High Spatial and Temporal Resolution Analysis of the Late Secretory Pathway and Exocytosis in S. cerevisiae

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Dynamic organization is a hallmark of life. Although changes in biological organization occur at all levels of physical scale and across all time scales, all are driven by the same underlying chemistry and processes. In this dissertation, I discuss just a few of these dynamic processes, focusing primarily on the mechanisms of vesicular trafficking, exocytosis (secretion), and cellular polarity regulation. Using budding yeast as a model organism for exploring these mechanisms, I present two main stories: one regarding a novel Rho-GAP protein which contributes to Rho3 distribution on the plasma membrane and another concerning a characterization of the timeline of events leading to the tethering and fusion of constitutive secretory vesicles with the plasma membrane. In Chapter 2, an attempt to define additional regulators of secretory vesicle transport resulted in the identification of a previously uncharacterized ORF, YHR182W, which has now been preliminarily described and named Rgd3. Rgd3 is a Rho-GAP which accelerates the GTP-hydrolysis of Rho3 and localizes to polarized vesicles, distinct from constitutive secretory vesicles. This protein is capable of directly associating with lipid membranes through an F-BAR domain and is polarized to the bud by Myo2 in a phosphorylation-state-dependent manner. While these Rgd3-bound vesicles have not been definitively identified, they appear to be transient and likely represent some component of the endosomal system. In Chapter 3, I define the timing of several events relative to secretory vesicle tethering. Constitutive secretory vesicles, marked by Sec4 and the exocyst complex, tether at the plasma membrane for approximately five seconds before membrane fusion, a time significantly shorter than previously shown. Several components, like Sec2 and Myo2, which are associated with secretory vesicles during transport, are quickly disposed of upon tethering, while others like Rho3, Sro7 and Sec1 localize to sites of exocytosis following the initial tethering of secretory vesicles. Though the order of events observed is consistent with previous models of secretion, further studies should seek to probe the timing of events relative to specific protein interactions and the exact ‘triggers’ that define protein recruitment and final membrane fusion. Despite the maturity of the field of secretory vesicle trafficking and the field of yeast biology as a whole, these two projects highlight some interesting biological questions still to be answered in this deceptively complex organism.
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192 pages
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Exocyst; Exocytosis; RhoGAP; Sec1/Munc18; Secretion; Yeast
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Bretscher, Anthony Paul
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Fromme, Chris
Emr, Scott David
Degree Discipline
Biochemistry, Molecular and Cell Biology
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Ph. D., Biochemistry, Molecular and Cell Biology
Degree Level
Doctor of Philosophy
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Government Document
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Attribution-NonCommercial 4.0 International
dissertation or thesis
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