]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...

A temperature-sensitive Saccharomyces cerevisiae mutant harboring a lesion in the ERG26 gene has been isolated. ERG26 encodes 4␣-carboxysterol-C3 dehydrogenase, one of three enzymatic activities required for the conversion of 4,4-dimethylzymosterol to zymosterol. Gas chromatography/mass spectrometry analyses of sterols in this mutant, designated erg26-1, revealed the aberrant accumulation of a 4-methyl-4-carboxy zymosterol intermediate, as well as a novel 4-carboxysterol. Neutral lipid radiolabeling studies showed that erg26-1 cells also harbored defects in the rate of biosynthesis and steady-state levels of mono-, di-, and triglycerides. Phospholipid radiolabeling studies showed defects in the rate of biosynthesis of both phosphatidic acid and phosphatidylinositol. Biochemical studies revealed that microsomes isolated from erg26-1 cells contained greatly reduced 4␣-carboxysterol-C3 dehydrogenase activity when compared with microsomes from wild type cells. Previous studies have shown that loss of function mutations in either of the fatty acid elongase genes SUR4/ELO3 or FEN1/GNS1/ELO2 can "bypass" the essentiality of certain ERG genes (Ladeveze, V., Marcireau, C., Delourme, D., and Karst, F. (1993) Lipids 28, 907-912; Silve, S., Leplatois, P., Josse, A., Dupuy, P. H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le Fur, G., Caput, D., Ferrara, P., and Loison, G. (1996) Mol. Cell. Biol. 16, 2719 -2727). Studies presented here have shown that this sphingolipiddependent "bypass" mechanism did not suppress the essential requirement for zymosterol biosynthesis. However, studies aimed at understanding the underlying physiology behind the temperature-sensitive growth defect of erg26-1 cells showed that the addition of several antifungal compounds to the growth media of erg26-1 cells could suppress the temperature-sensitive growth defect. Fluorescence microscopic analysis showed that GFP-Erg26p and GFP-Erg27p fusion proteins were localized to the endoplasmic reticulum. Two-hybrid analysis indicated that Erg25p, Erg26p, and Erg27p, which are required for the biosynthesis of zymosterol, form a complex within the cell.

Loss of function of either the RVS161 or RVS167 Saccharomyces cerevisiae amphiphysin-like gene confers similar growth phenotypes that can be suppressed by mutations in sphingolipid biosynthesis. We performed a yeast two-hybrid screen using Rvs161p as bait to uncover proteins involved in this sphingolipid-dependent suppressor pathway. In the process, we have demonstrated a direct physical interaction between Rvs167p and the two-hybrid interacting proteins, Acf2p, Gdh3p, and Ybr108wp, while also elucidating the Rvs167p amino acid domains to which these proteins bind. By using subcellular fractionation, we demonstrate that Rvs167p, Ybr108wp, Gdh3p, and Acf2p all localize to Rvs161p-containing lipid rafts, thus placing them within a single compartment that should facilitate their interactions. Moreover, our results suggest that Acf2p and Gdh3p functions are needed for suppressor pathway activity. To determine pathway mechanisms further, we examined the localization of Rvs167p in suppressor mutants. These studies reveal roles for Rvs161p and the very long chain fatty acid elongase, Sur4p, in the localization and/or stability of Rvs167p. Previous yeast studies showed that rvs defects could be suppressed by changes in sphingolipid metabolism brought about by deleting SUR4 (Desfarges, L., Durrens, P., Juguelin, H., Cassagne, C., Bonneu, M., and Aigle, M. (1993) Yeast 9, 267-277). Using rvs167 sur4 and rvs161 sur4 double null cells as models to study suppressor pathway activity, we demonstrate that loss of SUR4 does not remediate the steady-state actin cytoskeletal defects of rvs167 or rvs161 cells. Moreover, suppressor activity does not require the function of the actin-binding protein, Abp1p, or Sla1p, a protein that is thought to regulate assembly of the cortical actin cytoskeleton. Based on our results, we suggest that sphingolipid-dependent suppression of rvs defects may not work entirely through regulating changes in actin organization.

