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Abstract
The budding yeast Saccharomyces cerevisiae is a powerful and well-studied model organism that is responsible for much of our understanding in the fields of endocytosis, the secretory pathway, and membrane trafficking. Traditionally, it was thought that endomembrane systems were highly conserved from yeast to vertebrate cells, with cargo first being internalized at the plasma membrane (PM) before being trafficked to the early endosome via vesicle budding and subsequent fusion. From here, cargo could be recycled back to the PM via recycling endosomes, or remain in the endosome as it matures, eventually being degraded as the late endosome fuses to a lysosome (or the vacuole in yeast). In recent years, there has been confusion in defining the fundamental organelles that comprise the yeast endomembrane system. Some groups have shown that budding yeast has a minimal endomembrane that lacks early and recycling endosomal structures. While others have found specific cargos do sort through recycling endosomes in yeast. With the vast amount of information that has been discovered using the budding yeast endomembrane, the field must now come to understand these pathways in the context of these new paradigms. The aim of this dissertation is to clarify which organelle is accepting post-endocytic vesicles and to determine the fusion machinery that mediates their fusion. Central to membrane fusion is a family of proteins known as SNAREs (Soluble NSF Attachment protein Receptors), which are found on both vesicle and target membrane structures. These SNAREs are localized to distinct membranes and are responsible for specific vesicle fusion events, making them an excellent lens for studying the endomembrane system. Chapter 1 is a published review that synthesizes what is known about SNARE distribution and interactions in budding yeast. Chapter 2 describes a novel CRISPR-Cas9 strategy we developed to engineer marker-free SNARE mutants in budding yeast. Chapter 3 describes an unpublished study that uses yeast genetic, molecular and evolutionary biology to better understand how SNARE protein evolution and expansion gave rise to complex and modern endosomal systems.