ORGANIC ANION-TRANSPORTING ATPASE OF RAT-LIVER .2. FUNCTIONAL RECONSTITUTION OF ACTIVE-TRANSPORT AND REGULATION BY PHOSPHORYLATION

We have previously purified to homogeneity from rat liver plasma membr anes a 90-kDa glycoprotein with S-(2,4-dinitrophenyl)glutathione-stimu lated ATPase activity and other properties which identify it as the mu ltispecific organic anion transporter (MOAT) specific for the transpor t 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. 2 69, 27566-27573). In the present communication, we report the function al reconstitution of this protein into artificial proteoliposomes. The reconstituted protein catalyzed time and concentration-dependent up t ake of S-(2,4-dinitrophenyl) glutathione into the vesicles, The transp ort required the presence of ATP. Phosphorylation of the 90-kDa protei n by protein kinase C prior to reconstitution more than tripled the V- max of transport but did not change the K-m for S-(2,4-dinitrophenyl)g lutathione. The protein created and, at steady state, maintained a mor e 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 suffici ent to account for the hepatic secretory maximum of non-bile acid orga nic anions in the rat.