Identification and partial purification of PAPS translocase.
Sulfation of all macromolecules in higher organisms requires the high-energy donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). PAPS is synthesized via the sequential actions of two cytoplasmic enzymes, ATP sulfurylase and APS kinase, and then must be transferred across the Golgi membrane for utilization by lumenal sulfotransferases. Following the kinetic characterization of the PAPS translocase as a specific transporter that act through an antiport mechanism with PAP as the returning ligand [Ozeran, J.D., Westley, J., Schwartz, N.B. (1996) Biochemistry 35, 3685-3694 (accompanying paper)], the present study describes the identification and physical characterization of the PAPS translocase from rat liver Golgi membranes. The following evidence suggests the PAPS translocase is a membrane spanning protein of approximately 230 kDa: isolation by affinity chromatography on beta-methylene PAPS matrices of a 230 kDa Golgi membrane protein concomitant with PAPS translocase activity; demonstration that the 230 kDa protein possesses the only PAPS binding site accessible to the cytoplasmic face of intact Golgi membranes, while several other PAPS binding proteins are labeled in solubilized membrane preparations; reduction in size of the 230 kDa membrane protein and loss of PAPS translocase activity following protease treatment; estimation via hydrodynamic analysis of a molecular size of the membrane protein associated with PAPS translocase activity; and correlation of beta-methylene PAPS binding and labeling of the 230 kDa Golgi protein with PAPS translocase activity in artificial liposomes. These and the accompanying data have permitted the identification of the first of a potentially large class of Golgi membrane nucleotide-metabolite transporters.
Ozeran
J D
JD
Department of Pediatrics, University of Chicago, Chicago, Illinois 60637, USA.
Westley
J
J
Schwartz
N B
NB
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D017920:Antiporters / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport / Q000502:physiology; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000201:enzymology*; D017136:Ion Transport / Q000502:physiology; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D010724:Phosphoadenosine Phosphosulfate / Q000031:analogs & derivatives / Q000378:metabolism*; D051381:Rats; D013430:Sulfate Adenylyltransferase / Q000302:isolation & purification / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D017920:Antiporters; D003432:Cross-Linking Reagents; D008081:Liposomes; C099313:PAPS translocase protein, rat; D013431:Sulfates; C099314:beta-methylene PAPS; D010724:Phosphoadenosine Phosphosulfate; D000244:Adenosine Diphosphate; D013430:Sulfate Adenylyltransferase; D015238:Sulfotransferases; D014357:Trypsin