SACCHAROMYCES-CEREVISIAE CONTAINS 4 FATTY-ACID ACTIVATION (FAA) GENES- AN ASSESSMENT OF THEIR ROLE IN REGULATING PROTEIN N-MYRISTOYLATION AND CELLULAR LIPID-METABOLISM

Saccharomyces cerevisiae has been used as a model for studying the reg ulation of protein N-myristoylation. MyristoylCoA:protein N-myristoyl- transferase (Nmt1p), is essential for vegetative growth and uses myris toylCoA as its substrate. MyristoylCoA is produced by the fatty acid s ynthetase (Fas) complex and by cellular acylCoA synthetases. We have r ecently isolated three unlinked Fatty Acid Activation (FAA) genes enco ding long chain acylCoA synthetases and have now recovered a fourth by genetic complementation. When Fas is active and NMT1 cells are grown on media containing a fermentable carbon source, none of the FAA genes is required for vegetative growth. When Fas is inactived by a specifi c inhibitor (cerulenin), NMT1 cells are not viable unless the media is supplemented with long chain fatty acids. Supplementation of cellular myristoylCoA pools through activation of imported myristate (C14:0) i s predominantly a function of Faa1p, although Faa4p contributes to thi s process. Cells with nmt181p need larger pools of myristoylCoA becaus e of the mutant enzyme's reduced affinity for this substrate. Faa1p an d Faa4p are required for maintaining the viability of nmt1-181 strains even when Fas is active. Overexpression of Faa2p can rescue nmt1-181 cells due to activation of an endogenous pool of C14:0. This pool appe ars to be derived in part from membrane phospholipids since overexpres sion of Plb1p, a nonessential lysophospholipase/phospholipase B, suppr esses the temperature-sensitive growth arrest and C14:0 auxotrophy pro duced by nmt1-181. None of the four known FAAs is exclusively responsi ble for targeting imported fatty acids to peroxisomal beta-oxidation p athways. Introduction of a peroxisomal assembly mutation, pas1 Delta, into isogenic NMT1 and nmt1-181 strains with wild type FAA alleles rev ealed that when Fas is inhibited, peroxisomes contribute to myristoylC oA pools used by Nmt1p. When Fas is active, a fraction of cellular myr istoylCoA is targeted to peroxisomes. A NMT1 strain with deletions of all four FAAs is still viable at 30 degrees C on media containing myri state, palmitate, or oleate as the sole carbon source-indicating that S. cerevisiae contains at least one other FAA which directs fatty acid s to beta-oxidation pathways.