Biochemistry, Cell Biology and Molecular Biology of Lipids ... · PDF fileyeast Yeast 14,...

Yeast 14, 1471–1510 (1998)

Biochemistry, Cell Biology and Molecular Biology ofLipids of Saccharomyces cerevisiae

GU}NTHER DAUM1*, NORMAN D. LEES2, MARTIN BARD2 AND ROBERT DICKSON3

1Institut fur Biochemie und Lebensmittelchemie, Technische Universitat, Petersgasse 12/2, A-8010 Graz, Austria2Department of Biology, Indiana University Purdue University Indianapolis, 723 W. Michigan Street, Indianapolis,IN 46202, U.S.A.3Department of Biochemistry and the Lucille P. Markey Cancer Center, University of Kentucky Medical Center,Lexington, KY 40536-0298, U.S.A.

The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological andmolecular biological aspects of lipid synthesis. Most but not all genes encoding enzymes involved in fatty acid,phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many geneproducts have been functionally characterized. Less information is available about genes and gene productsgoverning the transport of lipids between organelles and within membranes, turnover and degradation of complexlipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes. Here wesummarize current knowledge about lipid biosynthetic pathways in S. cerevisiae and describe the characteristicfeatures of the gene products involved. We focus on recent discoveries in these fields and address questions on theregulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross-talk between organelles duringlipid synthesis and subcellular distribution of lipids. Finally, we discuss distinct functions of certain key lipids andtheir possible roles in cellular processes. ? 1998 John Wiley & Sons, Ltd.

— yeast; S. cerevisiae; lipids; phospholipids; sterols; sphingolipids; fatty acids

CONTENTS

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1471Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . 1472

Phospholipid synthesis . . . . . . . . . . . . . . . . . 1473A brief survey on the biosynthesis offatty acids in yeast . . . . . . . . . . . . . . . . . . . . 1480Subcellular location of phospholipids,sites of synthesis and general aspectsof phospholipid transport . . . . . . . . . . . . . . 1481

Sterols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1482Sterol biosynthesis: acetate to farnesylpyrophosphate. . . . . . . . . . . . . . . . . . . . . . . . 1482Sterol biosynthesis in yeast: farnesylpyrophosphate to ergosterol . . . . . . . . . . . . 1485Intracellular transport and location ofsterol in yeast . . . . . . . . . . . . . . . . . . . . . . . . 1489

Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . 1490

873-6952; e-mail: [email protected]

CCC 0749–503X/98/161471–40 $17.50? 1998 John Wiley & Sons, Ltd.

Sphingolipid biosynthesis pathway:genes, enzymes and phenotypes . . . . . . . . . 1490Cellular location, sites of synthesis andfunctions of sphingolipids . . . . . . . . . . . . . . 1494

General conclusions and future perspectives 1495Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 1497References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1497

*Correspondence to: G. Daum, Institut fur Biochemie undLebensmittelchemie Technische Universitat, Petersgasse 12/2,A-8010 Graz, Austria. Tel: +43-316-873-6462; fax: +43-316-

INTRODUCTION

Membranes of eukaryotic cells have several im-portant functions. First, they serve as a diffusionbarrier between the interior of the cell and itsenvironment, and between the lumen of organellesand the cytosol. Second, membranes harbour pro-teins that catalyse selective transport of moleculesor act as enzymes in metabolic and regulatorypathways. Third, membranes harbour receptorswhich contribute to recognition processes. Majorlipid components of eukaryotic membranesare phospholipids, sterols, sphingolipids and
glycoglycerolipids. Some lipids are recognized as
Received 20 August 1998Accepted 28 August 1998

yeast.

1472 . .

essential, although their specific function may notbe known.

The baker’s yeast, S. cerevisiae, is a convenientexperimental organism for studying the synthesisand intracellular transport of lipids. S. cerevisiaecan serve as a valuable model for multicellulareukaryotes because of similarity in subcellularstructures, and because much is known about thebiochemistry and molecular biology of yeast lipids.The availability of the complete yeast genomesequence has provided the opportunity to identifygene homologies between yeast and higher eu-karyotes. The ease with which deletion mutantscan be made in S. cerevisiae has facilitated studiesof the function of specific lipids and lipid metabo-lites. The results of these studies can be difficultto interpret, however, because genes involved inlipid synthesis and transport may exist in multiplecopies or because the absence of one lipid can becompensated for by overproduction of another. Asa consequence, mutants lacking one gene of lipidbiosynthesis or transport do not necessarily exhibitan identifiable phenotypic defect.

Although most biosynthetic routes of lipids inyeast are similar to those in higher eukaryotes,some specific steps are unique in yeast. Forexample, the biosynthesis of phosphatidylserine,which occurs by a base exchange mechanism inhigher eukaryotes, is accomplished in yeast by areaction requiring CDP-diacylglycerol. Anotherexample occurs in the biosynthesis of the yeast-specific sterol, ergosterol, where there are require-ments for certain enzymatic steps not found inthe cholesterol biosynthetic pathways in mammalsor the sitosterol biosynthetic pathway in plants.Finally, yeast sphingolipids are characterized bytheir inositol moiety, which is not found in thesphingolipids of animals.

Similar to the situation in multicellular eu-karyotes, lipid synthesis in yeast is regulated at thetranscriptional level. The most prominent exampleof the regulation of lipid synthesis in yeast is theresponse to inositol and choline that occursthrough cis- and trans-acting elements (for recentreviews, see refs 35, 82 and 198). In the case ofphospholipid biosynthetic genes, the response toinositol and choline in the growth medium isdictated by a 10 bp element called the UASINO.The INO2 and INO4 genes were identified aspositive regulators of transcription that are re-quired for derepression, whereas the OPI1 geneproduct is required for repression of phospholipidgene expression. In some cases posttranslational

? 1998 John Wiley & Sons, Ltd.

modifications appear to play a role in the regula-tion of the activity of lipid biosynthetic enzymes.Finally, the interplay of organelles appears tocontribute to the coordination of lipid biosyntheticprocesses.

