Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1995

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The Saccharomyces cerevisiae oxysterol binding protein homologue Kes1/Osh4 is a member of an enigmatic class of proteins found throughout Eukarya. This family of proteins is united by a ?-barrel structure that binds sterols and oxysterols. An N-terminal lid is thought to both sequester sterols inside the core and promote localization of Kes1 to regions of high membrane curvature via a predicted ArfGAP lipid packing sensor motif. Additionally, a phosphoinositide-binding region on a discrete surface of Kes1 has also been identified. In this thesis, structure-function analysis of Kes1 determined that phosphoinositide binding is required for membrane association in vitro, and in vivo phosphoinositide binding is required for localization to the Golgi. Ergosterol, the major sterol in S. cerevisiae, and membrane curvature had minimal effects on membrane association. This study also revealed a role for Kes1 in the regulation of both phosphatidylinositol-4-phosphate and phosphatidylinositol-3-phosphate homeostasis. Phosphoinositide and sterol binding by Kes1 are necessary for it to alter phosphatidylinositol-4-phosphate, but not phosphatidylinositol-3-phosphate homeostasis. Misregulation of phosphatidylinositol-4-phosphate homeostasis by Kes1 manifested itself in an inability of the v-SNARE Snc1 to traffic properly and was consistent with a defect in trans-Golgi/endosome trafficking. I went on to demonstrate a role for Kes1 in regulating the conversion of phosphatidylinositol-4-phosphate to phosphatidylinositol for the synthesis of sphingolipids, and I present a model for the role of Kes1 at the Golgi. Kes1 acts as a sterol sensor that regulates phosphatidylinositol-4-phosphate to sphingolipids metabolism, which ultimately regulates the delivery of proteins that assemble into lipid rafts for their transport from the Golgi to the plasma membrane. I also uncovered a previously unknown role for Kes1 in the regulation of the cytoplasm-to-vacuole and autophagy trafficking pathways, which is mediated by the ability of Kes1 to regulate phosphatidylinositol-3-phosphate homeostasis.

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Includes bibliographies. This thesis describes the isolation of putative promoter sequences that can produce differential expression of a gene during anaerobic wine fermentations, the use of these sequences in the development of expression vectors and the application of this work to the production of genetically engineered wine yeasts for commercial purposes.

A yeast two-hybrid assay suggested the possibility of an interaction between Cdc4p, a small EF-hand protein essential for cytokinesis, and Pik1p in S. pombe. This interaction was unexpected, as one function of Cdc4p is that of an essential light chain, bound to the first IQ-motif of type II myosins, whereas Pik1p is a phosphatidylinositol 4-kinase. The objective of this work was to analyze the effects of Pik1p lipid kinase activity on the cell cycle of S. pombe. Another goal of this study was to evaluate the functional significance of the interaction between Cdc4p and Pik1p. This was performed by generating two mutants of pik1: one that abolished lipid kinase activity (pik1-D709A) and one that abolished Pik1p Cdc4p-binding activity (pik1-R838A). Pik1p has a conserved IQ-motif in its C-terminal region. A mutation in this site (R838A), homologous to a residue which was mutated in myosin and abrogated the interaction with Cdc4p, prevented the interaction with Cdc4p in a yeast two-hybrid assay and ELISA. An increase in lipid kinase activity was observed in cell extracts upon ectopic expression of pik1-wt from an episome, which was abolished by a mutation in the lipid kinase domain of Pik1p (D709A), but not by the R838A mutation. However, little to no increase in lipid kinase activity was observed upon ectopic expression of pik1-wt and pik1-R838A in a strain carrying a conditionally lethal allele of cdc4 (cdc4-G107S). This mutation in Cdc4p was shown previously to prevent the interaction with Pik1p in yeast two-hybrid assays. Ectopic expression of pik1-wt suppressed cell proliferation, with disruption of actin cytoskeletal structures and contractile ring formation. These results were not observed with the ectopic expression of the pik1-R838A mutant or when pik1-wt was expressed in the cdc4-G107S strain. Ectopic expression of pik1-R838A resulted in cell shortening, likely through inhibition of growth, and many of the short cells showed an accumulation of the expressed Pik1p protein at the cell tips. Formation of the contractile ring appeared unaffected in cells with ectopic expression of the pik1-D709A mutant, but many of these cells had thick or more than one septum, characteristic of a septation defect. The ectopic expression phenotypes were dosage dependent since lower levels of expression greatly reduced the severity of the ectopic phenotypes. Pik1p lipid kinase activity is essential and, based on ectopic expression studies, is required for septation. There is a physical and functional interaction between Cdc4p and Pik1p which is not essential for cell viability, but suggests a role for Cdc4p in phosphoinositide metabolism.

A yeast two-hybrid assay suggested the possibility of an interaction between Cdc4p, a small EF-hand protein essential for cytokinesis, and Pik1p in S. pombe. This interaction was unexpected, as one function of Cdc4p is that of an essential light chain, bound to the first IQ-motif of type II myosins, whereas Pik1p is a phosphatidylinositol 4-kinase. The objective of this work was to analyze the effects of Pik1p lipid kinase activity on the cell cycle of S. pombe. Another goal of this study was to evaluate the functional significance of the interaction between Cdc4p and Pik1p. This was performed by generating two mutants of pik1: one that abolished lipid kinase activity (pik1-D709A) and one that abolished Pik1p Cdc4p-binding activity (pik1-R838A). Pik1p has a conserved IQ-motif in its C-terminal region. A mutation in this site (R838A), homologous to a residue which was mutated in myosin and abrogated the interaction with Cdc4p, prevented the interaction with Cdc4p in a yeast two-hybrid assay and ELISA. An increase in lipid kinase activity was observed in cell extracts upon ectopic expression of pik1-wt from an episome, which was abolished by a mutation in the lipid kinase domain of Pik1p (D709A), but not by the R838A mutation. However, little to no increase in lipid kinase activity was observed upon ectopic expression of pik1-wt and pik1-R838A in a strain carrying a conditionally lethal allele of cdc4 (cdc4-G107S). This mutation in Cdc4p was shown previously to prevent the interaction with Pik1p in yeast two-hybrid assays. Ectopic expression of pik1-wt suppressed cell proliferation, with disruption of actin cytoskeletal structures and contractile ring formation. These results were not observed with the ectopic expression of the pik1-R838A mutant or when pik1-wt was expressed in the cdc4-G107S strain. Ectopic expression of pik1-R838A resulted in cell shortening, likely through inhibition of growth, and many of the short cells showed an accumulation of the expressed Pik1p protein at the cell tips. Formation of the contractile ring appeared unaffected in cells with ectopic expression of the pik1-D709A mutant, but many of these cells had thick or more than one septum, characteristic of a septation defect. The ectopic expression phenotypes were dosage dependent since lower levels of expression greatly reduced the severity of the ectopic phenotypes. Pik1p lipid kinase activity is essential and, based on ectopic expression studies, is required for septation. There is a physical and functional interaction between Cdc4p and Pik1p which is not essential for cell viability, but suggests a role for Cdc4p in phosphoinositide metabolism.

Thesis (Ph.D)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009. / Committee Chair: Doyle, Donald; Committee Member: Barry, Bridgette; Committee Member: Bommarius, Andreas; Committee Member: Ledoux, Joe; Committee Member: Matsumura, Ichiro; Committee Member: Oyelere, Adegboyega. Part of the SMARTech Electronic Thesis and Dissertation Collection.