The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane

Abstract

To investigate the vital function(s) of the phosphoinositol-containing sphingolipids of Saccharomyces cerevisiae, we measured their intracellular distribution and found these lipids to be highly localized in the plasma membrane. Sphingolipids were assayed in organelles which had been uniformly labeled with [3H]inositol or 32P and by chemical measurements of alkali-stable lipid P, of long chain bases, and of very long chain fatty acids. We have developed an improved method for the preparation of plasma membranes which is based on the procedure of Duran et al. (Proc. Natl. Acad. Sci. USA 72:3952-3955, 1975). On the basis of marker enzyme and DNA assays carried out with a number of preparations, the plasma membranes contained less than 10% vacuolar membranes (alpha-mannosidase) and nuclei (DNA); the contamination by the endoplasmic reticulum (NADPH-cytochrome c reductase) varied from 0 to 20%. The plasma membrane preparations showed a 13-fold increase in the specific activity of vanadate-sensitive ATPase, compared with that in the homogenate, with a yield ranging from 50 to 80%. A comparison of the distribution of the ATPase with that of sphingolipids assayed by a variety of methods showed that 80 to 100% of the sphingolipids are localized in the plasma membrane; the sphingolipids constitute about 30% of the total phospholipid content of the plasma membrane. Minor amounts of sphingolipids that were found in isolated mitochondria and nuclei can be attributed to the presence of small amounts of plasma membrane in these fractions. These results suggest that one or more essential functions of these lipids is in the plasma membrane. Furthermore, sphingolipids may be useful chemical markers of the plasma membrane of S. cerevisiae.

A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of Saccharomyces cerevisiae to an antifungal plant defensin from dahlia ( Dahlia merckii )

“…In the yeast S. cerevisiae, three types of sphingolipids are synthesized: IPC, MIPC, and M(IP) 2 C. These three types of sphingolipids are located primarily in the plasma membrane (21,22), where they account for 30% of the plasma membrane phospholipids. M(IP) 2 C, the terminal sphingolipid, represents the major fraction among membrane sphingolipids (21). Sphingolipids associate with sterols in the plasma membrane to form patches (also called rafts) that are highly enriched in glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins (23).…”

Biochemistry, cell biology and molecular biology of lipids ofSaccharomyces cerevisiae

“…Using these approaches, it appears that IPC, MIPC and M(IP) 2 C are located primarily in the plasma membrane of S. cerevisiae. 94,199 It remains to be determined whether they are present in both the inner and outer leaflet of the plasma membrane or if there is an asymmetric distribution, as there is for many types of lipids present in the plasma membrane of higher eukaryotes. Sphingolipids are detected in other locations in S. cerevisiae cells, especially IPC, which is found in the Golgi as well as in the vacuole.…”

Sphingolipid transport in eukaryotic cells

“…M(IP) 2 C, the most complex sphingolipid found in yeast, accounts for 75% of the mass of total sphingolipids [80]. IPC, MIPC and M(IP) 2 C are primarily located in the yeast plasma membrane, where they constitute some 30% of total phospholipid [81]. Upon closer inspection [82], considerable amounts of IPC were found both in the Golgi and in the vacuole (the yeast equivalent of lysosomes).…”