Within a cell, not all membranes have the ca-pacity to synthesize their own lipids. Thus, lipidsmust migrate from their site of synthesis to thecellular membrane(s) of destination by efficientprocesses of transport from one hydrophobic com-partment to another. Although some lipids such asphosphatidylcholine or phosphatidylinositol arefound in all subcellular membranes, the mainten-ance of balanced levels of lipids within a subcellu-lar compartment depends on reliable mechanismsof lipid distribution within the cell. Other lipids(such as cardiolipin, sterols and sphingolipids) aremostly restricted to distinct subcellular compart-ments, thus requiring even more stringent mecha-nisms of intracellular transport and turnover. Onthe other hand, yeast cells are surprisingly flexibleand tolerant to changes in their lipid composition,at least under laboratory conditions, which makesinvestigations of lipid transport and assembly intocellular membranes even more complicated.

In this review, we describe, discuss and summar-ize the enzymatic steps, genes and gene productsnecessary for synthesis and transport of lipids inS. cerevisiae. We also discuss progress in under-standing the function of specific lipid species andareas of research that need to be addressed. Thereader is also referred to recent reviews dealingwith lipid synthesis and its regulation in

35,60,82,126,143,198

PHOSPHOLIPIDS

Phospholipids, which are regarded as a primarystructural element of the biological membranes,consist of a glycerol backbone esterified with fattyacids in the sn-1 and sn-2 positions, and a phos-phate group in the sn-3 position. One hydroxylgroup of the phosphate is linked to a polar headgroup, which is relevant for classifying the variousphospholipids and for the physical properties ofthese molecules. Fatty acids in the sn-1 position aremostly saturated whereas those in the sn-2 positionare unsaturated. The fatty acid composition ofS. cerevisiae phospholipids is fairly simple: themost abundant fatty acids are C-16:0 (palmiticacid), C-16:1 (palmitoleic acid), C-18:0 (stearicacid) and C-18:1 (oleic acid). Minor amounts of

Yeast 14, 1471–1510 (1998)

1473

C-14:0 (myristic acid) can be detected, and mostrecently C-26 fatty acids were recognized as typicalminor components.226,227 Other fatty acids, how-ever, may be present in minor quantities but still beof physiological importance. As an example, phos-pholipids containing C-26 fatty acid appear to beessential for nuclear function.226,227

Although phospholipids are indispensable asbulk components of yeast organelle membranes, itis not clear whether or not all individual classes ofphospholipids are essential. One exception is phos-phatidylinositol (PtdIns). Mutations in the PIS1gene encoding PtdIns synthase (Figure 1) result in


lethality.190 Defects in structural genes encodingenzymes involved in the biosynthesis of theaminoglycerophospholipids, phosphatidylserine(PtdSer), phosphatidylethanolamine (PtdEtn) andphosphatidylcholine (PtdCho), cause phenotypicchanges but are not lethal under laboratoryconditions.

Figure 1. Pathways of phospholipid synthesis in yeast. Metabolites: G-3-P: glycerol-3-phosphate;DHAP: dihydroxyacetone phosphate; ADHAP: acyldihydroxyacetone phosphate; ACoA: acyl-CoA;LPA: lysophosphatidic acid; PA: phosphatidic acid; CDP-DAG: CDP-diacylglycerol; DAG: diacyl-glycerol; Ins: inositol; Ser: serine; Etn: ethanolamine; EtnP: ethanolaminephosphate; Cho: choline;ChoP: cholinephosphate; CDP-Etn: CDP-ethanolamine; CDP-Cho: CDP-choline; PtdSer: phosphatidyl-serine; PtdIns: phosphatidylinositol; PtdIns-3-P: phosphatidylinositol-3-phosphate; PtdIns-3,4-PP:phosphatidylinositol-3*4*-biphosphate; PtdIns-4-P: phosphatidylinositol-4-phosphate; PtdIns-4,5-PP:phosphatidylinositol-4*5*-biphosphate; PtdEtn: phosphatidylethanolamine; MMPtdEtn: monomethylphosphatidylethanolamine; DMPtdEtn: dimethyl phosphatidylethanolamine; PtdCho: phosphatidyl-choline; SAM: S-adenosyl methionine; PtdGroP: phosphatidylglycerophosphate; PtdGro: phosphati-dylglycerol; CL: cardiolipin. Enzymes: GAT: glycerol-3-phosphate acyltransferase; AGAT:1-acylglycerol-3-phosphate acyltransferase; DHAPAT: dihydroxyacetone phosphate acyltransferase;ADHAPR: acyldihydroxyacetone phosphate reductase; PtdIns-3,4-K: phosphatidylinositol-3-phosphate4-kinase; PGPP: phosphatidylglycerophosphate phosphatase. Names of genes and gene products thathave not yet been identified on a molecular level are shown on a grey background. Genes and therespective gene products are listed in Table 1.

Phospholipid synthesisThe pathway of glycerophospholipid formation

in yeast is shown in Figure 1. This scheme isdivided into two sections representing the extra-

Yeast 14, 1471–1510 (1998)

1474 . .

mitochondrial and the mitochondrial spaces. It iswell accepted that the endoplasmic reticulum andmitochondria are the subcellular compartmentsthat contribute most to yeast phospholipid biosyn-thesis (for a review, see ref. 54). Recently, it wasrecognized that other subcellular compartments,such as the Golgi and lipid particles contribute tophospholipid synthesis as well.137,268,308 Biosyn-thesis and intracellular migration of phospholipidsare highly interconnected because extensive trans-fer of intermediates and cross-compartment co-ordination of partial reactions are required. Themost prominent example in that respect is thesequence of steps in aminoglycerophospholipidsynthesis.54,267,282,283 PtdSer, which is synthesizedin the endoplasmic reticulum, has to be transferredto the mitochondria and/or a Golgi/vacuolar com-partment to be decarboxylated to phosphatidyl-ethanolamine (PtdEtn).268,269,270 Some of thisPtdEtn migrates to the endoplasmic reticulumwhere methylation to phosphatidylcholine (Ptd-Cho) occurs. Another requirement for balancedphospholipid biosynthesis is the supply of phos-phatidic acid (PA) to its sites of metabolic conver-sion. PA formed in the endoplasmic reticulum andlipid particles9 is required as a precursor for thesynthesis of CDP-DAG, which appears to occur inthe endoplasmic reticulum and mitochondria.129

Thus, the biosynthesis of phospholipids and theirinter-organelle transport have to be linked andcoordinated processes.

