Phospholipid methylation and the regulation of phospholipid biosynthesis in Saccharomyces cerevisiae

Abstract

Phospholipid biosynthesis is a coordinately regulated process in the yeast Saccharomyces cerevisiae. The major membrane phospholipids phosphatidylcholine (PC) and phosphatidylinositol (PI) are synthesized via a bifurcating pathway. Several enzymes involved in synthesis of PC and PI are fully repressed only in the presence of both inositol and choline. At least three genetically defined loci (INO2, INO4, and OPI1) are involved in the coordinate regulation of this pathway at the level of structural gene mRNA abundance.;In the absence of free choline, PC is synthesized from phosphatidylethanolamine (PE) via three sequential methylation reactions. A new genetic locus, CHO2 is defined in this thesis. The CHO2 locus is unlinked to other loci affecting phospholipid synthesis. Biochemical analysis indicated that this locus likely encodes a PE methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE compared to wild type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for either choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for the viability of yeast cells. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. The CHO2 gene was cloned and used to create a null allele of cho2 by disrupting the CHO2 gene in vivo. The resultant null allele was phenotypically the same as the original cho2 mutant.;The CHO2 gene was used as a probe to study mRNA abundance under various growth conditions. Steady-state CHO2 mRNA levels were repressed by the presence of soluble phospholipid precursors. CHO2 mRNA was present at repressed levels in the non-derepressable ino2 and ino4 regulatory mutants, regardless of the growth medium. Conversely, CHO2 mRNA levels were elevated in all media in the constitutive opi1 regulatory mutant. Evidence described in this thesis indicated that ongoing PC synthesis is required for proper repression of enzymes involved in phospholipid biosynthesis.