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Abstract
The Golgi apparatus functions as the heart of the eukaryotic cellular endomembrane system, sorting newly made, ER-exported proteins and lipids while circulating and recycling older ones from other organelles. Many membrane traffic machinery proteins reside at the Golgi, with their localizations polarized across the set of cisternae comprising this organelle. A suite of resident Golgi enzymes is likewise polarized. These enzymes sequentially modify proteins and lipids passing through the Golgi, fine-tuning them for their appropriate functions at other organelles or the cell exterior. To perform these functions, the Golgi organizes itself through cisternal maturation, a process by which individual Golgi cisternae are assembled, biochemically altered over time, and then disassembled. Retrograde intra-Golgi vesicular transport pathways are essential for the fidelity of this maturation process, because they both polarize and recycle resident Golgi proteins by allowing younger cisternae to receive these proteins from older cisternae. In spite of this understanding, the number of intra-Golgi vesicular transport pathways and their molecular characteristics have not yet been identified.
In this thesis, I identify and characterize three molecularly distinct intra-Golgi vesicular trafficking pathways at the Saccharomyces cerevisiae Golgi. To accomplish this, I designed vesicle capture and tethering assays that concentrate distinct vesicle populations at the yeast bud neck based on cargo content or affinity with a vesicle tether. Both assay types reveal three populations of vesicles containing largely unique sets of transmembrane resident Golgi proteins. Each of these vesicle populations presumably belongs to a distinct intra-Golgi trafficking pathway. The first two pathways employ coat protein complex I (COPI) for vesicle formation and operate at early and intermediate stages of cisternal maturation. The early COPI pathway apparently uses the golgin Rud3 to tether its vesicles prior to their fusion with cisternae, and the intermediate COPI pathway likewise uses the Sgm1 golgin tether. At the late Golgi, an intra-Golgi trafficking pathway is operated by the clathrin coat and its AP-1 and Ent5 adapter proteins. This AP-1/Ent5 pathway utilizes the Imh1 golgin and Golgi-associated retrograde protein (GARP) complex to coordinately tether its vesicles. Cargo-based vesicle capture analysis further differentiates the intra-Golgi AP-1/Ent5 pathway from other transport pathways that traffic proteins between the late Golgi and prevacuolar endosome (PVE). The PVE-to-Golgi pathway notably utilizes Imh1 but apparently not GARP to tether its vesicles at cisternae. Altogether, the findings presented in this thesis grant us important insights into the molecular mechanisms of Golgi self-organization, particularly with regard to resident protein retention and polarization. Collectively, this work comprises the first systematic molecular characterization of intra-Golgi trafficking pathways.
A substantial amount of data presented in this thesis is from fluorescence time lapse microscopy of live yeast cells. Such experiments showcase the dynamic nature of cisternal maturation, and their data are naturally depicted as videos. Cryo-electron tomography data obtained for this thesis can also be represented as videos revealing a continuum of sections through the 3D volume. Representative videos for both experiment types have been archived online as supplementary files. As a reference, the first frame of each video and an accompanying legend are included at the end of each pertinent chapter.