Helms J B, Rothman J E
Rockefeller Research Laboratory, Sloan-Kettering Institute, New York, New York 10021.
Nature. 1992 Nov 26;360(6402):352-4. doi: 10.1038/360352a0.
A wide variety of membrane transformations important in intracellular transport are inhibited by the fungal metabolite brefeldin A (refs 1-4), implying that the target for this drug is central to the formation and maintenance of subcellular compartments. Brefeldin A added to cells causes the rapid and reversible dissociation of a Golgi-associated peripheral membrane protein (M(r) 110,000) which was found to be identical to one of the subunits of the coat of Golgi-derived (non-clathrin) coated vesicles, beta-COP, implying that brefeldin A prevents transport by blocking the assembly of coats and thus the budding of enclosed vesicles. In addition to the coatomer (a cytosol-derived complex of seven polypeptide chains, one of which is beta-COP), the non-clathrin (COP) coat of Golgi-derived vesicles contains stoichiometric amounts of a small (M(r) approximately 20,000) GTP-binding protein, the ADP-ribosylation factor (ARF). Binding of ARF to Golgi membranes is necessary before coatomer/beta-COP can bind these membranes (ref. 12; and D. J. Palmer et al., manuscript submitted), so the primary effect of brefeldin A seems to be on the reaction responsible for ARF binding. Indeed, like beta-COP, ARF is dissociated from the Golgi complex by treatment with brefeldin A and brefeldin A prevents ARF from associating in vitro, but the mechanism of this action by brefeldin A has been unclear. Here we report the discovery of an enzyme in a Golgi-enriched fraction that catalyses guanine nucleotide (GDP-GTP) exchange on ARF-1 protein, and which is inhibited by brefeldin A. We suggest that activation of ARF proteins for membrane localization by compartmentalized exchange enzymes is in general the first committed step in membrane transformation pathways.
真菌代谢产物布雷菲德菌素A可抑制细胞内运输中多种重要的膜转化过程（参考文献1 - 4），这表明该药物的作用靶点对于亚细胞区室的形成和维持至关重要。向细胞中添加布雷菲德菌素A会导致一种与高尔基体相关的外周膜蛋白（分子量110,000）迅速且可逆地解离，该蛋白被发现与高尔基体衍生的（非网格蛋白）被膜小泡的包被亚基之一β - 衔接蛋白相同，这意味着布雷菲德菌素A通过阻止包被的组装从而抑制封闭小泡的出芽来阻断运输。除了外被体（一种由七条多肽链组成的胞质衍生复合物，其中一条是β - 衔接蛋白），高尔基体衍生小泡的非网格蛋白（COP）包被还含有化学计量的一种小的（分子量约20,000）GTP结合蛋白，即ADP核糖基化因子（ARF）。在包被蛋白/β - 衔接蛋白能够结合这些膜之前，ARF与高尔基体膜的结合是必需的（参考文献12；以及D. J. 帕尔默等人，已提交的手稿），所以布雷菲德菌素A的主要作用似乎是针对负责ARF结合的反应。实际上，与β - 衔接蛋白一样，ARF通过用布雷菲德菌素A处理会从高尔基体复合物中解离，并且布雷菲德菌素A在体外阻止ARF的结合，但布雷菲德菌素A这种作用的机制尚不清楚。在此我们报告在富含高尔基体的组分中发现了一种酶，它催化ARF - 1蛋白上的鸟嘌呤核苷酸（GDP - GTP）交换，并且被布雷菲德菌素A抑制。我们认为通过区室化交换酶激活用于膜定位的ARF蛋白通常是膜转化途径中的首个关键步骤。
