REGULATION OF GTP BINDING PROTEINS

激活鸟嘌呤核苷酸交换蛋白失活的ARF-GDP (GEP)需要促进GDP的解离和GTP的约束，a 可被灯盏花素A(BFA)抑制的过程。BFA敏感且 在可溶性细胞和颗粒细胞中都观察到了不敏感的GEP 分数。早些时候，这个小组已经提纯了两个不敏感的可溶性BFA GEP。今年，从牛脑中提纯了一种BFA抑制的GEP 胞浆。从纯化的GEP中提取的4个胰蛋白酶多肽含有氨基酸 与Sec 7中的序列有47%相同的序列 酿酒酵母，与以下观点一致 BFA敏感的200 kDa蛋白可能是哺乳动物中Sec7的对应物， 它在酵母泡运输中起关键作用，而Sec7可能 作为一个或多个酵母ARF的GEP。细胞粘附素-1--一种丰富的蛋白质 在免疫系统的细胞中，已经被提出是人类 酿酒酵母sec7基因产物的同源物，这在 蛋白质运输。挽救的人或酵母ARF基因的过表达 带有sec7缺陷的酵母，恢复了分泌途径功能。要定义 改善ARF与Sec7相关蛋白的相互作用，效果 在大肠杆菌中合成的细胞粘附素-1对ARF活性的影响 已评估。细胞粘附素-1增强[~(35)S]GTP[Ys]或[~3H]GDP与 ARF(即，它充当ARF-GEP)。细胞粘附素-1在急性肾衰中的作用 [35S]GTP[Ys]绑定，加速[35S]GTP[Ys]释放，这是 没有它就微不足道了。细胞粘附素-1也增加了ARF与高尔基体的结合 但其作用不被BFA抑制，BFA是一种 可逆地抑制高尔基体功能。在这方面，它不同于 BFA敏感的ARF-GEP，它包含Sec7结构域，也由 今年的组别。

To activate inactive ARF-GDP, a guanine nucleotide-exchange protein (GEP) is required to promote dissociation of GDP and binding of GTP, a process that can 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. This year, a BFA-inhibited GEP was purified from bovine brain cytosol. Four tryptic peptides from the purified GEP had amino acid sequences that were 47% identical to sequences in Sec7 from Saccharomyces cerevisiae, consistent with the view that the BFA-sensitive 200-kDa protein may be a mammalian counterpart of Sec7, which plays a critical role in yeast vesicular transport, and Sec7 may be a GEP for one or more yeast ARFs. Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homologue of the S. cerevisiae Sec7 gene product, which is crucial in protein transport. Overexpression of human or yeast ARF genes rescued yeast with Sec7 defects, restoring secretory pathway function. To define better the interaction between ARF and Sec7-related proteins, effects of cytohesin-1, synthesized in Escherichia coli, on ARF activity were evaluated. Cytohesin-1 enhanced binding of [35S]GTP[yS] or [3H]GDP to ARF (i.e., it acted as an ARF-GEP). Addition of cytohesin-1 to ARF with [35S]GTP[yS]bound, accelerated [35S]GTP[yS] release, which was negligible without it. Cytohesin-1 also increased ARF binding to Golgi membranes, but its effect was not inhibited by BFA, a drug that reversibly inhibits Golgi function. In this regard, it differs from the BFA-sensitive ARF-GEP that contains a Sec7 domain also described by the group this year.