ROLES OF GTP-BINDING PROTEINS IN THE FORMATION OF INTRACELLULAR TRANSPORT VESICLES

分泌蛋白、膜蛋白和溶酶体蛋白的细胞内运输是由载体囊泡介导的，载体囊泡从供体囊室萌发并与靶囊室融合以运送货物蛋白。在囊泡出芽过程中，多种gtp结合蛋白参与其中。在本课题中，我们研究了小gtp结合蛋白ARF家族、它们的鸟嘌呤核苷酸交换因子及其效应物，以及一个高分子量gtp结合蛋白动力蛋白及其同源物。我们已经揭露了以下内容。我们已经确定了三个ARFs的鸟嘌呤核苷酸交换因子，分别为BIG1， BIG2和GBF1，它们催化ARFs上GDP交换GTP。所有的蛋白质在其中心区域都有一个Sec7结构域，这是鸟嘌呤核苷酸交换活性所必需的。我们已经证明，GBF1参与顺式高尔基网络中copi包被囊泡的形成，BIG2参与反式高尔基网络中网格蛋白/ ap -1包被囊泡的形成(2)。我们已经确定了一个新的ARF效应家族，命名为GGA1， GGA2和GGA3。所有蛋白均具有VHS结构域、GGAH结构域、富含脯氨酸的区域和AGEH结构域。我们发现，在这些结构域中，GGAH结构域参与与gtp结合的活性arf的结合，AGEH结构域参与与Rabaptin-5和γ-synergin的结合。此外，我们已经证明所有GGA蛋白都与反式高尔基网络相关，并调节网格蛋白包被囊泡的出芽(3)。动力蛋白II不仅参与了质膜的囊泡形成，还参与了反式高尔基网络的囊泡形成。然而，通过对dynamin突变体的分析，我们发现dynamin II不参与反式高尔基网络中囊泡的形成(4)。我们已经鉴定出一种新的动力蛋白同源物，命名为DVLP，并发现它通过分子内和分子间相互作用形成四聚体。

Intracellular transport of secretory, membrane and lysosomal proteins is mediated by carrier vesicles, which bud from a donor compartment and fuse with a target compartment to deliver cargo proteins. In the process of vesicle budding, a variety of GTP-binding proteins are involved. In this research project, we have studied on the ARF family of small GTP-binding proteins, their guanine nucleotide exchange factors and their effectors, and a high molecular weight GTP-binding protein, dynamin, and its homolog. We have revealed the followings.(1). We have identified three guanine nucleotide exchange factors for ARFs, designated BIG1, BIG2 and GBF1, which catalyze the exchange of GDP for GTP on ARFs. All the proteins have a Sec7 domain in their central region, which is essential for the guanine nucleotide exchange activity. We have shown that GBF1 is involved in the formation of COPI-coated vesicles from the cis-Golgi network and BIG2 in the formation of clathrin/AP-1-coated vesicles from the trans-Golgi network.(2). We have identified a novel family of ARF effectors, designated GGA1, GGA2 and GGA3. All the proteins have a VHS domain, GGAH domain, proline-rich region and an AGEH domain. We have found that, among these domains, the GGAH domain is involved in binding to GTP-bound active ARFs and the AGEH domain in binding to Rabaptin-5 and γ-synergin. Further, we have shown that all the GGA proteins are associated with the trans-Golgi network and regulate the budding of clathrin-coated vesicles.(3). Dynamin II has been proposed to be involved in the formation of vesicles not only from the plasma membrane but also from the trans-Golgi network. However, by analysis using dynamin mutants, we have shown that dynamin II is not involved in the formation of vesicles from the trans-Golgi network.(4). We have identified a novel dynamin homolog, designated DVLP, and found that it forms a tetramer by virtue of intramolecular and intermolecular interactions.