CHARACTERIZATION OF TRANSPORT THROUGH THE PERIPLASMIC HISTIDINE PERMEASE USING PROTEOLIPOSOMES RECONSTITUTED BY DIALYSIS

The superfamily of traffic ATPases (ABC transporters) includes bacteri al periplasmic transport systems (permeases) and various eukaryotic tr ansporters. The histidine permease of Salmonella typhimurium and Esche richia coli is composed of a membrane-bound complex containing four su bunits and of a soluble receptor, the substrate binding protein (HisJ) , and is energized by ATP. The permease was previously reconstituted i nto proteoliposomes by a detergent dilution method (1). Here we extens ively characterize the properties of this permease after reconstitutio n into proteoliposomes by dialysis and encapsulation of ATP or other r eagents by freeze-thawing. We show that histidine transport depends en tirely on both ATP and liganded HisJ, with apparent K-m values of 8 mM and 8 mu M, respectively, and is affected by pH, temperature, and sal t 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 b y histidine implies that the membrane-bound complex HisQ/M/P carries a substrate-binding site. The reconstituted permease activity correspon ds to about 40-70% turnover rate of the in vivo rate of transport.