Abstract
Sterols are essential lipids of most eukaryotic cells with multiple functions (structural, regulatory and developmental). Sterol profile of yeast cells is often determined during the studies of ergosterol synthesis mutants used to uncover a number of functions for various sterols in yeast cells. Molecular studies of ergosterol biosynthesis have been also employed to identify essential steps in the pathway against which antifungals might be developed. We present here a protocol for the isolation of non-saponifiable lipids (sterols) from Kluyveromyces lactis yeast cells and a chromatographic method for quantitative analysis of sterols in lipid extracts (HPLC) that can be performed in laboratories with standard equipment.
Keywords: Ergosterol biosynthesis, Kluyveromyces lactis, Lipid extraction, High-performance liquid chromatography
Background
Ergosterol, the primary membrane sterol found in yeast cells, serves a structural role in cellular membranes similar to that of cholesterol in mammalian systems. Sterols have been shown to be responsible for a number of important physical characteristics of membranes by affecting rigidity, fluidity and permeability of membranes. Through their interactions with phospholipids and sphingolipids, sterols are proposed to maintain the lateral heterogeneity of the protein and lipid distribution in the plasma membrane because of their putative role in inducing microdomains called lipid rafts (Dupont et al., 2011; Souza et al., 2011). Sterol biosynthesis in yeast is an energy-expensive, multistep aerobic process, requiring heme and molecular oxygen. Ergosterol and its biosynthetic steps are the major targets for antifungal compounds which have minor effects on cholesterol synthesis of the host organism (Daum et al., 1998). Both ergosterol and some of its biosynthetic intermediates (squalene, 7-dehydrocholesterol) belong to chemicals with a direct positive appeal to people. Therefore a simple and reliable method for sterol isolation is highly rewarding (Valachovic and Hapala, 2017). In this protocol, we describe the analytical method for sterol isolation used for determination of sterol profile in yeast cells.
Materials and Reagents
Equipment
Procedure
Data analysis
The peak identity was determined from the retention times of ergosterol standard and from characteristic spectra (Figure 1). The quantity of sterols could be calculated from calibration curves constructed for individual standards used. Figure 1. Chromatogram of sterols (A) Standard ergosterol (0.8 mmol/L) and (B) Sterol profile of K. lactis cells (ergosterol represents more than 80% of total amount of yeast sterols)
Notes
Recipes
Acknowledgments
This protocol was adapted from Valachovic and Hapala (2017) and used in our previous studies (Goffa et al., 2014; Konecna et al., 2016). This work was supported by the Slovak Research and Development Agency grant APVV-0282-10 and VEGA 2/0111/15.
References
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