Disruption of the Saccharomyces cerevisiae FAT1 gene decreases very long-chain fatty acyl-CoA synthetase activity and elevates intracellular very long-chain fatty acid concentrations

Activation of fatty acids to their coenzyme A derivatives is necessary for subsequent metabolism, Very long-chain fatty acids, which accumulate in tissues of patients with X-Linked adrenoleukodystrophy, are activated by very long-chain acyl-CoA synthetase (VLCS) normally found in peroxisomes and microsomes, We identified a candidate yeast VLCS gene (FAT1), previously identified as encoding a fatty acid transport protein, by its homology to rat liver peroxisomal VLCS, Disruption of this gene decreased, but did not abolish, cellular VLCS activity. Fractionation studies showed that VLCS activity, but not long-chain acyl-CoA synthetase activity, was reduced to about 40% of wild-type level in both 27,000 x g supernatant and pellet fractions. Separation of organelles in the pellet fraction by density gradient centrifugation revealed that VLCS activity was associated with peroxisomes and microsomes but not mitochondria, FAT1 deletion strains exhibited decreased growth on medium containing dextrose, oleic acid, and cerulenin, an inhibitor of fatty acid synthesis. FAT1 deletion strains grown on either dextrose or oleic acid medium accumulated very long-chain fatty acids. Compared wth wild-type yeast, C22:0, C24:0, and C26:0 levels were increased approximately 20-, 18-, and 3-fold in deletion strains grown on dextrose, and 2-, 7-, and B-fold in deletion strains grow on oleate. Long-chain fatty acid levels in wild-type and deletion strains were not significantly different.All biochemical defects in FAT1 deletion strains were restored to normal after functional complementation with the FAT1 gene. The level of VLCS activity measured in both wild-type and deletion yeast strains transformed with FAT1 cDNA paralleled the level of expression of the transgene. The extent of both the decrease in peroxisomal VLCS activity and the very long-chain fatty acid accumulation in the yeast FAT1 deletion model resembles that observed in cells from X-linked adrenoleukodystrophy patients. These studies suggest that the FAT1 gene product has VLCS activity that is essential for normal cellular very long-chain fatty acid homeostasis.