The role of vesicle docking proteins in biogenesis and mitotic disassembly of the Golgi apparatus

Maintenance of compartmentalization of the Golgi apparatus in the presence of continuous membrane traffic depends on two key events: correct sorting of specific components into a budding vesicle, and targeted fusion of a vesicle to its acceptor compartment. This thesis addresses questions in both key steps of membrane traffic in the early secretory pathway.; Sorting during vesicle formation was studied using targeting of GPP130 in polarized cells. In these cells, all basolateral sorting signals identified to date lie exclusively in the cytoplasmic domains of the proteins examined. Hence basolateral targeting is thought to be mediated exclusively by direct interactions of the cytoplasmic domains of the proteins with specific components of the vesicle coat machinery. We demonstrate that GPP130, a cis-Golgi protein that undergoes endosome-to-Golgi retrieval using the late endosome-bypass pathway in non-polarized cells, is an important counterexample. GPP130 was shown to cycle via the basolateral membrane in polarized MDCK cells. Significantly, the membrane-proximal lumenal domain of GPP130, which mediates GPP130 localization and trafficking in non-polarized cells, was both necessary and sufficient for basolateral cycling in MDCK cells. The use of lumenal determinants for both basolateral cycling and endosome-to-Golgi retrieval suggests that a novel receptor-mediated mechanism operates at both the TGN and distal sites to sort GPP130 along the late-endosome bypass retrieval pathway in polarized cells.; Mechanisms involved in vesicle fusion were studied with emphasis on their importance in Golgi biogenesis and the mitotic Golgi vesiculation. The prevalent model, based on in vitro studies, states that cell-cycle regulation of the giantin/p115/GM130 vesicle tether formation, postulated to be the first step in vesicle docking and fusion, mediates Golgi disassembly. The role of this complex in the maintenance of Golgi structure, however, had not been demonstrated in vivo. Therefore, we generated antibodies directed against the mapped binding sites in each protein of the complex and injected these into mammalian tissue culture cells. Surprisingly, the injected anti-p115 and anti-giantin antibodies caused proteasome-mediated degradation of the corresponding antigens. Reduction of p115 levels below detection led to COPI-dependent Golgi fragmentation and apparent accumulation of Golgi-derived vesicles. (Abstract shortened by UMI.)...