Abstract
Four unlinked fatty acid activation (FAA) genes encoding acyl-CoA synthetases have been identified in Saccharomyces cerevisiae and characterized by noting the phenotypes of isogenic strains containing all possible combinations of faa null alleles. None of these genes is required for vegetative growth when acyl-CoA production by the fatty acid synthetase (Fas) complex is active. When Fas is inhibited by cerulenin, exponentially growing cells are not viable on media containing a fermentable carbon source unless supplemented with fatty acids such as myristate, palmitate, or oleate. The functionally interchangeable FAA1 and FAA4 genes are responsible for activation of these imported fatty acids. Analysis of lysates prepared from isogenic FAA1FAA4 and faa1 delta faa4 delta strains indicated that Faa1p and Faa4p together account for 99% of total cellular myristoyl-CoA and palmitoyl-CoA synthetase activities. Genetic complementation studies revealed that rat liver acyl-CoA synthetase (RLACS) rescues the viability of faa1 delta faa4 delta cells in media containing a fermentable carbon source, myristate or palmitate, plus cerulenin. Rescue is greater at 37 degrees C compared with 24 degrees C, paralleling the temperature-dependent changes in RLACS activity in vitro as well as the enzyme's ability to direct incorporation of tritiated myristate and palmitate into cellular phospholipids in vivo. Complementation by RLACS is blocked by treatment of cells with triacsin C (1-hydroxy-3-(E,E,E,2',4',7'- undecatrienylidine)triazene). Even though Faa1p, Faa4p, and RLACS are all able to activate imported myristate and palmitate in S. cerevisiae, the sensitivity of Faa4p and RLACS, but not Faa1p, to inhibition by triacsin C suggests that the rat liver enzyme is functionally more analogous to Faa4p than to Faa1p. Finally, an assessment of myristate and palmitate import into FAA1FAA4 and faa1 delta faa4 delta strains, with or without episomes that direct overexpression of Faa1p, Faa4p or RLACS, indicated that fatty acid uptake is not coupled to activation in S. cerevisiae.
Highlights
At least two metabolic pathways in S. cerevisiae that produce myristoyl-CoA: de novo synthesis or activation of free myristate by acyl-CoA synthetases
When fatty acid synthetase (Fas) is inhibited with cerulenin, wild-type strains of S. cerevisiae require supplementation of media with fatty acids such as myristate, palmitate, or oleate to remain viable
The results indicate that if Fas is active and NMTl cells are grown on rich media containing a fermentable carbon source te.g, dextrose), none of the FAA genes are needed for vegetative growth
Summary
Vol 270, No 18, Issue of May 5, pp. 10861-10867, 1995 Printed in U.S.A. Complementation of Saccharomyces cerevisiae Strains Containing Fatty Acid Activation Gene (FAA) Deletions with a Mammalian Acyl-CoA Synthetase*. Four unlinked fatty acid activation (FAA) genes encoding acyl-CoA synthetases have been identified in Saccharomyces cerevisiae and characterized by noting the phenotypes of isogenic strains containing all possible combinations of faa null alleles. When Fas is inhibited with cerulenin, wild-type strains of S. cerevisiae require supplementation of media with fatty acids such as myristate, palmitate, or oleate to remain viable These fatty acids are imported and activated by cellular acyl-CoA synthetases (Duronio et al, 1992; Johnson et al, 1994b, 1994c). 20lelp, a cis ,1.9 desaturase, accounts for all de novo production of monoenoic fatty acids from saturated acyl-CoA substrates in S. cereuisiae (Bossie and Martin, 1989; Stukey et al, 1989, 1990; McDonough et al, 1992)
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