Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase.

Abstract

We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzymosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 309-amino acid polypeptide (calculated molecular mass, 36.48 kDa). The amino acid sequence shows a C-terminal endoplasmic reticulum retrieval signal KKXX and three histidine-rich clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The sterol profile of an ERG25 disruptant was consistent with the erg25 allele obtained by mutagenesis.In the synthesis of sterols, required components of eukaryotic membranes, an initial sterol (lanosterol in animals and fungi and cycloartenol in plants) undergoes three demethylations prior to formation of the end product sterol. The first demethylation occurs directly with lanosterol or cycloartenol and results in removal of the C-14 methyl group. The fungal demethylation is performed by the product of the ERG11 gene (Fig.

Functional identification of sterol‐4α‐methyl oxidase cDNAs from Arabidopsis thaliana by complementation of a yeast erg25 mutant lacking sterol‐4α‐methyl oxidation1

“…5) and 4,4-dimethyl-zymosterol (peak 2, Fig. 5) but no ergosterol as previously shown [9] (Fig. 5, Table 1).…”

“…S. cerevisiae. The methyl oxidase de¢cient strain, erg25-25c (Mat a ade5, his3, leu2-3, ura3-52, upc2, erg25-25c) used in this study has been previously described [9]. Sterol auxotrophs were grown an- E-mail address: enzymo@bota-ulp.u-strasbg.fr (A. Rahier).…”

“…Similarly, the EST clone 241A21T7 (accession number N65676) was shown to contain a 732 bp fragment (AF222719) and the deduced protein sequence had 35 and 40% identity with H. sapiens [8] and S. cerevisiae [9] SMO, respectively. However, this polypeptide did not contain a complete ORF.…”

“…In Saccharomyces, the ERG25 product, the SMO, is an essential enzyme since disruption of ERG25 is lethal as the erg25 strain requires ergosterol (or cholesterol) supplementation for viability [9]. Thus the pVT-VOID transformants were ¢rst plated onto an ergosterol containing medium then replicated onto a medium devoid of sterol.…”

Sterol Methyl Oxidases Affect Embryo Development via Auxin-Associated Mechanisms

“…These results suggest that the two SMO family proteins perform specific C-4 demethylation, with SMO1 participating in the first C-4a-methyl group removal and SMO2 participating in the second C-4 methyl group removal (Rahier, 2011). In yeast, ERG25 was demonstrated to be essential because the yeast erg25 mutant was lethal (Bard et al, 1996). In humans, SMO deficiency was reported to cause growth delays during infancy (He et al, 2011).…”