Defense Date


Document Type


Degree Name

Master of Science



First Advisor

Derek Prosser



Eukaryotes rely on vesicular transport of essential cellular products, such as proteins and lipids, into and out of the cell to maintain important cellular functions necessary for survival. The internalization of cellular products into the cell’s plasma membrane (PM) is referred to as endocytosis, and typically relies on polymerization of the coat protein clathrin to stabilize membrane curvature. This study focuses on the lesser-understood mechanisms behind endocytosis, namely those that do not make use of clathrin-coated vesicles (CCVs). In the absence or deficiency of available clathrin, eukaryotic cells can activate endocytic pathways independent of CCVs, which are concurrently but often less utilized than clathrin-mediated endocytosis (CME). To better understand how this is accomplished, several experiments were conducted using S. cerevisiae, or budding yeast, mutant cells expressing fluorescently tagged cargo as well as deletions of the four main adaptor proteins required for CME; this essentially rendered the CME pathway inviable. In these cells, overexpression of the cell wall regulatory proteins Mid2, Rom1 and Rho1 activate a clathrin-independent endocytic (CIE) pathway that depends on the actin-polymerizing protein Bni1 and subunits of the polarisome complex. Here, I will characterize endocytic roles for two other polarisome subunits, Msb3 and Msb4, that are known GTPase-activating proteins (GAPs) for the secretory Rab, Sec4. While Msb3 and Msb4 activity leads to Sec4 inactivation after delivery of a secretory vesicle to the PM, they also bind to inactive Rho1. Based on this dual binding activity, I hypothesize that Msb3 and Msb4 are poised to integrate secretory and CIE events. In support of this, I find that overexpression of wild-type Msb3 or Msb4 increases internalization of the endocytic cargo Ste3-GFP, but that GAP-inactive mutants cannot. Moreover, re-expression of wild-type Msb3 or Msb4, but not the GAP-inactive mutants, in msb3msb4∆ CME-deficient cells was sufficient to allow ROM1 overexpression to activate CIE. The observed effects were not due to defective assembly of the polarisome, since Spa2-GFP localization was not perturbed in cells lacking either or both MSB genes. Based on these findings, I propose a model where the polarity proteins Msb4 and Msb4 function in compensatory endocytosis to balance membrane delivery at the cell surface via exocytosis. This activity may regulate PM-cell wall balance, or facilitate membrane retrieval during cell growth. Overall, this study provides evidence in better elucidating how CIE occurs in yeast and potentially other eukaryotes.


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Available for download on Saturday, December 12, 2026