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In this work, I addressed issues pertaining to endomembrane transport, compartment organization, and the mechanisms behind compartmentation. First, I assessed the role of COPI in organizing the Golgi. COPI is known to play an important role in Golgi transport, but its precise function in Golgi organization and maturation was previously unclear. A puzzling question was how a single type of coated vesicle could generate the multiple molecularly distinct stages of Golgi maturation. I used an "anchor-away" technique to rapidly inactivate COPI in yeast. In addition to defects in secretion and Golgi-to-ER retrograde traffic, COPI inactivation blocked the normal maturation kinetics of early Golgi proteins. The continued cycling of late Golgi resident proteins revealed that COPI is a driving force behind early, but not late, Golgi maturation. COPI plays a part in recycling early Golgi proteins to younger cisternae. This work led to the proposal that AP-1 and clathrin-mediated transport is the most likely driver of late Golgi maturation. I also aimed to clarify the poorly understood organization of yeast endosomes. The prior understanding in the field was that yeast possesses early and late endosomes, similar to the well documented endosomal organization of mammalian cells. However, key features of the mammalian endosomal network, including a clearly defined early endosome and endosomal maturation, had not been shown in yeast. A spatiotemporal analysis of endosomal markers and endocytic cargo routes revealed three surprising findings: budding yeast lacks a mammalian-like early endosome, the yeast counterpart to the late endosome is a long-lived structure, and the yeast trans-Golgi network (TGN) serves the role of early and recycling endosomes. I directly visualized the targeting of endocytic material to the yeast TGN and showed that disrupting TGN exit blocks progress to downstream fates on the endocytic pathway. My results demonstrate that, remarkably, the TGN is the earliest destination for multiple types of endocytic cargoes. These findings support a new, streamlined model for the yeast endomembrane system that has implications for the evolutionary relationship between yeast and mammalian endosomes. The endomembrane system I describe for yeast resembles that of plants, in which the TGN also functions as an early endosome. It is possible that the early and recycling endosomal compartments and the endosomal maturation pathway represent evolutionarily novel features that permitted a more complex endosomal trafficking network in higher eukaryotes. These studies involved a considerable amount of video microscopy to track fluorescently labeled endomembrane structures and to optimize imaging and processing methods. Temporal analysis is essential to understanding maturation, protein localization, and compartmental behavior. The movies associated with each chapter are included as supplementary files online. For reference, the first frame and a description of each movie are provided at the end of their respective chapters.


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