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

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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.