We had previously isolated the temperature-sensitive erg26-1 mutant and characterized the sterol defects in erg26-1 cells (Baudry, K., Swain, E., Rahier, A., Germann, M., Batta, A., Rondet, S., Mandala, S., Henry, K., Tint, G. S., Edlind, T., Kurtz, M., and Nickels, J. T., Jr. (2001) J. Biol. Chem. 276, 12702-12711). We have now determined the defects in sphingolipid metabolism in erg26-1 cells, examined their effects on cell growth, and initiated studies designed to elucidate how might changes in sterol levels coordinately regulate sphingolipid metabolism in Saccharomyces cerevisiae. Using [ 3 H]inositol radiolabeling studies, we found that the biosynthetic rate and steady-state levels of specific hydroxylated forms of inositolphosphorylceramides were decreased in erg26-1 cells when compared with wild type cells. [ 3 H]Dihydrosphingosine radiolabeling studies demonstrated that erg26-1 cells had decreased levels of the phytosphingosine-derived ceramides that are the direct precursors of the specific hydroxylated inositol phosphorylceramides found to be lower in these cells. Gene dosage experiments using the sphingolipid long chain sphingoid base (LCB) hydroxylase gene, SUR2, suggest that erg26-1 cells may accumulate LCB, thus placing one point of sterol regulation of sphingolipid synthesis possibly at the level of ceramide metabolism. The results from additional genetic studies using the sphingolipid hydroxylase and copper transporter genes, SCS7 and CCC2, respectively, suggest a second possible point of sterol regulation at the level of complex sphingolipid hydroxylation. In addition, [ 3 H]inositol radiolabeling of sterol biosynthesis inhibitor-treated wild type cells and late sterol pathway mutants showed that additional blocks in sterol biosynthesis have profound effects on sphingolipid metabolism, particularly sphingolipid hydroxylation state. Finally, our genetic studies in erg26-1 cells using the LCB phosphate phosphatase gene, LBP1, suggest that increasing the levels of the LCB sphingoid base phosphate can remediate the temperature-sensitive phenotype of erg26-1 cells.Sphingolipids are ubiquitous membrane lipids that are found in all eukaryotic cells. They are structural components of lipid bilayers (1), have emerged as signaling molecules generated in response to a variety of growth modulators (2-5), and have been found to play an important role in regulating vesicular and lipid trafficking events in higher eukaryotes (6 -8). With regards to human pathology, a number of diseases have been attributed to the inappropriate trafficking and/or metabolism of sphingolipids. Human Niemann-Pick disorders are characterized by the intercellular mislocalization and accumulation of sphingolipid and cholesterol in lysosomes (9 -11), and a number of rather severe sphingolipid storage diseases result from the inability of a diseased cell to properly catabolize sphingolipids (12, 13).The mechanisms regulating sphingolipid metabolism at the molecular level are not that well understood. However, there is some evidence sug...

erg26-1ts cells harbor defects in the 4␣-carboxysterol-C3 dehydrogenase activity necessary for conversion of 4,4-dimethylzymosterol to zymosterol. Mutant cells accumulate toxic 4-carboxysterols and are inviable at high temperature. A genetic screen aimed at cloning recessive mutations remediating the temperature sensitive growth defect has resulted in the isolation of four complementation groups, ets1-4 (erg26-1 ts temperature sensitive suppressor). We describe the characterization of ets1-1 and ets2-1. Gas chromatography/mass spectrometry analyses demonstrate that erg26-1 ts ets1-1 and erg26-1 ts ets2-1 cells do not accumulate 4-carboxysterols, rather these cells have increased levels of squalene and squalene epoxide, respectively. ets1-1 and ets2-1 cells accumulate these same sterol intermediates. Chromosomal integration of ERG1 and ERG7 at their loci in erg26-1 ts ets1-1 and erg26-1 ts ets2-1 mutants, respectively, results in the loss of accumulation of squalene and squalene epoxide, re-accumulation of 4-carboxysterols and cell inviability at high temperature. Enzymatic assays demonstrate that mutants harboring the ets1-1 allele have decreased squalene epoxidase activity, while those containing the ets2-1 allele show weakened oxidosqualene cyclase activity. Thus, ETS1 and ETS2 are allelic to ERG1 and ERG7, respectively. We have mapped mutations within the erg1-1/ ets1-1 (G247D) and erg7-1/ets2-1 (D530N, V615E) alleles that suppress the inviability of erg26-1 ts at high temperature, and cause accumulation of sterol intermediates and decreased enzymatic activities. Finally using erg1-1 and erg7-1 mutant strains, we demonstrate that the expression of the ERG25/26/27 genes required for zymosterol biosynthesis are coordinately transcriptionally regulated, along with ERG1 and ERG7, in response to blocks in sterol biosynthesis. Transcriptional regulation requires the transcription factors, Upc2p and Ecm22p.