Identification, purification, and characterization of the rat liver Golgi membrane ATP transporter

Phosphorylation of secretory and integral membrane proteins and of proteogl ycans also occurs in the lumen of the Golgi apparatus, ATP, the phosphate d onor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golg i membrane has been previously demonstrated in vitro using intact Golgi mem brane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi membrane ATP transp orter. The transporter was purified to apparent homogeneity by a combinatio n of conventional ion exchange, dye color, and affinity chromatography, An similar to 70,000-fold purification (2% yield) was achieved starting from c rude rat liver Golgi membranes. A protein with an apparent molecular mass o f 60 kDa was identified as the putative transporter by a combination of col umn chromatography, photoaffinity labeling with an analog of ATP, and nativ e functional size determination on a glycerol gradient, The purified transp orter appears to exist as a homodimer within the Golgi membrane, and when r econstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3'-phosphate 8'-phosphosulfate transport. The transport activity was saturable with an apparent K-m very similar to that of intact Golgi vesicles.