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Dissertation
Structure/function analysis of the putative lipid transporter Arv1 and investigation of its role in polarized growth in Saccharomyces cerevisiae
Doctor of Philosophy (Ph.D.), Drexel University
Dec 2010
DOI:
https://doi.org/10.17918/00007809
Abstract
The function of the putative lipid transporter, Arv1, is essential at high temperatures and in mutants unable to esterify sterols in the yeast Saccharomyces cerevisiae. Deletion of ARV1 results in defects in sphingolipid metabolism, sterol trafficking, and GPI biosynthesis. Our studies focused on examining the role of Arv1 in yeast cell growth. We used structure/function analysis to dissect the multiple roles of Arv1 during yeast conjugation, growth under heat stress, and growth in the presence of antifungal drug compounds. Here we demonstrate that arv1 cells are mating defective because of a failure to effectively recruit the MAPK scaffold Ste5 to the plasma membrane. Failure to polarize phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) likely contributes to the mislocalization of Ste5 during the mating response in arv1. These defects ultimately lead to the inability of arv1 cells to generate shmoos or mate. We also show that arv1 cells are highly sensitive to antifungal drug compounds including the polyene compound nystatin and the azole compounds fluconazole and miconazole. Structure/function analysis of Arv1 identified critical residues required for Arv1 function during growth at high temperature and in the presence of antifungal drugs. Using targeted mutagenesis of conserved residues and charged-to-alanine scanning mutagenesis, L13, C27, 147, R58 H59, N63, H68 K71, K146 K147, and K205 H206 were found to be essential for Arv1 function. To gain a better understanding of Arv1 function, membrane topology studies were initiated; we found that the conserved Arv1 homology domain (AHD) is cytosolic and that Arv1 has a large ER luminal loop. Finally, we discovered that the increased sensitivity to azole compounds observed in arv1 cells may be attributed to decreased protein levels of the azole target Erg11. Importantly, the conserved AHD was shown to be required for all Arv1 functions tested.
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Details
- Title
- Structure/function analysis of the putative lipid transporter Arv1 and investigation of its role in polarized growth in Saccharomyces cerevisiae
- Creators
- Michelle Luan Villasmil
- Contributors
- Joseph T. Nickels (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xii, 163 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Drexel University
- Other Identifier
- 991021888820404721