A general precursor of all glycerolipids is phos-phatidic acid (PA). The first step of PA synthesis inyeast is acylation of glycerol-3-phosphate (G-3-P)or dihydroxyacetone phosphate (DHAP), respect-ively.172 1-Acyl-DHAP is then reduced to form1-acylglycerol-3-phosphate (lysophosphatidic acid;LPA),210 which is also the direct product of G-3-Pacylation. In a second acyltransferase reaction,LPA is converted to PA. While much is knownabout further conversion of PA to more complexphospholipids, biosynthesis of PA itself is not aswell understood. In S. cerevisiae, the highestspecific activity of the enzyme(s) that catalyses theacylation of G-3-P was found in the lipid particlefraction,9,42,308 a compartment consisting of ahydrophobic core of triacylglycerols and sterylesters that is enveloped by a phospholipid mono-layer with a small amount of embedded pro-teins.140 Recently, it was shown by subcellularanalysis of acyltransferase mutants183,260 that lipidparticles harbour two distinct acyltransferases.9

The first enzyme, a putative Gat1p that has neither


been isolated nor characterized on the gene level,acylates G-3-P and forms LPA as an intermediate.The second enzyme encoded by the SLC1 gene(sphingolipid compensation)183 is a 1-acylglycerol-3-phosphate acyltransferase (AGAT) which com-pletes the biosynthesis of PA. In an slc1 deletionstrain, LPA accumulates. Both enzymes, however,were detected not only in lipid particles, but also inthe endoplasmic reticulum.9 In addition to Gat1pand Slc1p, the microsomal fraction seems to con-tain another system of PA synthesis with at leastone additional GAT and AGAT, respectively. Thisobservation explains why mutations of GAT1 orSLC1 are not lethal. Redundancy of acylationsystems may also be the reason for experimen-tal difficulties in isolating acyltransferases andidentifying their respective genes.

A second important aspect of PA synthesis inyeast is the use of G-3-P and/or DHAP as sub-strate for the first step of acylation. In animal cells,the DHAP pathway of PA biosynthesis is obliga-tory for ether lipid biosynthesis.278 The possibleoccurrence of ether lipids in yeast has not beenunambiguously proven. Johnston and Paltauf110

demonstrated that yeast cells incorporate DHAPinto PA. DHAPAT activity was detected in S.carlsbergensis110 and S. cerevisiae.222 Controversialresults were presented regarding the questionof whether glycerol-3-phosphate acyltransferase(GAT) and dihydroxyacetone phosphate acyl-transferase (AGAT) are different enzymes orwhether one enzyme can utilize both substrates.Tillman and Bell260 suggested that acylation of theprecursors G-3-P and DHAP is catalysed by thesame protein. In contrast, Racenis et al.210 arguedthat GAT and DHAPAT activities might be attrib-uted to different enzymes because of different pHoptima and different degrees of sensitivity toinhibition by N-maleimide of the two enzymeactivities. The latter findings, however, are alsoconsistent with a single enzyme containing twoactive sites. Both GAT and DHAPAT activitieswere increased during respiratory growth, but thedegree of increase in the levels of the two activitieswere different.172,210 Employing elaborate cellfractionation techniques,306 GAT and DHAPATactivities were measured in highly purified or-ganelles (K. Athenstaedt, personal communi-cation). Using a gat1 mutant strain, it was shownthat Gat1p present in lipid particles and the endo-plasmic reticulum could accept both G-3-P andDHAP as substrates. Similarly, the additionalacyltransferase(s) present in the endoplasmic

Yeast 14, 1471–1510 (1998)

1475

reticulum9 can acylate both precursors. Morespecifically, however, yeast mitochondria harbourenzyme(s) that appear to prefer DHAP as a sub-strate for acylation (see Figure 1), suggesting thatat least one additional independent DHAPAT ispresent in this organelle. Enzymatic activity of1-acyl-DHAP reductase, which is required for theconversion of 1-acyl-DHAP to lysophosphatidicacid, was only detectable in lipid particles and theendoplasmic reticulum, but not in mitochondria.The subcellular distribution of enzymes involvedin PA synthesis requires migration of acylationintermediates between organelles. Such migrationmay occur by free exchange between subcellularcompartments, since PA, LPA and 1-acyl-DHAPare largely water-soluble.

A central metabolite in phospholipid biosynthe-sis is CDP-diacylglycerol (CDP-DAG). The yeastgene encoding CDP-diacylglycerol synthase,CDS1, was identified by its homology to thecorresponding Escherichia coli and Drosophilagenes.68,236 Consistent with its central role inde novo glycerolipid synthesis, deletion of CDS1 islethal, suggesting that CDS1 encodes the onlyCDP-DAG synthase in yeast or supplies an essen-tial subcellular pool of CDP-DAG. Cds1p, whichhas been purified to near homogeneity fromyeast,115 converts PA to CDP-DAG in a CTP-dependent reaction (see Figure 1). A high level ofCDP-DAG synthase activity favours synthesis ofPtdIns over PtdSer.234 Cells expressing low levelsof Cds1p excrete inositol into the medium.233

CDP-DAG synthase levels, and hence CDP-DAGlevels, affect the transcription of the INO1 andCHO1/PSS1 genes independent of the expressionof the regulator genes INO2 and INO4 or theinositol/choline levels in the medium. Reduction ofthe level of CDP-DAG synthase activity results inan increase of PtdSer synthase activity and theCHO1/PSS1 mRNA level. PIS1 message is un-affected by CDP-DAG synthase levels, but PtdInssynthase activity declines as the level of CDP-DAG synthase activity is lowered. Thus, levels ofCDP-DAG appear to affect both the level of geneexpression and enzyme stability. Since CDP-DAGsynthase activity was detected in the endoplasmicreticulum and mitochondria,115,129,308 its sub-cellular distribution may be of physiologicalrelevance. The availability of CDP-DAG inspecific compartments may contribute to the con-trol of the synthesis of different phospholipids. Forexample, enrichment of CDP-DAG in mitochon-dria might stimulate synthesis of phosphatidyl-


glycerol (PtdGro) and cardiolipin (CL) in thisorganelle, whereas formation of other phospho-lipids, such as PtdIns and PtdSer, might beenhanced by an excess of CDP-DAG in theendoplasmic reticulum.

