Sterol methylation in Saccharomyces cerevisiae

Area of Science:

• Molecular Biology
• Biochemistry
• Yeast Genetics

Background:

• S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) is crucial for ergosterol biosynthesis in yeast.
• Mutations in the erg6 gene confer nystatin resistance and affect this enzyme's activity.

Purpose of the Study:

• To investigate the lipid and sterol synthesis in Saccharomyces cerevisiae mutants deficient in delta 24-sterol-C-methyltransferase.
• To characterize the genetic locus erg6 and its enzyme activity distribution.

Main Methods:

• Genetic analysis of nystatin-resistant mutants to identify erg6 alleles.
• Biochemical assays to measure enzyme activity in different cellular fractions (mitochondria, microsomes, lipid layer).
• Sterol and lipid analysis of mutant and wild-type yeast strains, including chromatographic separation and detection.

Main Results:

• All nystatin-resistant mutants studied carried alleles of the erg6 gene, located near trp1 on chromosome 4.
• Enzyme activity for delta 24-sterol-C-methyltransferase was detected in multiple cellular fractions, influenced by assay conditions.
• Erg6 mutants accumulated sterols other than ergosterol, with undetectable levels (<10(-11) mol/10(8) cells) of ergosterol.
• Mutants exhibited significantly higher steryl ester concentrations, particularly during exponential growth.
• Changes in fatty acid composition, including increased oleic acid in phosphatidylethanolamine, were observed in specific erg6 mutants.

Conclusions:

• The erg6 gene is essential for ergosterol production in Saccharomyces cerevisiae.
• The cellular localization of delta 24-sterol-C-methyltransferase activity is complex and can be modulated.
• Ergosterol deficiency profoundly affects yeast lipid metabolism, leading to altered sterol esterification and fatty acid profiles.