Cytohesin-1 in 2001.
Cytohesin-1 in 2001.
复制标题
细胞粘附素-1，2001 年。
DOI：
10.1006/abbi.2001.2661
复制
发表时间：
2002
影响因子：
3.9
通讯作者：
Vaughan,Martha
中科院分区：
文献类型：
--
作者：
Moss,Joel;Vaughan,Martha
20-kDa ARF（ADP-核糖基化因子GTP酶）3是所有真核细胞中众多囊泡运输途径的关键组分（综述见参考文献10）。1和2）。它们还直接激活哺乳动物磷脂酶D1和磷脂酰肌醇4-磷酸5-激酶（3）。ARF被GTP结合激活，当结合的GTP水解为GDP时失活。由于这两个过程本质上都非常缓慢，ARF失活需要GTP酶激活蛋白（GAP）的作用，而激活依赖于鸟嘌呤核苷酸交换蛋白（GEP）。这些重要的调控分子家族的目录随着更多成员被认识而继续扩展。在ARF GEP的情况下，一些（例如，cytohesin-1）已经知道其他功能，一些直接通过蛋白质纯化或数据库搜索新的候选序列进行鉴定。在所有这些蛋白质中，GEP活性是200个氨基酸的所谓Sec 7结构域的特性。在布雷菲尔德菌素A（BFA）抑制的GEP中发现了Sec 7结构域的存在（4），此后不久，Chardin等人。(5)报道，重组合成的人细胞粘连素Sec 7结构域，他们命名为ARNO，即ARF核苷酸结合位点开放器，足以激活ARF。在人类基因组的草图中，13个不同的基因含有Sec 7结构域（6），这表明更多的ARF GEP仍有待鉴定。在200和190 kDa的两种蛋白质之前几年，在高尔基体膜中描述了被BFA抑制的GEP活性（7，8），这两种蛋白质中的每一种都表现出被BFA抑制的GEP活性，作为670-kDa复合物的组分纯化（4）。两者后来都被克隆和表征（9-11）。来自200-kDa蛋白质的肽的氨基酸序列表明，它可能是酵母Sec 7的同源物，Sec 7是酿酒酵母中囊泡运输所必需的蛋白质（12）。由于胞粘连蛋白-1中存在Sec 7结构域，因此研究并证明了其GEP活性（13）。Sec 7结构域包含BFA敏感性和GEP活性本身的决定因素。在BFA敏感和不敏感的Sec 7结构域之间一致不同的特定氨基酸的突变鉴定出似乎负责BFA敏感性的那些（14，15）。BFA抑制的动力学特征为非竞争性（16，17）。对与ARF-GDP相关的Sec 7结构域结构的详细研究表明，当ARF-GDP与Sec 7结构域相互作用时，形成BFA结合位点（15，16）。只有这样BFA才能结合，导致形成四聚体蛋白-核苷酸-抑制剂复合物，该复合物有效地将ARF从其功能循环中去除，直到当BFA浓度降低时复合物解离。毫无疑问，其他GEP相互作用同样会导致相关分子产生新的（或改变的）物理或催化性质。
The 20-kDa ARFs (ADP-ribosylation factor GTP-ases) 3 are critical components of numerous vesicular trafficking pathways in all eukaryotic cells (reviewed in Refs. 1 and 2). They also directly activate mammalian phospholipase D1 and phosphatidylinositol 4-phosphate 5-kinase (3). ARF is activated by GTP binding and inactivated when bound GTP is hydrolyzed to GDP. Because both processes are intrinsically very slow, ARF inactivation requires the action of a GTPase-activating protein (GAP) and activation depends on guanine nucleotideexchange proteins (GEPs). The catalog of these important families of regulatory molecules continues to expand as more members are recognized. In the case of ARF GEPs, some (eg, cytohesin-1) were already known for other functions, and some were identified directly by protein purification or database searching for new candidate sequences. In all of these proteins, GEP activity is a property of a so-called Sec7 domain of 200 amino acids. The presence of a Sec7 domain was noted in a brefeldin A (BFA)-inhibited GEP (4), and soon thereafter, Chardin et al.(5) reported that the recombinantly synthesized Sec7 domain of a human cytohesin that they named ARNO, for ARF nucleotide-binding-site opener, was sufficient for ARF activation. In the draft of the human genome, 13 different genes contain a Sec7 domain (6), suggesting that more ARF GEPs remain to be identified.A GEP activity that was inhibited by BFA was described in Golgi membranes (7, 8) several years before two proteins of 200 and 190 kDa, each of which exhibited GEP activity that was inhibited by BFA, were purified as components of a 670-kDa complex (4). Both were later cloned and characterized (9–11). Amino acid sequences of peptides from the 200-kDa protein suggested that it might be a homologue of yeast Sec7, a protein essential for vesicular transport in Saccharomyces cerevisiae (12). Because of the presence of a Sec7 domain in cytohesin-1, its GEP activity was investigated and demonstrated (13). The Sec7 domain contains determinants of BFA sensitivity and GEP activity itself. Mutation of specific amino acids that differ consistently between BFA-sensitive and-insensitive Sec7 domains identified those that appeared to be responsible for BFA sensitivity (14, 15). The kinetics of BFA inhibition characterized it as uncompetitive (16, 17). Detailed studies of the structure of the Sec7 domain associated with ARF–GDP showed that the BFA-binding site is formed when ARF–GDP interacts with the Sec7 domain (15, 16). Only then can BFA bind, resulting in the formation of a tetrameric protein–nucleotide–inhibitor complex that effectively removes ARF from its functional cycle until the complex dissociates when the concentration of BFA decreases. There seems little doubt that other GEP interactions similarly result in the creation of new (or altered) physical or catalytic properties for the molecules involved.