Phosphatidylinositol (PtdIns) is a phospholipidthat is essential for yeast. It is probably not thestructural requirement for membrane assemblythat makes PtdIns essential but rather its roles incellular signalling and as a membrane sensor.PtdIns is synthesized from CDP-DAG and inositol(Figure 1). Inositol required for this enzymaticreaction can either be synthesized endogenously ortaken up from the medium.292 Free endogenousinositol is derived from inositol-1-phosphate pro-duced from inositol-1-phosphate synthase (Ino1p),which utilizes glucose-6-phosphate as a precursor(for a review, see ref. 152). Inositol uptake fromthe medium is actively catalysed by two transport-ers, Itr1p and Itr2p.192 Transcription of the ITR1gene responds to regulation by inositol.131,191

When inositol is present in the medium the activityof Itr1p is reduced. The transcriptional regulationof ITR1 depends on the INO2, INO4 and OPI1genes. In addition, repression of ITR1 is regulatedat the posttranscriptional level,132 namely byinternalization of Itr1p through the endocytoticpathway and degradation in the vacuole. In thestationary phase, degradation of Itr1p occurs alsoin inositol-free medium, indicating that at leasttwo distinct mechanisms contribute to the post-translational regulation of the level of the trans-porter.215 It is not known whether the ITR2 geneis subject to the same control. Regulation ofphospholipid biosynthetic gene expression requiresinositol transport, since an itr1 itr2 double mutantis defective in repression of the INO1 gene.131

PtdIns synthase (Pis1p) has been purified fromS. cerevisiae,70 and the structural gene PIS1 hasbeen cloned.189,190 In yeast, PtdIns is essentialsince disruption of PIS1 is lethal.190 Transcriptionof PIS1 is not affected by the presence of inositolin the medium.71 Nevertheless, supplementation ofyeast cells with inositol increases the cellular levelof PtdIns, because the Km of Pis1p for inositol ismuch higher than the intracellular inositol concen-tration.116 The increase of PtdIns occurs at theexpense of PtdSer because both PtdIns synthase(Pis1p) and PtdSer synthase (Pss1p/Cho1p) com-pete for CDP-DAG, the common precursor.

One reason why PtdIns may be essential isbecause it is required for synthesis of GPI anchors.S. cerevisiae contains numerous GPI-anchored

Yeast 14, 1471–1510 (1998)

1476 . .

Table 1. Genes for S. cerevisiae phospholipid metabolism.

Gene Function of gene product Key references

CCT1/PCT1 Cholinephosphate:CTP cytidyltransferase 164, 273CDS1 CDP-diacylglycerol synthase (CTP:phosphatidic acid cytidylyltransferase) 115, 236CKI1 Choline kinase 101, 120, 164CPT1 Cholinephosphate:CTP cytidylyltransferase 164, 166CRD1 Cardiolipin synthase 39, 109, 276DPP1 Diacylglycerol pyrophosphatase 262ECT1/MUQ1 Ethanolamine phosphate:CTP cytidylyltransferase 170, 184EPT1 CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase 97, 165, 167GAT1* Glycerol-3-phosphate acyltransferase 9, 260GAT2* Glycerol-3-phosphate acyltransferase 9LPP1 Phosphatidate phosphatase 263MSS4 Phosphatidylinositol-4-phosphate 5-kinase 51, 58, 98PEM1/CHO2 Phosphatidylethanolamine N-methyltransferase 124, 255PEM2/OPI3 Phospholipid N-methyltransferase 162, 255PGS1/PEL1 Phosphatidylglycerophosphate synthase 38, 104, 105, 106PIK1 Phosphatidylinositol-4-kinase 72, 78PIS1 Phosphatidylinositol synthase 70, 190PSD1 Phosphatidylserine decarboxylase (mitochondrial) 43, 269PSD2 Phosphatidylserine decarboxylase (extramitochondrial) 268, 270PSS1/CHO1 Phosphatidylserine synthase 12, 121, 248SLC1 1-Acylglycerol-3-phosphate acyltransferase 9, 183STT4 Phosphatidylinositol 4-kinase 271, 302VPS34 Phosphatidylinositol-3-kinase 95, 252

*Uncharacterized genes.

proteins36,47,86 but only two of them, Gas1p andá-agglutinin, have been studied in detail. In con-trast to mammalian cells, GPI anchoring seems tobe essential for yeast.145 Another reason for theessential role of PtdIns is that it serves as asubstrate for the synthesis of inositol-containingsphingolipids (see below in this review).

A small portion of PtdIns is converted topolyphosphoinositides (Figure 1). Two yeast phos-phatidylinositol 4-kinases, gene products of PIK1and STT4, have been characterized by function.Pik1p is associated with the yeast nucleus and isessential for growth.72,78 Conditional mutantshave a cytokinesis defect, and it was suggested thatPik1p might control cytokinesis through the actincytoskeleton. STT4 was originally discovered in ascreen for sensitivity to staurosporin.302 Stt4p showsmarked homology to protein kinase C (Pkc1p), andan interaction of the two gene products was pro-posed. Deletion of STT4 is lethal under normalgrowth conditions, but Yoshida et al.302 reportedthat an stt4 deletion defect can be rescued by highosmotic strength (1 sorbitol). This effect, however,

271
may be strain-dependent because Trotter et al.

were not able to reproduce this result in a differentgenetic background. Whereas Yoshida et al.302 con-cluded that Stt4p may be involved in the Pkc1protein kinase pathway, Trotter et al.271 demon-strated that, in an stt4 mutant strain, the transportof PtdSer from the endoplasmic reticulum to aGolgi/vacuole fraction is disturbed (see below).Thus, the PtdIns branch of phospholipid biosynthe-sis indirectly affects the biosynthetic pathway ofaminoglycerophospholipids.

The example of Stt4p, however, is not the onlyone that demonstrates the effect of PtdIns kinaseson the intracellular trafficking of macromolecules.The VPS34 gene, which is required for sorting ofvacuolar proteins in yeast,95,252 encodes a phos-phatidylinositol 3-kinase (Figure 1). The roleof phosphatidylinositol 3-phosphate (PtdIns-3-P)formed by Vps34p in vesicle-mediated trafficking isstill a matter of dispute. It was hypothesized thatphosphorylated PtdIns may be important tomaintain the appropriate curvature of transportvesicles.59 Another possible role for PtdIns-3-P isas a signal or a tag for vesicle docking on a target
membrane.
Yeast 14, 1471–1510 (1998)

1477

Recently, Mss4p was identified as aphosphatidylinositol-4-phosphate 5-kinase51,58,98

by homology to the human enzyme. Mss4p islocalized on the plasma membrane of S. cerevisiae.The MSS4 gene is essential, and a temperature-sensitive mss4 mutant was found to be defective inthe organization of the actin cytoskeleton.

