Purification of the Golgi adenosine 3'-phosphate 5'-phosphosulfate transporter, a homodimer within the membrane.
Sulfation of proteoglycans, secretory and membrane proteins, and glycolipids occurs in the lumen of the Golgi apparatus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the sulfate donor in these reactions, must be transported from the cytosol, its site of synthesis, into the lumen of the Golgi apparatus. We have identified and purified to apparent homogeneity the rat liver Golgi membrane PAPS transporter by a combination of conventional and affinity chromatography as well as photoaffinity radiolabeling with adenosine 3',5'-bisphosphate, a competitive inhibitor of PAPS transport. The transporter, a 75-kDa protein, was purified 70,000-fold over homogenate (6% yield) and transported PAPS into phosphatidylcholine liposomes selectively and in a saturable manner (apparent Km of 1.7 microM). Radiation target-inactivation analyses of the transport activity in rat liver Golgi vesicles, together with the above described biochemical approaches, demonstrate that the PAPS transporter within the Golgi membrane is a homodimer.
Mandon
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Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01655-0103.
Milla
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ME
Kempner
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Hirschberg
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CB
D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000528:radiation effects; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004307:Dose-Response Relationship, Radiation; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000378:metabolism* / Q000528:radiation effects; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D051381:Rats
D000345:Affinity Labels; D002352:Carrier Proteins; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D010724:Phosphoadenosine Phosphosulfate