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A yeast strain lacking lipid particles bears a defect in ergosterol formation
Daniel Sorger1, Karin Athenstaedt, Claudia Hrastnik
1Institut für Biochemie, Technische Universität Graz, Petersgasse12/2, A-8010 Graz, Austria.
The Journal of Biological Chemistry
|May 25, 2004
Summary
Triacylglycerols (TAG) synthesis drives lipid particle growth more than steryl esters (STE) in yeast. Lack of STE reduces Erg1p stability, while TAG presence offers protection against Erg1p inhibitors.
Area of Science:
- Cell Biology
- Biochemistry
- Yeast Genetics
Background:
- Lipid particles in Saccharomyces cerevisiae store triacylglycerols (TAG) and steryl esters (STE).
- Four key enzymes, Dga1p, Lro1p, Are1p, and Are2p, are responsible for TAG and STE synthesis.
- A quadruple mutant lacking these enzymes is devoid of lipid particles, serving as a model for studying storage lipid functions.
Purpose of the Study:
- To investigate the relative contributions of TAG and STE synthesis to lipid particle formation.
- To explore the localization and stability of lipid particle proteins in the absence of storage lipids.
- To understand the physiological consequences of altered lipid metabolism on cellular processes and drug sensitivity.
Main Methods:
- Construction and analysis of a quadruple yeast mutant (dga1lro1are1are2) lacking TAG and STE synthases.
- Complementation of the quadruple mutant with inducible expression of DGA1, LRO1, or ARE2.
- Biochemical fractionation to determine protein localization (microsomal vs. lipid particle fractions).
- Analysis of gene transcription and protein stability.
- Sensitivity assays using terbinafine, an inhibitor of squalene epoxidase (Erg1p).
Main Results:
- TAG synthesis promotes lipid particle proliferation more effectively than STE synthesis.
- Proteins normally found on lipid particles localize to microsomes in the quadruple mutant, indicating a link between lipid particles and the endoplasmic reticulum.
- Erg1p stability is reduced in the quadruple mutant, primarily due to the absence of STE, and this is not due to transcriptional downregulation.
- The quadruple mutant exhibits increased sensitivity to terbinafine, suggesting a protective role for TAG and/or intact lipid particles.
- Reduced STE synthesis leads to decreased ergosterol incorporation into the plasma membrane, despite higher total cellular free ergosterol.
Conclusions:
- TAG synthesis is a major driver of lipid particle biogenesis in yeast.
- Lipid particle proteins are closely associated with the endoplasmic reticulum, and their localization/stability is influenced by specific lipid components.
- Steryl esters play a crucial role in the stability of Erg1p and the supply of ergosterol to the plasma membrane.
- The presence of TAG and/or lipid particles confers a protective effect against Erg1p inhibition.