Besides the above-mentioned kinases, whose en-zymatic activities have been functionally proven, anumber of putative PtdIns kinases were identifiedby homology. The most promising candidate isTor2p.223 Similar to Mss4p, Tor2p appears to berequired for the organization of the actin cyto-skeleton. Furthermore, biochemical evidence forthe formation of phosphatidylinositol 3,5-bisphosphate in yeast has been presented,67 but therespective kinase has not yet been identified.

Phosphatidylserine (PtdSer) is only a minorcomponent of total cell phospholipids, but animportant intermediate in the de novo synthesis ofthe two main yeast phospholipids, PtdEtn andPtdCho. In contrast to mammalian PtdSer syn-thases,130 which form PtdSer through a base-exchange mechanism, yeast phosphatidylserinesynthase, which is encoded by the PSS1/CHO1gene (for a review, see ref. 297), forms PtdSer fromCDP-DAG and serine (Figure 1). PtdSer, by itself,appears to be non-essential and disruption of thePSS1/CHO1 structural gene is not lethal whencells are grown in the presence of ethanolamine/choline.12 In pss1/cho1 mutants, PtdEtn and Ptd-Cho can be formed via the so-called Kennedy orCDP-ethanolaminecholine pathway (see below). Apss1/cho1 null mutant has no detectable PtdSer,which suggests that there is only one copy of thePSS1/CHO1 gene in yeast.121,248 Expression of thePSS1/CHO1 gene is maximal in the absence ofinositol and choline and is repressed several-foldby the presence of these two substances in thegrowth medium.13 Moreover, derepression of thePSS1/CHO1 gene was found to be sensitive tomutations in the INO2 and INO4 activator genesand the OPI1 repressor gene.14 Thus, PSS1/CHO1expression underlies the same response to inositoland choline supplementation as INO1.

The Pss1p of yeast is located in the endoplasmicreticulum. Recently, it was recognized that aspecific subfraction of the yeast endoplasmic re-ticulum called MAM (mitochondria associatedmembrane) is highly enriched in Pss1p.77 Thisfinding appears to be important because the ma-jority of cellular PtdSer is converted to PtdEtn,and the major site of PtdSer decarboxylation is the


mitochondrion.129 Thus, juxtaposition of sites ofsynthesis and metabolic conversion of PtdSer maypre-determine its route of intracellular migration.Membrane association was suggested as a pre-requisite for PtdSer transport from the endo-plasmic reticulum to mitochondria.1,77,275 Mostlikely, surface proteins of the organelle(s) are in-volved in this process, although they have not yetbeen characterized at a molecular level. A secondPtdSer decarboxylase present in the Golgi/vacuolar compartment268,270 also requires PtdSerfrom the endoplasmic reticulum. Recently, the firstcomponent involved in this translocation processwas identified. This component is a PtdIns4-kinase (see above), the STT4 gene product.271 Itis not clear at present if PtdIns-4-P formed byStt4p acts as an intracellular signal required for theexit of PtdSer from the endoplasmic reticulum or ifit is involved in formation of active transportvesicles as has been suggested for PtdIns-3-Pformed by Vps34p.

The major route of phosphatidylethanolamine(PtdEtn) synthesis in yeast is the de novo pathwaythrough decarboxylation of PtdSer (Figure 1). Themajority of PtdSer decarboxylase (PSD) activityhas been localized to the mitochondrial fraction.129

It came as a surprise that the function of themitochondrial PSD, Psd1p, is dispensable for thecell43,269 even in the absence of ethanolamine,which is substrate for PtdEtn biosynthesis throughthe Kennedy pathway (see Figure 1). Inactivationof the PSD1 structural gene had no effect on cellviability and little impact on the cellular lipidcomposition. Residual PSD activity in psd1 de-letion strains was attributed to Psd2p, which ac-counts for 5–10% of the cellular PSD activity invitro.268,270 The intracellular location of Psd2p isnot completely clear. A Golgi/vacuole compart-ment has been suggested to harbour Psd2p, and aGolgi targeting/retention signal similar to thatfound in the Golgi located Kex2p protease hasbeen identified in Psd2p. Inactivation of both PSDenzymes results in a complete loss of cellular PSDactivity and renders cells auxotrophic for ethanol-amine. The relative roles of the two forms of PSDare not known. Whereas transcription of PSD1 isunder control of inositol and choline,43 no suchregulation has been observed for PSD2.270 Theresponse of the major cellular PSD activity attribu-table to PSD1 is dependent on wild-type alleles ofthe INO2, INO4 and OPI1 regulatory genes, dem-onstrating that it is co-regulated with the otherenzymes in the pathway.134

Yeast 14, 1471–1510 (1998)

1478 . .

An alternative route for the synthesis of PtdEtnin yeast is the Kennedy pathway originally de-scribed by Kennedy and Weis.117 This route is asalvage pathway for yeast cells which are eitherunable to synthesize PtdSer or lack PSD activity(see above), provided that sufficient ethanolamine/choline is present in the growth medium.35 Alter-natively, ethanolamine can be provided by theaction of phospholipases type D160,285 or by deg-radation of long-chain sphingoid base phos-phates.156 Some of the genes involved in theethanolamine branch of the Kennedy pathway(Figure 1) also catalyse reactions involved in theutilization of choline through the choline branchof this pathway. The first step of the CDP-ethanolamine pathway, phosphorylation of etha-nolamine by choline kinase (Cki1p),101,296 iscatalysed by such an enzyme with dual substratespecificity. Choline kinase, which exists as anoligomer in its native form, was recently purified tohomogeneity from the cytosol.120 It is not known,at present, whether or not additional enzymesmay catalyse phosphorylation of ethanolamine.Ethanolaminephosphate is then converted toCDP-ethanolamine by the ethanolaminephosphatecytidylyltransferase (Ect1p/Muq1p).170,184 In anect1/muq1 deletion strain, conversion ofcholinephosphate to CDP-choline appears to beunaffected. Finally, PtdEtn is formed in the endo-plasmic reticulum by CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase(Ept1p).165 Although Ept1p can utilize CDP-ethanolamine, CDP-monomethylethanolamine,CDP-dimethylethanolamine, and CDP-cholineas substrates in vitro97,167 the almost exclusivesubstrate in vivo is CDP-ethanolamine.161,163

