Inositol lipid-dependent functions in Saccharomyces cerevisiae: analysis of phosphatidylinositol phosphates.
Inositol phospholipids regulate many cellular processes, including cell survival, membrane trafficking, and actin polymerization. Quantification of inositol lipids is one of the essential techniques needed for studies that aim to decipher inositol lipid-dependent cellular functions. The study of phosphoinositides in most organisms is hampered by a lack of facile genetic tools. However, the essential elements of most inositol lipid signaling pathways appear to be conserved across eukaryote phylogeny. They can therefore readily be elucidated (both genetically and biochemically) in the budding yeast Saccharomyces cerevisiae. Because of the low abundance of polyphosphoinositides in cells, many analytical methods start by radioactively labeling intact cells and then extracting the lipids with chloroform/methanol/ water mixtures based on those first devised half a century ago. Yeast present special extraction problems because the cell wall must be broken in order to facilitate solvent access and maximize lipid yield. Once lipids have been extracted, fatty acids are removed and the resulting water-soluble glycerophosphoinositol phosphates are analysed by anion-exchange HPLC. This chapter describes how to extract and quantify the inositol lipids of S. cerevisiae cells that have been radiolabeled to isotopic equilibrium with [3H]myo-inositol.
Dove
Stephen K
SK
Phosphoinositide Lab, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK. s.k.dove@bham.ac.uk
Michell
Robert H
RH
D000215:Acylation; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents / Q000737:chemistry; D008744:Methylamines / Q000737:chemistry; D018129:Phosphatidylinositol Phosphates / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011868:Radioisotopes; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D013194:Staining and Labeling
D007202:Indicators and Reagents; D008744:Methylamines; D018129:Phosphatidylinositol Phosphates; D011868:Radioisotopes; C027451:methylamine