| dc.contributor.author | Howe, Alicia G. | en_US |
| dc.date.accessioned | 2014-10-21T12:36:05Z | |
| dc.date.available | 2005 | |
| dc.date.issued | 2005 | en_US |
| dc.identifier.other | AAINR08412 | en_US |
| dc.identifier.uri | http://hdl.handle.net/10222/54743 | |
| dc.description | Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic cells, comprising 50% of total cellular phospholipid, and thus plays a major role in cellular and organellar biogenesis. In these studies, we exploited the genetic tractability of Saccharomyces cerevisiae to explore the biological roles of PC in eukaryotic cells. We used both choline deprivation and a temperature sensitive allele of PCT1 (CTP:phosphocholine cytidylyltransferase, pct1ts to regulate the CDP-choline pathway for PC synthesis and coupled this with an inactivated phosphatidylethanolamine methylation pathway to determine how cells respond to cessation of PC synthesis. PC biosynthesis decreased to negligible levels within 1 hour upon shift to the non-permissive temperature for pct1 ts, and cell growth rate visibly slowed. Cells remained viable for approximately 8 h after PC synthesis was prevented, and viability was significantly reduced at time points beyond 8 h only if cells had been grown at 37°C, not 25°C. Immunofluorescence microscopy localized Pct1 to the nucleus and nuclear membrane. Pct1 activity is regulated by Sec14, a cytoplasm/Golgi localized PC/phosphatidylinositol binding protein that regulates Golgi-derived vesicle transport partially through regulation of Pct1 activity. Our nuclear localization suggests that Sec14 regulation of Pct1 may be indirect. | en_US |
| dc.description | Sec14 is essential in Saccharomyces cerevisiae, and mutations in the enzymes of the CDP-choline pathway for PC synthesis bypass the requirement for Sec14 activity. Increased expression of Scs2, an ER localized protein related to mammalian VAP, was found to prevent growth of a bypass strain containing a temperature sensitive Sec14 mutant and a genetically inactivated choline kinase at the non-permissive temperature for sec14 ts. We have also identified multicopy suppressors of bypass at the non-permissive temperature by screening a high copy yeast library in the sec14ts cki1 strain. The screen identified a number of proteins involved in yeast polarized cell growth, including the small GTPase Cdc42 and the downstream kinases Ste20 and ClA4. Suppression of growth was not due to increased PC synthesis, and invertase secretion was not affected by increased copy number of suppressor genes. (Abstract shortened by UMI.) | en_US |
| dc.description | Thesis (Ph.D.)--Dalhousie University (Canada), 2005. | en_US |
| dc.language | eng | en_US |
| dc.publisher | Dalhousie University | en_US |
| dc.publisher | | en_US |
| dc.subject | Biology, Molecular. | en_US |
| dc.title | Using yeast genetics to understand the roles of phosphatidylcholine synthesis in the regulation of cell growth and death. | en_US |
| dc.type | text | en_US |
| dc.contributor.degree | Ph.D. | en_US |
