Organic anion-transporting ATPase of rat liver. II. Functional reconstitution of active transport and regulation by phosphorylation.

We have previously purified to homogeneity from rat liver plasma membranes a 90-kDa glycoprotein with S-(2,4-dinitrophenyl)glutathione-stimulated ATPase activity and other properties which identify it as the multispecific organic anion transporter (MOAT) specific for the transport into bile of non-bile acid organic anions (Pikula, S., Hayden, J. B., Awasthi, S., Awasthi, Y. C., and Zimniak, P. (1994) J. Biol. Chem. 269, 27566-27573). In the present communication, we report the functional reconstitution of this protein into artificial proteoliposomes. The reconstituted protein catalyzed time- and concentration-dependent uptake of S-(2,4-dinitrophenyl)glutathione into the vesicles. The transport required the presence of ATP. Phosphorylation of the 90-kDa protein by protein kinase C prior to reconstitution more than tripled the Vmax of transport but did not change the Km for S-(2,4-dinitrophenyl)glutathione. The protein created and, at steady state, maintained a more than 200-fold and 500-fold S-(2,4-dinitrophenyl)glutathione gradient across the membrane for the unphosphorylated and phosphorylated form, respectively. The transport activity of the 90-kDa protein is sufficient to account for the hepatic secretory maximum of non-bile acid organic anions in the rat.