Randazzo P A, Yang Y C, Rulka C, Kahn R A
Section of Regulatory Mechanisms, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 May 5;268(13):9555-63.
Recent evidence has implicated ADP-ribosylation factor (ARF) proteins as critical regulators of the protein secretory pathway, particularly in the endoplasmic reticulum-Golgi pathway. We have examined whether Golgi membranes contain activators of ARF and the consequences of ARF activation and acylation on its membrane association. Two means were used to assess ARF activation. First, guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to protein was found to be greater when ARF and Golgi were incubated together than when either was incubated alone. These data suggested that ARF GTP gamma S was formed. This was confirmed by showing that the GTP gamma S-bound protein functioned as a cofactor for cholera toxin-stimulated ADP-ribosylation of Gs alpha, a reaction for which activated ARF is a necessary cofactor. Trypsin treatment of Golgi, an inhibitory ARF peptide, and brefeldin A each inhibited Golgi-mediated activation by approximately 70%, demonstrating that a specific protein interaction is required for the majority of the ARF activation. This ARF-activating protein is a strong candidate for the molecular target for brefeldin A. The ubiquitous nature of ARF proteins and their importance in both the exocytic and endocytic pathways may explain the effects of brefeldin A on both exocytic and endocytic membrane traffic in animal cells. A protease-insensitive activation of ARF by Golgi could also be demonstrated and was the dominant activity observed in submicromolar concentrations of magnesium. We believe this to be the lipid-mediated process described previously for purified ARF proteins. ARF activation resulted in tight association of ARF with phospholipid vesicles. Vesicle association required amino-terminal myristoylation of ARF whereas activation did not. These studies indicate that the brefeldin A-sensitive ARF-activating protein and other factors that determine the level of activation of ARF in animal cells are fundamental regulators of membrane traffic in animal cells.
近期证据表明，ADP核糖基化因子（ARF）蛋白是蛋白质分泌途径的关键调节因子，尤其是在内质网-高尔基体途径中。我们研究了高尔基体膜是否含有ARF激活剂，以及ARF激活和酰化对其膜结合的影响。采用了两种方法来评估ARF激活。首先，发现当ARF与高尔基体一起孵育时，鸟苷5'-3-O-（硫代）三磷酸（GTPγS）与蛋白质的结合比单独孵育任何一种时都更强。这些数据表明形成了ARF GTPγS。通过显示与GTPγS结合的蛋白质作为霍乱毒素刺激的Gsα ADP核糖基化的辅因子发挥作用得到了证实，对于该反应，活化的ARF是必需的辅因子。用胰蛋白酶处理高尔基体、一种抑制性ARF肽和布雷菲德菌素A，每种都使高尔基体介导的激活抑制约70%，表明大多数ARF激活需要特定的蛋白质相互作用。这种ARF激活蛋白是布雷菲德菌素A分子靶点的有力候选者。ARF蛋白的普遍存在及其在胞吐和内吞途径中的重要性，可能解释了布雷菲德菌素A对动物细胞胞吐和内吞膜运输的影响。还可以证明高尔基体对ARF的蛋白酶不敏感激活，并且这是在亚微摩尔浓度的镁中观察到的主要活性。我们认为这是先前针对纯化的ARF蛋白描述的脂质介导过程。ARF激活导致ARF与磷脂囊泡紧密结合。囊泡结合需要ARF的氨基末端肉豆蔻酰化，而激活则不需要。这些研究表明，布雷菲德菌素A敏感的ARF激活蛋白和其他决定动物细胞中ARF激活水平的因素是动物细胞膜运输的基本调节因子。
