The sphingolipid metabolites ceramide and sphingosine-1-phosphate are second messengers with opposing roles in mammalian cell growth arrest and survival; their relative cellular level has been proposed to be a rheostat that determines the fate of cells. This report demonstrates that this rheostat is an evolutionarily conserved stress-regulatory mechanism that inf luences growth and survival of yeast. Although the role of sphingosine-1-phosphate in yeast was not previously examined, accumulation of ceramide has been shown to induce G 1 arrest and cell death. We now have identified a gene in Saccharomyces cerevisiae, LBP1, that regulates the levels of phosphorylated sphingoid bases and ceramide. LBP1 was cloned from a yeast mutant that accumulated phosphorylated long-chain sphingoid bases and diverted sphingoid base intermediates from sphingolipid pathways to glycerophospholipid biosynthesis. LBP1 and its homolog, LBP2, encode very hydrophobic proteins that contain a novel-conserved sequence motif for lipid phosphatases, and both have long-chain sphingoid base phosphate phosphatase activity. In vitro characterization of Lbp1p shows that this phosphatase is Mg 2؉ -independent with high specificity for phosphorylated long-chain bases, phytosphingosine and sphingosine. The deletion of LBP1 results in the accumulation of phosphorylated long-chain sphingoid bases and reduced ceramide levels. Moreover, deletion of LBP1 and LBP2 results in dramatically enhanced survival upon severe heat shock. Thus, these phosphatases play a previously unappreciated role in regulating ceramide and phosphorylated sphingoid base levels in yeast, and they modulate stress responses through sphingolipid metabolites in a manner that is reminiscent of their effects on mammalian cells.Branching pathways of sphingolipid metabolism may mediate growth arrest, stress, or proliferative responses depending on the cell type and the nature of the stimulus. Ceramide is emerging as an important regulatory component of stress responses and programmed cell death, known as apoptosis (1-5). In contrast, another sphingolipid metabolite, sphingosine-1-phosphate (SPP), has been implicated as a second messenger in cellular proliferation (6) and antagonizes ceramide-mediated apoptosis (7). Thus, it has been suggested that the relative intracellular levels of ceramide and SPP are a critical factor for cell survival. Although the ceramide͞SPP rheostat may be an inherent characteristic of mammalian cells, external stimuli can reset this ratio (7-9). A variety of stress stimuli, including Fas ligand, TNF-␣, IL-1, growth factor withdrawal, anticancer drugs, oxidative stress, heat shock, and ionizing radiation, increase ceramide levels (1, 2, 10, 11), whereas platelet-derived growth factor and other growth factors stimulate rapid, transient elevations in SPP levels (6). The mechanisms that regulate the levels of these sphingolipid second messengers are under intense investigation with most of the attention focused on degradative pathways: sphingomyelinase, which...

Infections due to Candida albicans are usually treated with azole antifungals such as fluconazole, but treatment failure is not uncommon especially in immunocompromised individuals. Relatedly, in vitro studies demonstrate that azoles are nonfungicidal, with continued growth at strain-dependent rates even at high azole concentrations. We hypothesized that upregulation of ERG11, which encodes the azole target enzyme lanosterol demethylase, contributes to this azole tolerance in Candida species. RNA analysis revealed that ERG11 expression in C. albicans is maximal during logarithmic-phase growth and decreases as the cells approach stationary phase. Incubation with fluconazole, however, resulted in a two-to fivefold increase in ERG11 RNA levels within 2 to 3 h, and this increase was followed by resumption of culture growth. ERG11 upregulation also occurred following treatment with other azoles (itraconazole, ketoconazole, clotrimazole, and miconazole) and was not dependent on the specific medium or pH. Within 1 h of drug removal ERG11 upregulation was reversed. Azole-dependent upregulation was not limited to ERG11: five of five ERG genes tested whose products function upstream and downstream of lanosterol demethylase in the sterol biosynthetic pathway were also upregulated. Similarly, ERG11 upregulation occurred following treatment of C. albicans cultures with terbinafine and fenpropimorph, which target other enzymes in the pathway. These data suggest a common mechanism for global ERG upregulation, e.g., in response to ergosterol depletion. Finally, azole-dependent ERG11 upregulation was demonstrated in three additional Candida species (C. tropicalis, C. glabrata, and C. krusei), indicating a conserved response to sterol biosynthesis inhibitors in opportunistic yeasts.

