title: Characterization of transport through the periplasmic histidine permease using proteoliposomes reconstituted by dialysis.
abstract: The superfamily of traffic ATPases (ABC transporters) includes bacterial periplasmic transport systems (permeases) and various eukaryotic transporters. The histidine permease of Salmonella typhimurium and Escherichia coli is composed of a membrane-bound complex containing four subunits and of a soluble receptor, the substrate-binding protein (HisJ), and is energized by ATP. The permease was previously reconstituted into proteoliposomes by a detergent dilution method (1). Here we extensively characterize the properties of this permease after reconstitution into proteoliposomes by dialysis and encapsulation of ATP or other reagents by freeze-thawing. We show that histidine transport depends entirely on both ATP and liganded HisJ, with apparent Km values of 8 mM and 8 microM, respectively, and is affected by pH, temperature, and salt concentration. Transport is irreversible and accumulation reaches a plateau at which point transport ceases. The permease is inhibited by ADP and by high concentrations of internal histidine. The inhibition by histidine implies that the membrane-bound complex HisQ/M/P carries a substrate-binding site. The reconstituted permease activity corresponds to about 40-70% turnover rate of the in vivo rate of transport.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Liu
authors.forename: G F
authors.initials: GF
authors.lastname: Ames
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism; D026923:Amino Acid Transport Systems, Basic; D000818:Animals; D001426:Bacterial Proteins; D001693:Biological Transport, Active; D002850:Chromatography, Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D005960:Glucosides; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011510:Proteolipids; C020485:proteoliposomes; C018619:octyl-beta-D-glucoside; C043828:histidine permease, Bacteria; D000251:Adenosine Triphosphatases; D008274:Magnesium