Balch W E, Kahn R A, Schwaninger R
Department of Cellular Biology, Scripps Research Institute, La Jolla, California 92037.
J Biol Chem. 1992 Jun 25;267(18):13053-61.
We describe the potential role of ADP-ribosylation factor (ARF) in vesicular trafficking using an in vitro assay that efficiently reconstitutes transport between the endoplasmic reticulum (ER) and the cis-Golgi compartment in mammalian semi-intact cells, a population of cells in which the plasma membrane is physically perforated to reveal intact ER and Golgi compartments. We demonstrate that peptides identical to the amino-terminal domain of ARF, which inhibit ARF cofactor activity in cholera toxin-catalyzed ADP-ribosylation of G alpha S (Kahn, R. A., Randazzo, P., Serafini, T., Weiss, O., Rulka, C., Clark, J., Amherdt, M., Roller, P., Orci, L., and Rothman, J. E. (1992) J. Biol. Chem. 267, 13039-13046), inhibit transport of the vesicular stomatitis virus G protein between the ER and cis-Golgi compartment. Inhibition of transport was rapid (t1/2 = 30-60 s) and irreversible. Half-maximal inhibition was observed at concentrations of 15 and 22 microM with peptides identical to the amino-terminal domain of the human ARF4 (hARF4) protein and the human ARF1 protein, respectively. Kinetic analysis of vesicular stomatitis virus G protein transport suggested that the hARF4 peptide inhibits a late vesicle fusion step. In addition, incubation of semi-intact cells in the presence of the myristoylated form human ARF1 (hARF1myr) protein, but not the nonmyristoylated form of ARF1, inhibited transport. In contrast to peptide, the hARF1myr blocked an early transport step, similar to that observed with guanosine 5'-3-O-(thio)triphosphate. These results suggest that ARF and components facilitating ARF function play an important role in the cyclical fission and fusion of transport vesicles mediating ER to Golgi trafficking.
我们利用一种体外测定方法描述了ADP核糖基化因子（ARF）在囊泡运输中的潜在作用，该方法能有效地在哺乳动物半完整细胞中重建内质网（ER）和顺式高尔基体区室之间的运输，这类细胞群体的质膜在物理上被穿孔以暴露出完整的内质网和高尔基体区室。我们证明，与ARF氨基末端结构域相同的肽，其在霍乱毒素催化的GαS的ADP核糖基化中抑制ARF辅因子活性（卡恩，R.A.，兰达佐，P.，塞拉菲尼，T.，魏斯，O.，鲁尔卡，C.，克拉克，J.，阿姆赫特，M.，罗勒，P.，奥尔西，L.和罗斯曼，J.E.（1992年）《生物化学杂志》267，13039 - 13046），抑制了水泡性口炎病毒G蛋白在内质网和顺式高尔基体区室之间的运输。运输抑制迅速（t1/2 = 30 - 60秒）且不可逆。分别用人ARF4（hARF4）蛋白和人ARF1蛋白的氨基末端结构域相同的肽，在浓度为15和22 microM时观察到半数最大抑制。对水泡性口炎病毒G蛋白运输的动力学分析表明，hARF4肽抑制了后期囊泡融合步骤。此外，在肉豆蔻酰化形式的人ARF1（hARF1myr）蛋白存在下孵育半完整细胞，但非肉豆蔻酰化形式的ARF1则无此作用，抑制了运输。与肽不同，hARF1myr阻断了早期运输步骤，类似于用鸟苷5'-3-O-(硫代)三磷酸观察到的情况。这些结果表明，ARF和促进ARF功能的成分在介导内质网到高尔基体运输的运输囊泡的周期性裂变和融合中起重要作用。
