and the addition of alpha-ketoglutarate to vesicle suspensions under non-energized conditions resulted in an increase in pH. These results indicate that KgtP is an alpha-ketoglutarate-proton symporter.
authors.affiliation: Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854.
authors.forename: W
authors.initials: W
authors.lastname: Seol
authors.forename: A J
authors.initials: AJ
authors.lastname: Shatkin
mesh_terms: D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D029382:Dicarboxylic Acid Transporters; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010957:Plasmids; D013379:Substrate Specificity
chemical_list: D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids; D026901:Membrane Transport Proteins; C068628:KgtP protein, E coli