KINETICS OF PAPS TRANSLOCASE - EVIDENCE FOR AN ANTIPORT MECHANISM

In order to gain an understanding of the mechanisms involved in the tr ansfer of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) from the cytoso l where it is synthesized to the Golgi lumen where it serves as the un iversal sulfate donor for sulfate ester formation in higher organisms, we have undertaken a kinetic characterization of the PAPS translocase from rat liver Golgi. Analyzing the PAPS translocase activity in both intact Golgi vesicles and in a reconstituted liposome system, we have determined a number of physical and kinetic parameters. Strong compet itive inhibition in zero-trans uptake experiments only with beta-methy lene PAPS and adenosine 3',5'-bisphosphate (PAP) suggest the transport er is highly specific for the 3'-phosphate. The demonstration of trans acceleration as observed by stimulation of transport activity under e xchange conditions suggests that the translocase is a carrier with dis tinct binding sites accessible from both faces of the membrane. The be havior of the PAPS translocase in the presence of equilibrium concentr ations of PAP supports the function of an antiport mechanism. Thus the translocase is characterized by its kinetic properties as a specific transporter of PAPS which acts through an antiport mechanism with PAP as the returning ligand. This characterization of the transport activi ty has proved instrumental in the identification of a similar to 230 k Da Golgi membrane protein as the PAPS translocase protein [Ozeran, J. D., Westley, J., & Schwartz, N.B. (1996) Biochemistry 35, 3695-3703 (a ccompanying paper)].