GTP BINDING PROTEINS AND ADENYLATE CYCLASE

ADP-核糖基化因子（ARF）是约20 Kda的鸟嘌呤核苷酸- 结合蛋白最初通过其增强免疫应答的能力来鉴定， 体外霍乱毒素催化的ADP-核糖基化，随后显示， 参与高尔基体和其他细胞中的囊泡运输 隔间 通过cDNA和基因组克隆， 被确认了身份 布雷菲德菌素A（BFA）破坏高尔基体膜， 抑制可溶性高分子量蛋白质与高尔基体的结合 分数 我们研究了BFA对ARF 1，3， 5在ATP再生系统存在下与高尔基体部分连接， 可溶性高分子量辅助蛋白（SAP）级分， 推测含有被其他人鉴定为包衣体的复合物， 参与囊泡运输。 所有情况下的ARF结合均为 依赖于GTP γ S并由ATP再生系统增加。 SAP增强了ARF 1和3的结合，但不增强ARF 5。 BFA 抑制SAP依赖性，但不是SAP非依赖性， ARF 1和3。 它对产生的结合增量没有影响， ATP再生系统 B36是BFA的非活性衍生物， 抑制ARF 1和3 SAP依赖性结合。 ARF 5的结合， 是SAP独立的，不受BFA的影响。 这些观察结果 这与哺乳动物ARF在其 依赖于辅助蛋白与高尔基体的相互作用，也许， 其他细胞膜，BFA特异性抑制SAP- 依赖性ARF结合。

ADP-ribosylation factors (ARFs) are about 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 ARFs 1,3, 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 GTPgammaS and increased by the ATP-regenerating system. Binding of ARFs 1, and 3, but not ARF 5, was enhanced by SAP. BFA inhibited the SAP-dependent, but not the SAP-independent , binding of ARFs 1 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 ARFs 1 and 3. Binding of ARF 5, 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.