Similar to the utilization of free ethanolamine,yeast cells can incorporate choline into PtdChothrough the Kennedy pathway (see Figure 1). Thispathway is not only active when exogenous cholineis present but also functions continuously to re-cycle degradation products of PtdCho.45 Theenzymes involved in the choline branch of thissalvage pathway are choline kinase (Cki1p),cholinephosphate cytidylyltransferase (Pct1p/Cct1p) and CDP-choline:1,2-diacylglycerolcholinephosphotransferase (Cpt1p). As mentionedabove, Cki1p296 can phosphorylate both cholineand ethanolamine. At high choline concentrations,the conversion of choline to cholinephosphatebecomes the rate-limiting step in this pathway.164

Cholinephosphate cytidylyltransferase (Cct1p/Pct1p)273 can utilize cholinephosphate and N,N-


dimethylethanolaminephosphate in vitro, butN-methylethanolaminephosphate and ethanol-aminephosphate are poor substrates. Most of theactivity of Cct1p/Pct1p in yeast cells is associatedwith membranes, suggesting that yeast cells pos-sess a mechanism that promotes membrane associ-ation of the cytidylyltransferase, perhaps similar tothat in mammalian cells.118 Under choline limita-tion, cholinephosphate cytidylyltransferase activitybecomes rate-limiting for the biosynthesis of Ptd-Cho through the Kennedy pathway.164 Expressionof CCT1/PCT1 is not dramatically affected byinositol and choline. The ultimate enzymatic stepin the Kennedy pathway for the genesis of de novosynthesized PtdCho is catalysed by CDP-choline:1,2-diacylglycerol cholinephosphotrans-ferase.166 Genetic approaches revealed that thisenzyme activity can be attributed to two pro-teins, namely to the gene product of CPT1 andto CDP-ethanolamine:1,2-diacylglycerol ethanol-aminephosphotransferase, Ept1p.96,97 Whereascells disrupted either in CPT1 or EPT1 retainapproximately 50% of measurable cholinephos-photransferase activity in vitro,97,163 Cpt1p con-tributes 95% of PtdCho synthesis through theKennedy pathway in vivo. Expression of CPT1underlies an inositol-induced mechanism oftranscriptional regulation.164,179

As mentioned above, expression of the CKI1and CPT1 genes is regulated at the transcriptionallevel by inositol, whereas expression of CCT1 isonly affected moderately by choline or a combina-tion of choline and inositol. Another regulatorymechanism of the Kennedy pathway appears tooccur through the action of Sec14p, which is theyeast PtdIns/PtdCho transfer protein. SEC14 wasoriginally discovered as a gene whose product isinvolved in protein secretion and acts at the levelof Golgi function.16 The lethality of a defect in theSEC14 gene is suppressed in second-site mutationsthat block PtdCho biosynthesis through theKennedy pathway.45 This observation, and thefact that Sec14p inhibits cholinephosphatecytidylyltransferase activity in vitro,242 suggestedthat Sec14p might regulate the PtdIns:PtdChoratio of Golgi membranes. Strains carrying muta-tions in the CDP-choline pathway, such as cki1, incombination with a temperature-sensitive sec14mutation, exhibit a dramatically increased cholinesecretion phenotype.201 Thus, Sec14p appears tobe involved in formation of phosphatidic acid (PA)through an interplay with phospholipase D1(Pld1p)-catalysed degradation of PtdCho.251

Yeast 14, 1471–1510 (1998)

1479

Alternatively, it was proposed that Sec14p may berequired to maintain a sufficient pool of diacyl-glycerol in the Golgi to support the production ofsecretory vesicles.114

In the absence of exogenous choline, PtdCho issynthesized primarily by a three-step methylationof PtdEtn (Figure 1). These reactions are catalysedby two independent methyltransferases, PtdEtnmethyltransferase (Pem1p/Cho2p) and phospho-lipid methyltransferase (Pem2p/Opi3p), whichare localized to the endoplasmic reticulum.112,123

Mutations in the PEM1/CHO2 gene cause a dra-matically decreased level of PtdCho and lead toan accumulation of PtdEtn.124,255 In a pem1/cho2deletion strain, the activity of PtdEtn methyltrans-ferase activity is greatly reduced but the ability ofPem2p/Opi2p to catalyse the first methylation ofPtdEtn allows formation of some PtdCho, albeitsomewhat inefficiently.255 When the PEM2/OPI3locus was deleted, phospholipid methyltransferaseactivity was completely lost and phosphatidyl-monomethylethanolamine accumulated. SincePem2p/Opi3p can replace Pem1p/Cho2p to a cer-tain extent, and mono- and dimethylated PtdEtnappear to replace PtdCho sufficiently, single muta-tions of pem1/cho2 and pem2/opi3 were found notto be auxotrophic for choline.162,255 Oppositeresults regarding the choline auxotrophy124,298

may have been the result of different strain back-grounds. The double mutant pem1/cho2 pem2/opi3,however, is clearly auxotrophic for choline.255

Both PEM1/CHO2 and PEM3/OPI3 aremembers of the group of genes regulated byinositol.125

Under standard growth conditions, phosphati-dylglycerol (PtdGro) is a minor phospholipidcomponent of yeast subcellular membranes.Cardiolipin (diphosphatidylglycerol; CL) com-prises 10–15% of mitochondrial phospholipids,308

especially when mitochondria are fully developedin the presence of non-fermentable carbon sources.PtdGro and CL are closely related biosynthetically(Figure 1). First, phosphatidylglycerophosphate(PtdGro-P) is synthesized from CDP-DAG andglycerol-3-phosphate by the action of phosphati-dylglycerophosphate synthase (for a review, seeref. 171). The PGS1 gene encoding this enzyme hasonly recently been identified.38 Surprisingly, thesame gene (named PEL1 at that time) has beenstudied before and was thought to encode a minorPtdSer synthase activity in yeast.104,105,106 Strainsmutated in PEL1 were isolated as petite lethals;they exhibited a temperature-sensitive growth phe-


notype on glucose, were not able to grow onsynthetic medium with a non-fermentable carbonsource, and had very low levels of cytochrome coxidase. These properties were confirmed recentlywith well-defined pgs1 deletion strains that wereshown to lack PtdGro and CL.38 These resultsdemonstrated that the PGS1/PEL1 gene encodesthe major, or even the only, PtdGro-P synthase ofyeast, and that neither PtdGro nor CL is abso-lutely essential for cell viability. PtdGro-P synthaseactivity is stimulated by factors affecting mito-chondrial development79 and regulated throughcross-pathway control by inositol.83,235

