| The Golgi apparatus mediates processing and sorting of newly synthesized proteins in the secretory pathway. This thesis concerns two aspects of biogenesis of the Golgi apparatus: the capacity of the organelle to form de novo, and the pathway and mechanism of pH-sensitive, retrieval-based protein localization to the Golgi.; Despite its complex structure, the Golgi undergoes substantial and reversible structural transformations under a variety of conditions, most notably at mitosis when the organelle extensively vesiculates. Reassembly of the Golgi apparatus might involve a stable template comprised of Golgi matrix proteins or it might occur by self-assembly. Three observations presented here support a self-assembly mode. First, by imposing a reversible endoplasmic reticulum (ER) export blockade, it was shown that the Golgi apparatus reassembled after complete redistribution and dispersal of its components, including matrix proteins, into the membranes of the ER. Second, by analyzing membrane protein localization in 3D, it was found that the Golgi apparatus underwent a nearly complete disassembly and dispersal of all its components during mitotic vesiculation with no evidence that differential sorting leaves matrix proteins enriched in remnant structures that mediate reassembly. Third, using RNA interference to block expression of key matrix proteins, normal Golgi structure and disassembly/reassembly were observed in cells lacking GM130, giantin, or GRASP65.; In the face of anterograde membrane flow maintenance of the Golgi apparatus requires retrieval mechanisms. The cis Golgi protein GPP130 is a useful retrieval marker as it redistributes to endosomes upon lumenal pH disruption and is retrieved to the Golgi upon restoration of acidic lumenal pH. However, the identity of the endosomes and the involved cycling route are unknown. It is also unknown whether any other cis Golgi proteins participate in this pathway. We demonstrate that although the Golgi protein GP73 did not associate with GPP130, it was similar to GPP130 in that it exhibited reversible redistribution to endosomes upon lumenal pH disruption and its targeting was mediated by a lumenal coiled-coil stem domain. In addition, both proteins were found to undergo endosome-to-Golgi retrieval in the recently discovered late endosome bypass pathway. (Abstract shortened by UMI.)... |
