FAH1P, A SACCHAROMYCES-CEREVISIAE CYTOCHROME B(5) FUSION PROTEIN, ANDITS ARABIDOPSIS-THALIANA HOMOLOG THAT LACKS THE CYTOCHROME B(5) DOMAIN BOTH FUNCTION IN THE ALPHA-HYDROXYLATION OF SPHINGOLIPID-ASSOCIATED VERY LONG-CHAIN FATTY-ACIDS

A search of the Saccharomyces cerevisiae genome data base for cytochro me b(5)-like sequences identified a 1.152-kilobase pair open reading f rame, located on chromosome XIII at locus YMR272C (FAH1). That gene en codes a putative 384-amino acid protein with an amino-terminal cytochr ome b(5) domain, The b(5) core domain shows a 52% identity and 70% sim ilarity to that of the yeast microsomal cytochrome b(5) and a 35% iden tity and 54% similarity to the b(5) core domain of OLE1, the S. cerevi siae Delta-9 fatty acid desaturase, Expression of the S. cerevisiae FA H1 cytochrome b(5) domain in Escherichia coli produces a soluble prote in that exhibits the typical oxidized versus reduced differential abso rbance spectra of cytochrome b(5). Sequence analysis of Fah1p reveals other similarities to Ole1p, Both proteins are predicted to have two h ydrophobic domains, each capable of spanning the membrane twice, and b oth have the HX(2-3)(XH)H motifs that are characteristic of membrane-b ound fatty acid desaturases, These similarities to Ole1p suggested tha t Fah1p played a role in the biosynthesis or modification of fatty aci ds. Disruption of the FAH1 gene in S. cerevisiae did not give any visi ble phenotype, and there was no observable difference in content or di stribution of the most abundant long chain saturated and unsaturated 1 4-18-carbon fatty acid species, Northern blot analysis, however, showe d that this gene is expressed at much lower levels (similar to 150-fol d) than the OLE1 gene, suggesting that it might act on a smaller subse t of fatty acids, Analysis of sphingolipid-derived very long chain fat ty acids revealed an approximately 40 fold reduction of alpha-HO 26:0 and a complementary increase in 26:0 in the gene-disrupted fah1 Delta strain, GAL1 expression of the S. cerevisiae FAH1 genes in the fah1 De lta strain restores alpha-HO 26:0 fatty acids to wild type levels, Als o identified are a number of homologs to this gene in other Species, E xpression of an Arabidopsis thaliana FAH1 gene, which does not contain the cytochrome b(5) domain, in the fah1 Delta strain produced an appr oximately 25-fold increase in alpha-HO 26:0 and reduced the levels of its 26-carbon precursor, suggesting that it functions in very long cha in fatty acid hydroxylation using an alternate electron transfer mecha nism.