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.
Highlights
Periplasmic permeases are complex transport systems composed of a soluble substrate-binding protein, the receptor, and a membrane-bound complex containing four subunits [2, 3]. Transport through these systems is energized by ATP hydrolysis mediated by one of the membrane-bound components that is evolutionarily conserved in a large superfamily of transporters, the traffic ATPases [4] or ABC transporters [5]
A model for transport through these permeases was proposed in which liganded HisJ interacts with HisQ/M/P, initiating ATP hydrolysis and the consequent substrate translocation [10]
A previous examination of the periplasmic histidine permease reconstituted into PLS by a dilution method had provided some basic insights into its properties [1]
Summary
HisQ/M/P, HisQ1⁄7HisM1⁄7HisP membranebound complex; PLS, reconstituted proteoliposomes; PAGE, polyacrylamide gel electrophoresis; octylglucoside, octyl--D-glucopyranoside; ATP-␥-35S, 35S-adenosine 5Ј-O-(3-thio)triphosphate; CF, 5(6)-carboxyfluorescein; MOPS, 4-morpholinepropanesulfonic acid. A model for transport through these permeases was proposed in which liganded HisJ interacts with HisQ/M/P, initiating ATP hydrolysis and the consequent substrate translocation [10]. Several in vitro reconstituted assay systems have been developed for the biochemical analysis and purification of the histidine permease [1, 11, 12]. A detergent dilution procedure has been shown to be effective for several systems [1, 13,14,15] This procedure is cumbersome, tends to give unreproducible PLS preparations, and, because it can only produce small batches of PLS that are unstable, it has not been useful for extended biochemical analyses. Evidence is provided for the existence of a substrate-binding site in HisQ/M/P
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