Regulation of Golgi-derived vesicular transport by the phospholipid transfer protein, Sec14, and an oxysterol binding protein homologue, Osh4/Kes1.
Date
2007
Authors
Fairn, Gregory D.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
Phosphatidylcholine (PC) is the most abundant phospholipid within eukaryotes, comprising over 50% of the cellular phospholipid, and thus plays a crucial role in the formation of biological membranes. Conversely, phosphatidylinositol 4-phosphate (PI-4P) is a rare phospholipid in eukaryotes comprising <0.1% of the total phospholipid; however, PI-4P is also essential for eukaryotic growth and viability. In this thesis, I exploited the genetic tractability of the budding yeast Saccharomyces cerevisiae to explore the regulation of PC and PI-4P homeostasis in eukaryotic cells.
Sec 14 is essential for growth of Saccharomyces cerevisiae, with loss of function altering both PC and PI-4P metabolism. Consequences resulting from loss of Sec14 function include a block in secretion from the trans-Golgi, a subsequent accumulation of aberrant membranous structures and vesicles, and ultimately death. Mutations in the enzymes in the CDP-choline pathway for PC biosynthesis and Kes1, an oxysterol binding protein homologue and PI-4P binding protein, bypass the essential requirement for Sec14. Using high-throughput genetic analysis I determined that sec14Delta cki1Delta and sec14Delta kes1Delta strains had additional genetic requirements for life, not seen with wild-type cells. The screens identified components of the Transport Protein Particle II (TRAPP II) and the Rab protein Ypt31 as being required for the viability of the sec14 cki1 strain. A yeast strain with an enfeebled Golgi PI 4-kinase, Pik1, also displayed a heightened genetic requirement for TRAPP II and Ypt31. The results suggested that Kes1 attenuates PI-4P signaling at the Golgi, as ablation of KES1 alleviates secretion defects and the lethality associated with loss of Sec14 or Pik1 function. Saccharomyces cerevisiae cells contain nearly as many Kes1 molecules as PI-4P molecules, suggesting Kes1 could attenuate PI-4P signaling simply by binding all of the available PI-4P. Analysis of the cellular phosphoinositides revealed that cells lacking a functional Kes1 also have an increase in PI-4P production. Microscopic analysis using a fluorescent PI-4P probe revealed that the increased PI-4P production occurs in the Golgi. Thus the genetic, cellular and biochemical evidence suggests that Kes1 negatively regulates Golgi-derived secretion by regulating the production and availability of PI-4P produced by Pik1.
Thesis (Ph.D.)--Dalhousie University (Canada), 2007.
Sec 14 is essential for growth of Saccharomyces cerevisiae, with loss of function altering both PC and PI-4P metabolism. Consequences resulting from loss of Sec14 function include a block in secretion from the trans-Golgi, a subsequent accumulation of aberrant membranous structures and vesicles, and ultimately death. Mutations in the enzymes in the CDP-choline pathway for PC biosynthesis and Kes1, an oxysterol binding protein homologue and PI-4P binding protein, bypass the essential requirement for Sec14. Using high-throughput genetic analysis I determined that sec14Delta cki1Delta and sec14Delta kes1Delta strains had additional genetic requirements for life, not seen with wild-type cells. The screens identified components of the Transport Protein Particle II (TRAPP II) and the Rab protein Ypt31 as being required for the viability of the sec14 cki1 strain. A yeast strain with an enfeebled Golgi PI 4-kinase, Pik1, also displayed a heightened genetic requirement for TRAPP II and Ypt31. The results suggested that Kes1 attenuates PI-4P signaling at the Golgi, as ablation of KES1 alleviates secretion defects and the lethality associated with loss of Sec14 or Pik1 function. Saccharomyces cerevisiae cells contain nearly as many Kes1 molecules as PI-4P molecules, suggesting Kes1 could attenuate PI-4P signaling simply by binding all of the available PI-4P. Analysis of the cellular phosphoinositides revealed that cells lacking a functional Kes1 also have an increase in PI-4P production. Microscopic analysis using a fluorescent PI-4P probe revealed that the increased PI-4P production occurs in the Golgi. Thus the genetic, cellular and biochemical evidence suggests that Kes1 negatively regulates Golgi-derived secretion by regulating the production and availability of PI-4P produced by Pik1.
Thesis (Ph.D.)--Dalhousie University (Canada), 2007.
Keywords
Biology, Molecular.