Characterization, quantitation, and subcellular localization of inositol-containing sphingolipids of the yeast, Saccharomyces Cerevisiae

In yeast, as in higher eukaryotic cells, sphingolipids are essential membrane components. The yeast, Saccharomyces cerevisiae, contains three classes of sphingolipids, inositolphosphorylceramide (InsP Cer), mannosylinositolphosphorylceramide (ManInsP Cer) and mannosyldiinositolphosphorylceramide (ManP Ins₂P Cer). As a prerequisite to localize these sphingolipids in subcellular membranes, authentic standards of the respective lipids were isolated and characterized using biochemical methods and electrospray ionization mass spectrometry. The complete set of yeast subcellular membranes was isolated at high purity, and sphingolipids were extracted. InsP Cer, ManInsP Cer, and ManP Ins₂P Cer were separated by thin‐layer chromatography, stained and densitometrically scanned along with the respective standards. These methods enable a complete overview of the subcellular distribution of yeast sphingolipids to be obtained, as far as is known, for the first time. InsP Cer was highly enriched in Golgi and vacuolar membranes, whereas the largest amounts of ManInsP Cer and ManPIns2PCer were found in the plasma membrane. The presence of inositol‐containing sphingolipids in organelles of the protein‐secretory pathway strongly supports the notion that protein secretion and intracellular trafficking of sphingolipids are linked processes.