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 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...

Characterization of the Adenosine Triphosphatase Activity of the Periplasmic Histidine Permease, a Traffic ATPase (ABC Transporter)

“…Chemicals were purchased from the following sources: CF (high purity mixed isomers), Molecular Probes, Eugene, OR; protamine (free base, from salmon), Sigma; n-octyl-␤-D-glucopyranoside, n-decanoylsucrose, and C 12 E 8 , Calbiochem; n-dodecyl-␤-D-maltoside, Boehringer Mannheim; Deriphat, Miranol, and Hega-11 (undecanoyl-N-hydroxyethylglucamide), Anatrace, Maumee, OH; bafilomycin A 1 , Calbiochem; ouabain, and N-ethylmaleimide, Sigma; and AMP-PNP, AMP-PCP, and ATP␥S, Calbiochem. (6), the ATPase activity is stimulated by liganded HisJ. The finding that HisJ can stimulate the activity when present on either side of the PLS is due to the fact that under the conditions of reconstitution HisQMP 2 is embedded in PLS in roughly equal amounts in the right side-out and inside-out orientations (6).…”

“…ATP Hydrolysis Assay-Membrane vesicles and PLS were prepared as described earlier (6), from strain TA1889 unless specified differently. ATPase reaction mixtures contained, in a final volume of 330 l, either membrane vesicles (10 l, containing 20 mg/ml protein) or PLS (100 l, containing 0.4 and 10 mg/ml protein and phospholipid, respectively; or 20 l of PLS reconstituted from purified HisQMP 2 , containing 0.2 and 10 mg/ml protein and phospholipid, respectively), and 50 mM MOPS/K ϩ buffer, pH 7.5.…”

“…(6), the ATPase activity is stimulated by liganded HisJ. The finding that HisJ can stimulate the activity when present on either side of the PLS is due to the fact that under the conditions of reconstitution HisQMP 2 is embedded in PLS in roughly equal amounts in the right side-out and inside-out orientations (6). Because HisP has been shown to be accessible from the periplasmic side of the membrane bilayer (26), it is possible that external ATP can reach the cytoplasmic ATPhydrolyzing domain of HisP.…”

“…The superfamily includes bacterial periplasmic permeases, the yeast STE6 gene product, the mammalian P-glycoprotein (multidrug resistance protein or MDR), 1 the human cystic fibrosis transmembrane conductance regulator (CFTR), and the mammalian heterodimeric transporter (TAP1/TAP2) involved in antigen processing (3,4). The periplasmic histidine permease in Salmonella typhimurium and the maltose permease in Escherichia coli have been extensively characterized; both are good model systems for this superfamily (5)(6)(7).…”

“…ATP hydrolysis provides the energy for the transport process (11). The histidine permease has been reconstituted into PLS that transport histidine when ATP is trapped internally and HisJ is added externally (6,12). As usually assumed in current working models, the liganded receptor sends a signal that initiates ATP hydrolysis and results in subsequent ligand translocation (13,14).…”