The superfamily of traffic ATPases (ABC transporters) includes bacterial periplasmic transport systems (permeases) and eukaryotic transporters. The histidine permease of Salmonella typhimurium is composed of a membrane-bound complex (HisQMP 2 ) containing four subunits, and of a soluble receptor, the histidine-binding protein (HisJ). Transport is energized by ATP. In this article the ATPase activity of HisQMP 2 has been characterized, using a novel assay that is independent of transport. The assay uses Mg 2؉ ions to permeabilize membrane vesicles or proteoliposomes, thus allowing access of ATP to both sides of the bilayer. HisQMP 2 displays a low level of intrinsic ATPase activity in the absence of HisJ; unliganded HisJ stimulates the activity and liganded HisJ stimulates to an even higher level. All three levels of activity display positive cooperativity for ATP with a Hill coefficient of 2 and a K 0.5 value of 0.6 mM. The activity has been characterized with respect to pH, salt, phospholipids, substrate, and inhibitor specificity. Free histidine has no effect.

The histidine-binding protein, HisJ, is the soluble receptor for the periplasmic histidine permease of Salmonella typhimurium. The receptor binds the substrate in the periplasm, interacts with the membrane-bound complex, transmits a transmembrane signal to hydrolyze ATP, and releases the ligand for translocation. HisJ, like other periplasmic receptors, has two lobes that are apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation), burying deeply the ligand. Such receptors are postulated to interact with the membrane-bound complex with high affinity in their liganded conformation, and, upon substrate translocation, to undergo a reduction in affinity and therefore be released. Here we show that in contrast to the current postulate, liganded and unliganded receptors have equal affinity for the membrane-bound complex. The affinity is measured both by chemical cross-linking and co-sedimentation procedures. An ATPase activity assay is also used to demonstrate the interaction of unliganded receptor with the membrane-bound complex. These findings support a new model for the transport mechanism, in which the soluble receptor functions independently of the commonly accepted high-low affinity switch.

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 K m values of 8 mM and 8 M, 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.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). This superfamily includes several eukaryotic proteins, such as the multidrug resistance protein (P-glycoprotein or MDR), the cystic fibrosis transmembrane conductance regulator (CFTR), and the STE6 gene product from yeast (6, 7).The histidine permease of Salmonella typhimurium and Escherichia coli comprises the receptor, the periplasmic histidine-binding protein HisJ (8), and a membrane-bound complex (HisQ/M/P) 1 containing two hydrophobic subunits, HisQ and HisM, and two identical copies of the ATP-binding (conserved) component, HisP (9). A model for transport through these permeases was proposed in which liganded HisJ interacts with HisQ/M/P, thus 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). Of these, the most responsive to in vitro analysis is reconstitution into PLS. A detergent dilution procedure has been shown to be effective for several systems (1, 13-15). However, 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.Here we extensively characterize the histidine permease using a simple and reliable dialysis method for reconstitution into PLS. Var...