The next reaction in CL synthesis is dephospho-rylation of PtdGro-P yielding PtdGro. The geneencoding PtdGro-P phosphatase has not yet beencloned, neither has the gene product been charac-terized. As the ultimate step in this biosyntheticsequence, CL is formed by reaction of PtdGro witha second molecule of CDP-DAG through catalysisperformed by CL synthase.221 The gene encodingCL synthase, CRD1 (formerly named CLS1), wasrecently identified.39,109,276 Crd1p was shown toform an oligomeric complex whose biogenesisand/or activity is influenced by the assembly ofcytochrome c oxidase.304 Strains deleted of CRD1are able to grow on all carbon sources, althoughthe growth rate on non-fermentable carbonsources is decreased. The lack of CL synthaseactivity in crd1 mutants results in a lack of detect-able CL and accumulation of PtdGro when cellsare cultivated aerobically on non-fermentable car-bon sources. Levels and activities of cytochromesare normal in cdr139,276 and the mitochondrialmembrane potential is not grossly compromised.Thus, CL is not essential for mitochondrial func-tion and can, at least in part, be replaced byPtdGro-P and/or PtdGro.

CL and PtdGro are typical mitochondrial phos-pholipids, although yeast peroxisomes were shownto contain amounts of CL that cannot be attrib-uted to cross-contamination with mitochon-dria.306,308 CL synthase and PtdGro-P synthaseactivity are mainly present in the mitochondrialinner membrane but some of the latter enzymaticactivity was also localized to microsomalmembranes.129,308

Although it is beyond the scope of this review todiscuss degradation and turnover of glycerophos-pholipids in detail, the role of diacylglycerol(DAG) has to be briefly addressed. In principle,DAG may be derived from degradation of phos-pholipids by catalysis of phospholipases type C.

Yeast 14, 1471–1510 (1998)

1480 . .

One such protein, Plc1p was characterizedas a phosphatidylinositol-specific phospholipaseC.73,202,300,301 Alternatively, phosphatidate phos-phatase (Lpp1p) and diacylglycerol pyrophos-phatase (Dpp1p) appear to be involved in DAGformation.34 Diacylglycerol pyrophosphate(DGPP) was recently detected in minor quantitiesin yeast.295 It was suggested that this lipid isinvolved in a lipid signalling pathway. Dpp1pcatalyses both the diphosphorylation of DGPP,yielding phosphatidic acid (PA), and the dephos-phorylation of PA, yielding DAG.262 The PAphosphatase activity of Dpp1p is not the only onein yeast, since a mutant with a DPP1 deletioncontains residual PA phosphatase activity butlacks DGPP phosphatase activity. Recently, theLPP1 gene263 was shown to encode another PAphosphatase distinct from Dpp1p. Lpp1p alsoexhibits lyso-PA and DGPP phosphatase activi-ties. Strains with deletions in DPP1, LPP1 and thedpp1 lpp1 double mutant are viable and do notexhibit any obvious growth defects.

DAG is also the substrate for the synthesis oftriacylglycerol (TAG), which is a storage form offatty acids in lipid particles of S. cerevisiae.42,140

TAG can be used as a source of fatty acids whenendogenous fatty acid synthesis is blocked, e.g. inthe presence of the inhibitor cerulenin.53 The fattyacids derived from TAG are not used as a primaryenergy source in yeast, unlike in mammalian cells,because S. cerevisiae has no mitochondrialâ-oxidation, and peroxisomal â-oxidation induced


by growth of cells on oleic acid is not very active,although it is sufficient to maintain growth. Incontrast, fatty acids set free from TAG are incor-porated into phospholipids that are required formembrane formation. Little is known about TAGsynthase in yeast. Some activity of this enzymehas been attributed to lipid particles,42 but morerecently the microsomal fraction was shown tocontain the highest TAG synthase activity (K.Athenstaedt, personal communication). Thegene encoding TAG synthase has not yet beenidentified, neither has the enzyme been isolated.

Table 2. Genes for S. cerevisiae fatty acid metabolism.


ACB1 Acyl-CoA binding protein 220ACC1 Acetyl-CoA carboxylase 2, 91ACP1 Mitochondrial acyl carrier protein 31, 225CEM1 Mitochondrial beta-ketoacyl-[acyl-carrier protein] synthase 89, 224ELO1 Fatty acid elongase (long-chain fatty acids) 64, 261ELO2/FEN1 Fatty acid elongase (synthesis of C-24 fatty acids) 194ELO3/SUR4 Fatty acid elongase (conversion of C-24 to C-26 fatty acids) 194FAH1/SCS7 Hydroxylation of the C-26 fatty acid in ceramide 85, 174FAS1 Fatty acid synthase (beta subunit) 41, 229FAS2 Fatty acid synthase (alpha subunit) 176, 229MCT1 Malonyl CoA:acyl carrier protein transferase 224OAR1 Mitochondrial 3-oxoacyl-[acyl-carrier-protein] reductase 224OLE1/MDM2 Fatty acid desaturase 173, 253SUR2/SYR2 Hydroxylation of sphinganine or sphinganine-containing

dihydroceramides at the C-4 position to yield phytoceramide84, 85

A brief survey on the biosynthesis of fatty acids inyeast

The fatty acid composition can greatly influencethe physical state of phospholipids and therebyaffect membrane fluidity and permeability. Thefatty acid pattern of yeast phospholipids appearsto be fairly simple. The major species are C-16 andC-18 fatty acids without or with one double bond.Other fatty acids, present in minor amounts inyeast, may be of physiological importance but theyhave not been systematically analysed.