Certain mammalian growth modulators, such as tumor necrosis factor ~, interleukin-lj3, and -/-interferon, induce an antiproliferative response-terminal differentiation, apoptosisis, or cell cycle arrest-through a novel signal transduction pathway mediated by the lipid ceramide as a second messenger. Both a ceramide-activated protein phosphatase and a ceramide-activated protein kinase have been implicated in transmitting the signals elicited by ceramide. We have determined that ceramide addition to the yeast Saccharomyces causes a similar antiproliferative response, resulting in arrest of cells in the G 1 phase of the cell cycle. We have also determined that yeast cells contain a ceramide-activated protein phosphatase composed of regulatory subunits encoded by TPD3 and CDC55 and a catalytic subunit encoded by SIT4. Because mutation of any one of these three genes renders strains resistant to ceramide inhibition, we conclude that the G 1 effects of ceramide are mediated at least in part by the yeast ceramide-activated protein phosphatase. These results highlight the conservation of signaling systems in yeast and mammalian cells and provide a novel approach to dissecting this ubiquitous signal transduction pathway.

SummaryThe most important group of antifungals is the azoles (e.g. miconazole), which act by inhibiting lanosterol demethylase in the sterol biosynthesis pathway. Azole activity can be modulated through structural changes in lanosterol demethylase, altered expression of its gene ERG11 , alterations in other sterol biosynthesis enzymes or altered expression of multidrug transporters. We present evidence that azole activity versus Saccharomyces cerevisiae is also modulated by Ca 2 + + + + -regulated signalling.

The regulation of lipid biosynthesis in the yeast Saccharomyces cerevisiae by fumonisin B1 was examined. Fumonisin B1 inhibited the growth of yeast cells. Cells supplemented with fumonisin B1 accumulated free sphinganine and phytosphingosine in a dose-dependent manner. The cellular concentration of ceramide was reduced in fumonisin B1-supplemented cells. Ceramide synthase activity was found in yeast cell membranes and was inhibited by fumonisin B1. Fumonisin B1 inhibited the synthesis of the inositol-containing sphingolipids inositol phosphorylceramide, mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide. Fumonisin B1 also caused a decrease in the synthesis of the major phospholipids synthesized via the CDP-diacylglycerol-dependent pathway and the synthesis of neutral lipids. The effects of fumonisin B1 and sphingoid bases on the activities of enzymes in the pathways leading to the synthesis of sphingolipids, phospholipids, and neutral lipids were also examined. Other than ceramide synthase, fumonisin B1 did not affect the activities of any of the enzymes examined. However, sphinganine and phytosphingosine inhibited the activities of inositol phosphorylceramide synthase, phosphatidylserine synthase, and phosphatidate phosphatase. These are key enzymes responsible for the synthesis of lipids in yeast. The data reported here indicated that the biosynthesis of sphingolipids, phospholipids and neutral lipids was coordinately regulated by fumonisin B1 through the regulation of lipid biosynthetic enzymes by sphingoid bases.

