Isolation and reconstitution of the chloride transporter of clathrin-coated vesicles.
Clathrin-coated vesicle acidification is mediated by an endomembrane proton translocating ATPase. This pump is electrogenic, and significant pH gradient formation requires the parallel movement of chloride through a chloride transporter in order to maintain net electroneutrality. We have solubilized, isolated and achieved 270-fold purification of this chloride transporter by means of selective detergent solubilization with cholate and polyoxyethelene 9-lauryl ether (C12E9), hydroxylapatite chromatography, and glycerol gradient centrifugation. Stabilization of the solubilized transporter requires 5 mM dithiothreitol. The partially purified transporter was co-reconstituted with the purified clathrin-coated vesicle proton translocating complex to yield preparations of proteoliposomes capable of valinomycin-independent proton pumping, as assessed by ATP-generated acridine orange quenching. In addition, the chloride transporter was independently reconstituted and was shown to catalyze diisothiocyano-disulfonic acid stilbene-sensitive 36Cl uptake. The anionic conductive selectivity of the reconstituted transporter (chloride = bromide greater than nitrate) exactly matched that of the transporter of native clathrin-coated vesicles. These studies demonstrate that the chloride transporter of vacuolar acidification systems is structurally and functionally dissociable from co-existing proton pumps and allow for investigations of pump-transporter interactions in a resolved system.
Xie
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Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235-9040.
Crider
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BP
Stone
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D000818:Animals; D001921:Brain / Q000201:enzymology; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002712:Chlorides / Q000378:metabolism*; D002845:Chromatography; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D017886:Durapatite; D011992:Endosomes; D006882:Hydroxyapatites; D007700:Kinetics; D013379:Substrate Specificity
D002352:Carrier Proteins; D002712:Chlorides; D006882:Hydroxyapatites; D017886:Durapatite