Protocol

Lipidomics

Volume 580 of the series Methods in Molecular Biology™ pp 221-232

Date:

Monitoring Sterol Uptake, Acetylation, and Export in Yeast

  • Vineet ChoudharyAffiliated withDepartment of Medicine, Division of Biochemistry, University of Fribourg
  • , Roger SchneiterAffiliated withDepartment of Medicine, Division of Biochemistry, University of Fribourg

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Summary

Sterols are essential lipid components of eukaryotic membranes. They are synthesized in the endoplasmatic reticulum (ER) from where they are efficiently transported to the plasma membrane, which harbors ~90% of the free sterol pool of the cell. The molecular mechanisms that govern this lipid transport, however, are not well characterized and are challenging to analyze. Saccharomyces cerevisiae offers the opportunity to circumvent some of the technical limitations associated with studying this forward transport of sterols from the ER to the plasma membrane, because the organism can also take up sterols from the environment, incorporate them into the plasma membrane and transport them back to the ER, where the free sterol is converted to steryl esters. This reverse sterol transport, however, occurs only under anaerobic conditions, where the cells become sterol auxotroph, or in mutant cells that cannot synthesize heme. The reverse sterol transport pathway, however, is more amenable to experimental studies, because arrival of the sterol in the ER membrane can be monitored unambiguously by following the formation of steryl esters. Apart from sterol acylation, we have recently described a reversible sterol acetylation cycle that is operating in the lumen of the ER. Acetylation occurs on both cholesterol and pregnenolone, a steroid precursor, and serves as a signal for export of the acetylated sterols into the culture media. The time-dependent appearance of acetylated sterols in the culture supernatant thus provides a new means to monitor the forward transport of chemically modified sterols out of the ER.

Key words

Sterol Cholesterol Steroids Pregnenolone Sterol Acetylation Lipid Transport Membranes Yeast Saccharomyces cerevisiae