A number of eukaryotic transcription factors are held in a latent state by being embedded in, or tethered to, cellular membranes. Mga2p of Saccharomyces cerevisiae is an endoplasmic reticulum (ER)-localized transcription factor that plays an overlapping role with homologous Spt23p in upregulating expression of OLE1, a gene required for the synthesis of essential oleic acid. Previous studies have documented that proteasome-dependent processing of ER bound 120 kDa Mga2p and Spt23p proteins generates transcriptionally competent 90 kDa polypeptides. In the case of Spt23p90, it is held at the membrane prior to release via a self-interaction with the unprocessed Spt23p120 anchor. It is currently thought that the highly conserved Rsp5p ubiquitin ligase provides the signal for partial degradation of both proteins. Cells lacking Rsp5p function require oleic acid for growth, and Spt23p processing is suppressed in rsp5 Delta cells and in wild-type RSP5 cells upon expression of Rsp5p dominant-negative mutants. We report here that Rsp5p is dispensable for Mga2p90 generation but not for release of the processed product from the ER. In addition, we demonstrate that polyubiquitinated Mga2p120 accumulates in cells lacking Npl4p or proteasome function and Rsp5p is required for Mga2p120 polyubiquitination. Finally, we provide evidence that Mga2p90 and Mga2p120 dimerize and that Rsp5p binds heterodimeric Mga2p complexes both in vitro and in vivo. In light of these experiments, we propose that Rsp5p facilitates Mga2p90 release from the ER by promoting polyubiquitination and Npl4p-proteasome-mediated degradation of the interacting Mga2p120 ER bound anchor.

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]dihydrosphingosine radiolabeling studies demonstrated that mutant cells had reduced rates of biosynthesis and lower steadystate levels of complex sphingolipids while accumulating certain hydroxylated ceramide species. Phospholipid radiolabeling studies showed that arv1⌬ cells harbored defects in the rates of biosynthesis and steadystate levels of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. Neutral lipid radiolabeling studies indicated that the rate of biosynthesis and steady-state levels of sterol ester were increased in arv1⌬ cells. Moreover, these same studies demonstrated that arv1⌬ cells had decreased rates of biosynthesis and steady-state levels of total fatty acid and fatty acid alcohols. Gas chromatography/mass spectrometry analyses examining different fatty acid species showed that arv1⌬ cells had decreased levels of C18:1 fatty acid. Additional gas chromatography/mass spectrometry analyses determining the levels of various molecular sterol species in arv1⌬ cells showed that mutant cells accumulated early sterol intermediates. Using fluorescence microscopy we found that GFP-Arv1p localizes to the endoplasmic reticulum and Golgi. Interestingly, the heterologous expression of the human ARV1 cDNA suppressed the sphingolipid metabolic defects of arv1⌬ cells. We hypothesize that in eukaryotic cells, Arv1p functions in the sphingolipid metabolic pathway perhaps as a transporter of ceramides between the endoplasmic reticulum and Golgi.Sphingolipids serve as constituents of membrane bilayers (1) and have emerged as potent signaling metabolites (2-5). Sphingolipid synthesis in eukaryotes begins with the condensation of serine and palmitoyl-CoA, a reaction catalyzed by the Lcb1p/ Lcb2p serine palmitoyltransferase subunits (Fig. 1) (6 -10). Ceramide serves as the backbone for all complex sphingolipids, and its biosynthesis is the point where animal and fungal sphingolipid biosynthesis begin to diverge (11). In higher eukaryotes, there are over three hundred different types of complex sphingolipids found in membranes (1), whereas in the yeast S. cerevisiae there are three, inositolphosphorylceramide (IPC), 1 mannose inositolphosphorylceramide (MIPC), and mannose diinositolphosphorylceramide (MIP 2 C). However, IPC, MIPC, and MIP 2 C lipids can differ in their hydroxylation state, giving rise to fifteen possible complex sphingolipids (11).The accumulation of free cholesterol in animal cells causes toxicity (12). One mechanism by which cells relieve this toxic effect is through the use of the membrane-associated SREBP transcription factors, which precisely regulate the genes that are required for sterol biosynthesis and LDL receptor expression (13,14). Worgall et al. (15) recently showed that SREBP activation was regulated by ceramide. Others have shown that animal cells regulate sphingolipid metabolism in response to subtle and transient changes in cholesterol (16,17). In S. cerevisiae, sphingolipid metabolism appears to be coordinately regulated with phospholipid an...