Identification, purification, and characterization of the rat liver golgi membrane ATP transporter.
Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An approximately 70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3'-phosphate 5'-phosphosulfate transport. The transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles.
Puglielli
L
L
Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118, USA.
Mandon
E C
EC
Hirschberg
C B
CB
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000302:isolation & purification; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000737:chemistry; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000737:chemistry* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000737:chemistry / Q000648:ultrastructure; D008099:Liver / Q000737:chemistry* / Q000648:ultrastructure; D010761:Phosphorus Radioisotopes; D051381:Rats
D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D001386:Azides; D010761:Phosphorus Radioisotopes; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate