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Isolation and characterization of mevinolin resistant mutants of Saccharomyces cerevisiae
M Bard1, N D Lees, A S Burnett
1Department of Biology, Indiana University-Purdue Universtiy, Indianapolis, 46223.
Abstract:
Mutant of Saccharomyces cerevisiae resistant to mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A (HMGCoA) reductase (EC1.1.1.34) were isolated and one mutant (MV71) was extensively characterized. While growth of resistant strains in the presence of mevinolin was growth. Diploids produced by mutant/wild-type matings showed levels of mevinolin resistance which indicated incomplete dominance. Sterol synthesis in the presence of mevinolin was inhibited in strain MV71 but to a lesser degree than seen in the wild-type strain. All mevinolin resistant mutants also demonstrated a slight resistance to the antibiotic nystatin. The subcellular location of HMGCoA reductase activity in MV71 and the wild-type strain were determined and it was shown that yeast HMGCoA reductase is not regulated by a dephosphorylation mechanism as has been shown for mammalian reductases. In vivo and in vitro studies of strain MV71 and the wild-type indicated that mevinolin resistance did not result in changes in HNGCoA reductase activity as has been demonstrated in mammalian systems. Based on growth data, sterol analysis, and the lack of detection of HMGCoA reductase activity differences between strain MV71 and the wild-type, mevinolin resistance is concluded to result possibly from a mutation in HMG2, one of the two functional yeast HMGCoA reductase genes, which accounts for a minor (up to 17%) amount of total cellular reductase activity.
Insights
Researchers isolated mevinolin-resistant Saccharomyces cerevisiae mutants, finding resistance possibly stems from a mutation in the HMG2 gene, affecting a minor portion of cellular 3-hydroxy-3-methylglutaryl-coenzyme-A reductase activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- Mevinolin is a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A (HMGCoA) reductase, a key enzyme in sterol synthesis.
- Understanding HMGCoA reductase regulation in yeast is crucial for metabolic studies and potential drug development.
Purpose of the Study:
- To isolate and characterize mevinolin-resistant mutants of Saccharomyces cerevisiae.
- To investigate the genetic basis and biochemical consequences of mevinolin resistance in yeast.
- To compare the regulation of HMGCoA reductase in yeast versus mammalian systems.
Main Methods:
- Isolation and genetic analysis of mevinolin-resistant yeast mutants.
- Growth assays in the presence of mevinolin.
- Sterol synthesis analysis.
- Determination of subcellular localization and activity of HMGCoA reductase.
- In vivo and in vitro enzymatic studies.
Main Results:
- A mevinolin-resistant mutant (MV71) was isolated and characterized.
- Mevinolin resistance showed incomplete dominance in diploid crosses.
- Sterol synthesis was less inhibited by mevinolin in the resistant mutant compared to wild-type.
- Mevinolin-resistant mutants exhibited slight resistance to nystatin.
- Yeast HMGCoA reductase is not regulated by dephosphorylation; resistance did not alter reductase activity.
- Resistance is possibly due to a mutation in the HMG2 gene.
Conclusions:
- Mevinolin resistance in Saccharomyces cerevisiae likely arises from a mutation in the HMG2 gene.
- This mutation affects a minor component of the total cellular HMGCoA reductase activity.
- Yeast HMGCoA reductase regulation differs from mammalian systems, particularly regarding dephosphorylation mechanisms.