Date of Graduation

Spring 2017

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Kyoungtae Kim

Keywords

protein recycling, endocytosis, yeast, intracellular trafficking, Snc1, actin, cell biology

Subject Categories

Biology

Abstract

Protein recycling is an essential cellular process that involves endocytosis, intracellular trafficking, and exocytosis. In mammalian systems, membrane lipids (including cholesterol, sphingolipids, and phospholipids) play a pivotal role in protein recycling. To address this role in budding yeast, Saccharomyces cerevisiae, I utilized GFP-Snc1, a fluorescent marker for reporting the recycling pathway. Moderate to significant GFP-Snc1 recycling defects occurred upon overexpression or inactivation of phospholipid, ergosterol, and sphingolipid biosynthesis enzymes, indicating that homeostasis of membrane lipid levels is prerequisite for proper protein recycling. Expression of a truncated version of GFP-Snc1 that cannot be recycled from the plasma membrane revealed that abnormalities in Snc1 localization in membrane lipid overexpression or underexpression mutants are not due to defects in the synthetic/secretory pathway, but rather in the intracellular trafficking pathway. Membrane lipid imbalance resulted in an accumulation of the late endosome marker Vps10-GFP, indicating trafficking from the endosomes to the Golgi may be being hindered, preventing recycling to the plasma membrane. To elucidate the possible mechanism for this trafficking hindrance, I stained the actin cytoskeleton, then quantified the percentage of cells with visible actin cables. Compared to wild-type cells, membrane lipid mutant cells exhibited lower levels of actin cables, indicating the actin cytoskeleton is disrupted upon membrane lipid imbalance. Taken together, my results show that impairment of proper recycling may be due to disruption of the actin cytoskeleton, which causes trafficking hindrance between the endosomes and Golgi.

Copyright

© Sara Elizabeth Woodman

Available for download on Saturday, December 01, 2018

Open Access

Included in

Biology Commons

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