Characterization of transport through the periplasmic histidine permease using proteoliposomes reconstituted by dialysis.
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.
Liu
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Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Ames
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GF
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*
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