Kahn R A, Randazzo P, Serafini T, Weiss O, Rulka C, Clark J, Amherdt M, Roller P, Orci L, Rothman J E
Laboratory of Biological Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1992 Jun 25;267(18):13039-46.
Deletion of the amino-terminal 17 residues from human ADP-ribosylation factor (ARF) resulted in a protein ([delta 1-17]mARF1p) devoid of ARF activity but which retained the ability to bind guanine nucleotides with high affinity. Unlike the wild type, the binding of guanine nucleotides to this deletion mutant was found to be independent of added phospholipids. A chimeric protein was produced, consisting of 10% (the amino-terminal 17 amino acids) human ARF1p and 90% ARL1p, an ARF-like protein (55% identical protein sequence) from Drosophila. This chimera was found to have ARF activity, lacking in the parental ARL1 protein. Thus, the amino terminus of ARF1p was shown to be a critical component of ARF activity. A synthetic peptide, derived from the amino terminus of ARF1p, has no ARF activity. Rather, the peptide was found to be a specific inhibitor of ARF activities. This peptide was also found to be a potent and specific inhibitor of both an in vitro intra-Golgi transport assay and the guanosine 5'-3-O-(thio)triphosphate-stimulated accumulation of coated vesicles and buds from Golgi preparations. We conclude that ARF is required for the budding of coated vesicles from the Golgi stacks and serves a regulatory role in protein secretion through the Golgi in eukaryotic cells.
从人ADP-核糖基化因子（ARF）中删除氨基末端的17个残基，产生了一种蛋白质（[δ1-17]mARF1p），该蛋白质缺乏ARF活性，但保留了以高亲和力结合鸟嘌呤核苷酸的能力。与野生型不同，发现鸟嘌呤核苷酸与这种缺失突变体的结合不依赖于添加的磷脂。构建了一种嵌合蛋白，它由10%（氨基末端的17个氨基酸）的人ARF1p和90%的ARL1p组成，ARL1p是一种来自果蝇的ARF样蛋白（蛋白质序列有55%相同）。发现这种嵌合体具有ARF活性，而亲本ARL1蛋白缺乏该活性。因此，ARF1p的氨基末端被证明是ARF活性的关键组成部分。一种源自ARF1p氨基末端的合成肽没有ARF活性。相反，该肽被发现是ARF活性的特异性抑制剂。还发现该肽是体外高尔基体内部运输测定以及鸟苷5'-3-O-（硫代）三磷酸刺激的高尔基体制剂包被囊泡和芽积累的有效且特异性抑制剂。我们得出结论，ARF是高尔基体堆叠产生包被囊泡所必需的，并且在真核细胞中通过高尔基体的蛋白质分泌过程中起调节作用。
