FATTY-ACID ELONGATION IN YEAST - BIOCHEMICAL CHARACTERISTICS OF THE ENZYME-SYSTEM AND ISOLATION OF ELONGATION-DEFECTIVE MUTANTS

Elongation of long-chain fatty acids was investigated in yeast mutants lacking endogenous de novo fatty acid synthesis. In this background, in vitro fatty acid elongation was dependent strictly on the substrate s malonyl-CoA, NADPH and a medium-chain or long-chain acyl-CoA primer of 10 or more carbon atoms. Maximal activity was observed with primers containing 12-14 carbon atoms, while shorter-chain-length acyl-CoA we re almost (octanoyl-CoA) or completely (hexanoyl-CoA, acetyl-CoA) inac tive. In particular, acetyl-CoA was inactive as a primer and as extend er unit. The Michaelis constants for octanoyl-CoA (0.33 mM), decanoyl- CoA (0.83 mM) lauroyl-CoA (0.05 mM), myristoyl-CoA (0.4 mM) and palmit oyl-CoA (0.13 mM) were determined and were comparable for fatty acid s ynthesis and elongation. In contrast, the affinity of malonyl-CoA was 17-fold lower for elongation (K-m = 0.13 mM) than for the fatty acid s ynthase (FAS) system. With increasing chain length of the primer (grea ter than or equal to 12:0), fatty acid elongation becomes increasingly sensitive to substrate inhibition. Due to the activation of endogenou s fatty acids, ATP exhibits a stimulatory effect at suboptimal but not at saturating substrate concentrations. In the yeast cell homogenate, the specific activity of fatty acid elongation is about 10-20-fold lo wer than that of de novo fatty acid synthesis. The same elongation act ivity is observed in respiratory competent and in mitochondrially defe ctive cells. The products of ill vitro fatty acid elongation are fatty acids of 15-17 or 22-26 carbon atoms, depending on whether tridecanoy l-CoA or stearoyl-CoA is used as a primer. In vitro, the elongation pr oducts are converted in part, by alpha-oxidation, to their odd-chain-l ength lower homologues or are hydrolyzed to fatty acids. In contrast, no odd-chain-length elongation products or very-long-chain fatty acids (VLCFA) shorter than 26:0 are observed in vivo. Hence, VLCFA synthesi s exhibits a higher processivity in vivo than in the cell homogenate. In addition: the in vivo process appears to be protected against side reactions such as hydrolysis or alpha-oxidation. Yeast mutants defecti ve in 12:0 or 13:0 elongation were derived from fits-mutant strains ac cording to their failure to grow on 13:0-supplemented media. In vivo, 12:0 elongation was reduced to 0-10% of the normal level, while 16:0 e longation and VLCFA synthesis were unimpaired. It is concluded that ye ast contains either two different elongation systems, or that the resp ective mutation interferes differentially with medium-chain and long-c hain fatty acid elongation. The yeast gene affected in the elongation- defective mutants was isolated and, upon sequencing, identified as the known ELO1 sequence. It encodes a putative membrane protein of 32-kDa molecular mass with no obvious similarity to any of the known FAS com ponent enzymes.