Mandon E C, Milla M E, Kempner E, Hirschberg C B
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01655-0103.
Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10707-11. doi: 10.1073/pnas.91.22.10707.
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.
蛋白聚糖、分泌蛋白和膜蛋白以及糖脂的硫酸化发生在高尔基体腔内。3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）是这些反应中的硫酸供体，它必须从其合成部位胞质溶胶转运到高尔基体腔内。我们通过传统色谱法和亲和色谱法相结合，以及用3',5'-二磷酸腺苷（一种PAPS转运的竞争性抑制剂）进行光亲和放射性标记，鉴定并纯化出了大鼠肝脏高尔基体膜PAPS转运体，使其达到表观均一性。该转运体是一种75 kDa的蛋白质，相对于匀浆纯化了70000倍（产率6%），并以可饱和的方式选择性地将PAPS转运到磷脂酰胆碱脂质体中（表观Km为1.7 microM）。大鼠肝脏高尔基体囊泡中转运活性的辐射靶失活分析，与上述生化方法一起，证明高尔基体膜内的PAPS转运体是一个同二聚体。
