Abstract
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 (HisQMP2) 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 HisQMP2 has been characterized, using a novel assay that is independent of transport. The assay uses Mg2+ ions to permeabilize membrane vesicles or proteoliposomes, thus allowing access of ATP to both sides of the bilayer. HisQMP2 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 K0. 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 activity is inhibited by orthovanadate, but not by N-ethylmaleimide, bafilomycin A1, or ouabain. Several nucleotide analogs, ADP, 5'-adenylyl-beta, gamma-imidodiphosphate, adenosine 5'-(beta,gammaimino)triphosphate, and adenosine 5'-O-(3-thio)triphosphate, inhibit the activity. Unliganded HisJ does not compete with liganded HisJ for the stimulation of the ATPase activity of HisQMP2.
Highlights
The superfamily of traffic ATPases comprises both prokaryotic and eukaryotic transport proteins which share a conserved nucleotide-binding domain [1, 2]
In this paper we describe a detailed study of the ATPase activity of the histidine permease reconstituted in PLS, using a novel assay in which the lipid bilayer has been rendered permeable to both ATP and the receptor
Total E. coli phospholipids (3 mg/ml final concentration) were added when the assay was performed with either membrane vesicles or PLS reconstituted with purified HisQMP2
Summary
Bacterial Strains and Growth Media—The following E. coli K12 strains were used: TA1889, which carries deletion unc702 (that eliminates the F0F1-ATPase) and harbors plasmid pFA17 (which contains the S. typhimurium hisQ, hisM, and hisP genes under the temperaturesensitive control of the PL promoter [18]); GA298 which is derived from TA1889 by loss of pFA17.
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