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.

) that either contain or lack sphingolipids, depending on whether they are cultured with a sphingolipid long-chain base. Strains lacking sphingolipid were unable to grow at low pH, at 37°C, or with high salt concentrations in the medium; these environmental stresses are known to inhibit the growth of some S. cerevisiae strains with a defective plasma membrane H+-ATPase. We found that sphingolipids were essential for proton extrusion at low pH and furthermore found that cells lacking sphingolipid no longer exhibited net proton extrusion at normal pH after a 1-min exposure to pH 3. Cells lacking sphingolipid appeared to rapidly become almost completely permeable to protons at low pH. The deleterious effects of low pH could be partially prevented by 1 M sorbitol in the suspension of cells lacking sphingolipid. Proton extrusion at normal pH (pH 6) was significantly inhibited at 39C only in cells lacking sphingolipid. Thus, the product of an SLC suppressor gene permits life without sphingolipids only in a limited range of environments. Outside this range, sphingolipids appear to be essential for maintaining proton permeability barriers and/or for proton extrusion.We are studying the function(s) of sphingolipids in Saccharomyces cerevisiae because this organism is well suited to molecular genetic analysis and because it has only a few sphingolipids, with phosphoinositol as a distinguishing feature of the polar head groups (17,18). This hydrophobic portion is composed of the long-chain base phytosphingosine, amide linked to an a-OH-C26 fatty acid (17,19).One strategy that we have adopted for studying sphingolipid function(s) is to compare the phenotype of a strain containing sphingolipids with the phenotype of the same strain that has been manipulated so as to lack sphingolipids. To implement this strategy, we isolated strains that are able to grow without making sphingolipids (5). Such strains have two essential mutations. First, the LCBJ gene is deleted so that no serine palmitoyltransferase is produced (2, 11). Since this enzyme catalyzes the first step in sphingolipid longchain base synthesis, the strain cannot make sphingolipids unless supplied with a long-chain base such as phytosphingosine (10, 20). Second, the strains carry a semidominant mutation, termed SLC (for sphingolipid compensation), that enables the cell to suppress or bypass the lcbl defect and to grow in the absence of exogenous phytosphingosine. Thus, SLC strains should be of value in unraveling sphingolipid function(s), since when cultured without phytosphingosine, they grow but make no detectable sphingolipid, whereas when grown with phytosphingosine, they make a normal complement of sphingolipids (5). We demonstrate here that in certain restrictive environments, SLC strains exhibit a sphingolipid requirement for the maintenance of a proton permeability barrier and/or for glucose-primed net proton extrusion.* Corresponding author. MATERIALS AND METHODSYeast strains and culture conditions. Strains 1A4 and 1A7, carrying the lcbl::URA3 deletion alle...

Phosphatidylinositol catabolism in Saccharomyces cerevisiae is known to result in the formation of extracellular glycerophosphoinositol (GroPIns). We now report that S. cerevisiae not only produces but also reutilizes extracellular GroPIns and that these processes are regulated in response to inositol availability. A wild-type strain uniformly prelabeled with [ 3 H]inositol displayed dramatically higher extracellular GroPIns levels when cultured in medium containing inositol than when cultured in medium lacking inositol. This difference in extracellular accumulation of GroPIns in response to inositol availability was shown to be a result of both regulated production and regulated reutilization. In a strain in which a negative regulator of phospholipid and inositol biosynthesis had been deleted (an opi1 mutant), this pattern of extracellular GroPIns accumulation in response to inositol availability was altered. An inositol permease mutant (itr1 itr2), which is unable to transport free inositol, was able to incorporate label from exogenous glycerophospho[ 3 H]inositol, indicating that the inositol label did not enter the cell solely via the transporters encoded by itr1 and itr2. Kinetic studies of a wild-type strain and an itr1 itr2 mutant strain revealed that at least two mechanisms exist for the utilization of exogenous GroPIns: an inositol transporter-dependent mechanism and an inositol transporterindependent mechanism. The inositol transporter-independent pathway of exogenous GroPIns utilization displayed saturation kinetics and was energy dependent. Labeling studies employing [ C]glycerophospho[ H] inositol indicated that, while GroPIns enters the cell intact, the inositol moiety but not the glycerol moiety is incorporated into lipids.The extracellular production of glycerophosphoinositol (GroPIns) represents a major pathway of phosphatidylinositol (PI) metabolism in growing cultures of Saccharomyces cerevisiae (1, 2). GroPIns accumulates in the growth medium at levels equivalent to about 25% of the amount of cellular PI, in contrast to much lower extracellular levels of the deacylated forms of the major yeast phospholipids phosphatidylcholine and phosphatidylethanolamine (1). In pulse-chase turnover experiments, Angus and Lester (1) have shown that GroPIns accounts for approximately 50% of the phosphorus and inositol lost from PI during growth. Furthermore, the release of GroPIns is regulated by glucose in the medium (2). Thus, the extracellular accumulation of GroPIns was shown to be specific, regulated, and a major route of PI turnover in a growing yeast culture. Recently, Hawkins et al. (9) confirmed that the addition of glucose to stationary-phase cultures results in the extracellular production of not only GroPIns but also low levels of GroPIns 4-phosphate and GroPIns 4,5-bisphosphate.GroPIns, GroPIns 4-phosphate, and GroPIns 4,5-bisphosphate are produced from PI, PI 4-phosphate, and PI 4,5-bisphosphate, respectively, by a phospholipase A and a lysophospholipase acting sequentially or by a phospholipas...

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