REGULATION OF GTP BINDING PROTEINS

为了激活失活的ARF-GDP， 核苷酸交换蛋白（GEP）是促进GDP增长所必需的 解离和GTP结合。这个过程有时可能是 布雷菲德菌素A（Brefeldin A，BFA）BFA敏感和不敏感的GEP 在可溶性和颗粒细胞组分中均观察到。 早些时候，这个小组已经纯化了两种可溶性的对BFA不敏感的GEP， 和来自牛脑胞质溶胶的BFA抑制的GEP。序列 使用来自BFA敏感的GEP的肽克隆cDNA 对于approx。200-kDa蛋白质。推导的氨基酸序列 含有190个氨基酸的所谓Sec 7结构域。第7集A 参与蛋白质分泌和细胞粘连素-1，a 免疫系统细胞中丰富的蛋白质已被提出 是酵母Sec 7的哺乳动物对应物它被证明是相当 细胞粘连素-1具有不受抑制的ARF GEP活性 在BFA。在杆状病毒感染的Sf 9中合成His标记的GEP 使用BFA敏感性GEP克隆的细胞被BFA抑制， 证实BFA敏感性是该ARF的固有属性 GEP。目前存在两大ARF GEP家族， 清晰，更大，接近。200-kDa蛋白质被 BFA和接近。50-kDa GEPs不是。还显示 酵母Sec 7的His标记Sec 7结构域 BFA抑制GEP对酵母ARF 1和ARF 2的活性， 在蛋白质分泌中起作用，但不是酵母ARF 3。因此，Sec 7 结构域还包含至少一些ARF的决定因素 的特异性阿法普汀是在埃克斯顿的实验室中鉴定和克隆的。 以显性负性ARF 3为诱饵酵母双杂交筛选， HL-60细胞cDNA文库。他们认为39-kDa的阿法肽 可能与高尔基体的功能有关。影响的研究 arfaptin对霍乱毒素和磷脂酶D ARF活性影响 得出的结论是，它作为一种抑制剂的行动， 活化ARF，对肉豆蔻酰化I类ARF最有效，但 不干扰ARF-GDP的GEP激活。

To activate inactive ARF-GDP, a guanine nucleotide-exchange protein (GEP) is required to promote GDP dissociation and GTP binding. This process can sometimes be inhibited by brefeldin A (BFA). BFA-sensitive and insensitive GEPs have been observed in both soluble and particulate cell fractions. Earlier, this group had purified two soluble BFA-insensitive GEPs and a BFA-inhibited GEP from bovine brain cytosol. Sequences of peptides from the BFA-sensitive GEP were used to clone a cDNA for the approx. 200-kDa protein. The deduced amino acid sequence contained a so-called Sec7 domain of 190 amino acids. Sec7 is an essential yeast gene involved in protein secretion and cytohesin-1, a protein abundant in cells of the immune system had been proposed to be a mammalian counterpart of yeast Sec7. It was shown rather quickly that cytohesin-1 has ARF GEP activity that is not inhibited by BFA. A His-tagged GEP synthesized in baculovirus-infected Sf9 cells using the BFA-sensitive GEP clone was inhibited by BFA, confirming that BFA sensitivity is an intrinsic property of this ARF GEP. The existence of two major families of ARF GEPs is now clear, the larger, approx. 200-kDa proteins that are inhibited by BFA and the approx. 50-kDa GEPs that are not. It was also shown that the His-tagged Sec7 domain from yeast Sec7 has BFA-inhibited GEP activity toward yeast ARF1 and ARF2, which function in protein secretion, but not yeast ARF3. Thus, the Sec7 domain contains also at least some of the determinants of ARF specificity. Arfaptin was identified and cloned in Exton's lab in a yeast two-hybrid screen with dominant negative ARF3 as bait and HL-60 cell cDNA library. They suggested that the 39-kDa arfaptin might be involved in Golgi function. Investigation of the effects of arfaptin on ARF activiation of cholera toxin and phospholipase D led to the conclusion that it acts as an inhibitor of the action of activated ARF, most effectively on myristoylated class I ARFs, but does not interfere with GEP activation of ARF-GDP.