Tsai S C, Adamik R, Haun R S, Moss J, Vaughan M
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 May 25;268(15):10820-5.
ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance in vitro cholera toxin-catalyzed ADP-ribosylation and subsequently shown to participate in vesicular transport in the Golgi and other cellular compartments. By cDNA and genomic cloning, at least six mammalian ARFs were identified. Brefeldin A (BFA) disrupts Golgi membranes and inhibits binding of soluble high molecular weight proteins to Golgi fractions. We examined the effects of BFA on binding of ARF1, -3, and -5 to a Golgi fraction in the presence of an ATP-regenerating system and a fraction of soluble, high molecular weight, accessory proteins (SAP), presumably containing complexes identified by others as coatomers that are involved in vesicular transport. ARF binding in all instances was dependent on guanosine 5'-O-(3-thiotriphosphate) and increased by the ATP-regenerating system. Binding of ARF1 and -3, but not ARF5, was enhanced by SAP. BFA inhibited the SAP-dependent, but not the SAP-independent, binding of ARF1 and -3. It had no effect on the increment in binding produced by an ATP-regenerating system. B36, an inactive derivative of BFA, did not inhibit SAP-dependent binding of ARF1 and -3. Binding of ARF5, which was SAP-independent, was not affected by BFA. These observations are consistent with the conclusion that mammalian ARFs differ in their dependence on accessory proteins for interaction with Golgi and, perhaps, other cellular membranes and that BFA specifically inhibits SAP-dependent ARF binding.
ADP-核糖基化因子（ARFs）是一类分子量约为20 kDa的鸟嘌呤核苷酸结合蛋白，最初是因其能增强体外霍乱毒素催化的ADP-核糖基化作用而被鉴定出来的，随后发现它们参与高尔基体及其他细胞区室的囊泡运输。通过cDNA和基因组克隆，至少鉴定出了六种哺乳动物ARFs。布雷菲德菌素A（BFA）会破坏高尔基体膜，并抑制可溶性高分子量蛋白与高尔基体组分的结合。我们研究了在存在ATP再生系统和一部分可溶性高分子量辅助蛋白（SAP）的情况下，BFA对ARF1、-3和-5与高尔基体组分结合的影响，这些辅助蛋白可能含有被其他人鉴定为参与囊泡运输的包被蛋白复合物。在所有情况下，ARF的结合都依赖于鸟苷5'-O-(3-硫代三磷酸)，并且ATP再生系统会使其增加。SAP增强了ARF1和-3的结合，但没有增强ARF5的结合。BFA抑制了ARF1和-3的SAP依赖性结合，但不抑制其SAP非依赖性结合。它对ATP再生系统产生的结合增加没有影响。BFA的无活性衍生物B36不抑制ARF1和-3的SAP依赖性结合。ARF5的结合不依赖于SAP，不受BFA影响。这些观察结果与以下结论一致：哺乳动物ARFs在与高尔基体以及可能与其他细胞膜相互作用时对辅助蛋白的依赖性不同，并且BFA特异性抑制SAP依赖性ARF结合。
