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Updated: May 17, 2026
Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry
Published on: March 8, 2020
A genetic and pharmacological analysis of isoprenoid pathway by LC-MS/MS in fission yeast
Tomonori Takami1, Yue Fang, Xin Zhou
1Division of Molecular Pharmacology and Pharmacogenomics, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan.
Abstract:
Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In hmg1-1, a mutant of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ergosterol (a final sterol product), and squalene (an intermediate pathway product), were decreased to approximately 80% and 10%, respectively, compared with that of wild-type cells. Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level.
Insights
Fission yeast models reveal that statins and antifungals impact the isoprenoid pathway. Decreased intermediate products, not just ergosterol, likely cause observed phenotypes.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- The isoprenoid pathway is crucial for cellular function, but its study in mammals is limited by genetic manipulation challenges.
- Statins are the primary drugs targeting this pathway in mammals, with limited tools to study enzyme inhibition effects.
- Fission yeast offers a tractable model with available mutants and inhibitors (statins, azoles, terbinafine) for isoprenoid pathway research.
Purpose of the Study:
- To investigate the impact of inhibiting the isoprenoid pathway in fission yeast using genetic mutants and chemical inhibitors.
- To quantify the levels of key intermediate and final products within the isoprenoid pathway under various inhibition conditions.
- To elucidate the relationship between ergosterol levels, intermediate isoprenoid products, and observed cellular phenotypes.
Main Methods:
- Utilized fission yeast mutants (hmg1-1, Δerg6, Δsts1) and wild-type cells.
- Treated cells with statins (pravastatin) and antifungal compounds (miconazole, terbinafine).
- Quantified isoprenoid pathway products, including ergosterol, squalene, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate, using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS).
Main Results:
- The hmg1-1 mutant and pravastatin treatment significantly reduced squalene and ergosterol levels, with a more pronounced effect on squalene.
- Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) led to greater decreases in farnesyl pyrophosphate and geranylgeranyl pyrophosphate compared to ergosterol, but less than squalene.
- Mutants affecting the final steps of ergosterol biosynthesis (Δerg6, Δsts1) showed distinct changes in intermediate products compared to the hmg1-1 mutant.
- Azoles and terbinafine had minimal impact on ergosterol levels in wild-type cells.
Conclusions:
- Phenotypes associated with hmg1-1 mutation and pravastatin treatment in fission yeast are likely attributable to reduced levels of isoprenoid pyrophosphates or other pathway intermediates.
- The study highlights the utility of fission yeast as a model for studying the isoprenoid pathway and drug effects.
- Ergosterol levels alone may not fully explain the cellular consequences of isoprenoid pathway disruption.