Genes involved in the formation of fatty acids inyeast (Table 2) are ACC1, encoding acetyl-CoAcarboxylase,2,91 and FAS1 and FAS2, encoding theâ- and á-subunits of the fatty acid synthase com-plex.41,176 An acyl-CoA binding protein, Acb1p, isthought to bind the acyl chain and hand it overto cellular processes that consume acyl-CoA.220

Yeast 14, 1471–1510 (1998)

1481

Mutations of FAS1 and FAS2 are lethal unlessmutants are supplemented with fatty acids.228

ACC1 mutations are lethal even in the presence offatty acids in the culture medium.2,91 The existenceof a second, mitochondrial, fatty acid-synthesizingsystem has been reported recently. Four genespossibly encoding for components of this machin-ery, ACP1, CEM1, OAR1 and MCT1, have beendescribed.31,89,90,224,225 Deletion of each genecauses a dramatic decrease in the lipoic acid con-tent of cells and a respiratory-deficient phenotype.

Pre-existing fatty acid in yeast can be modifiedby desaturation, elongation and hydroxylation.Mono-unsaturation of fatty acids is catalysed by asingle essential desaturase, Ole1p.173,253 The en-coding gene, OLE1/MDM2, has been demon-strated to be also involved in partitioning ofmitochondria between mother and daughter cells.The ole1/mdm2 mutants are viable when fed un-saturated fatty acids. Elongation of fatty acids isaccomplished by introducing malonyl-CoA as aC-2 unit into pre-existing fatty acids.64 This reac-tion is catalysed by the product of the ELO1gene.64,261 Strains deleted of ELO1 cannot growon media containing C-14:0 as the only fatty acid,but are rescued by adding C-16:0 and C-18:0 fattyacids to the media. Subsequently, two genes struc-turally related to ELO1, namely ELO2 and ELO3,were identified which encode components of thevery long chain (C-24 and C-26) fatty acid elonga-tion system.194 The major hydroxylated fattyacid in S. cerevisiae is the C-26 species, which isrequired for sphingolipid biosynthesis (see thisreview article). Alpha-hydroxylation of fatty acidsis catalysed by Scs7p,85,174 and long chain base

84,85
hydroxylation at C-4 by Sur2p.
Subcellular location of phospholipids, sites ofsynthesis and general aspects of phospholipidtransport

The major phospholipids, PtdCho, PtdEtn anPtdIns, are present in all subcellular membranes ofS. cerevisiae, although in variable amounts. Twoexceptions are PtdSer and CL, which are presentonly at low concentrations in most membranes,but are major phospholipid components of theplasma membrane and the inner mitochondrialmembrane, respectively (for a review see ref. 306).

Classically, the endoplasmic reticulum has beenthought of as the major site of lipid biosynthesis,with minor contributions from the mitochondria.This view needs revision. Besides these two com-


partments, the Golgi and lipid particles are nowrecognized to harbour certain enzymes for yeastphospholipid formation (see above). The questionremains, however, how phospholipids are trans-ported from their site(s) of synthesis to theirproper subcellular location. It is not only thesupply of the quantitatively predominant phos-pholipids (PtdCho, PtdEtn and PtdIns) to all or-ganelles which necessitates efficient mechanisms ofintracellular lipid transport, but also the migrationof precursor phospholipids to their site of meta-bolic conversion that is essential for the balancedsynthesis of cellular phospholipids. The exampleof PtdSer and PtdEtn translocation betweenorganelles has already been mentioned above.

Phospholipids appear to be transported within acell by vesicle flow, membrane contact and mono-mer transport, with the aid of so-called lipidtransfer proteins (for recent reviews, see refs 267,280, 282). Whereas there is no proof at present forthe contribution of lipid-transfer proteins to thesubcellular distribution of lipids in vivo, exper-imental efforts have tried to distinguish betweenvesicle flow and membrane contact as mechanismsof lipid translocation. In the past, most of thesestudies have focused on the intracellular move-ment of aminoglycerophospholipids (see ref. 54),because translocation of PtdSer and PtdEtn be-tween organelles (endoplasmic reticulum and mito-chondria) can be studied by metabolic conversionassays. Decarboxylation of PtdSer to PtdEtn bymitochondrial Psd1p provides such a means offollowing the import of PtdSer into mitochondriain vivo or in vitro. Most experimental evidencefavours membrane contact as the mechanism oftransport of PtdSer from MAM, a specializedsubfraction of the endoplasmic reticulum, to mito-chondria.1,54,77 As was shown for mammalianMAM,238 proteolytic treatment of yeast mitochon-dria negatively affected translocation of PtdSer.275

Fusogenic proteins similar to those described formammalian cells by Corazzi’s group33,211 andwhich are partially inactivated by exogenous pro-teases, may be involved in this process. None ofthe putative components, however, has been char-acterized on a molecular level, neither have therespective genes been identified. The first geneproduct involved in PtdSer transport from theendoplasmic reticulum to a Golgi/vacuole com-partment (the PtdIns-4-kinase, Stt4p) has beendescribed recently.271 The mechanism of PtdSertranslocation between these two compartments,however, may not involve membrane contact.

Yeast 14, 1471–1510 (1998)

1482 . .

Transport of phospholipids to the plasma mem-brane of yeast is even less well understood. While ithas become clear that sphingolipids utilize theprotein secretory machinery93,206 to reach theirdestination at the cell periphery, no such evidencehas been presented for phospholipids. Rather,preliminary evidence80 has indicated that, intemperature-sensitive secretory mutants (sec mu-tants), PtdCho and PtdIns are transported to theplasma membrane at a normal rate. This result isin line with the hypothesis of Atkinson10 thatdifferent types of vesicles are responsible for pro-tein export and membrane growth. The existenceof specific phospholipid transporting vesicles, how-ever, still needs to be verified experimentally.

STEROLS

Sterols are essential lipid components of eukaryo-tic membranes and have been shown to be respon-sible for a number of important physicalcharacteristics of membranes. The specific sterolpresent in eukaryotic species can vary and therehave been many studies using a variety of bio-chemical and biophysical techniques to demon-strate that sterols are important regulators ofmembrane permeability and fluidity. Althoughother membrane lipids also play a role in definingthese properties, eukaryotic cells are unable tomaintain viability without sterol. More recently,preliminary studies have described some physio-logical properties and effects of sterols on aerobicmetabolism,244,245 completion of the cell cycle,52

sterol uptake150 and sterol transport.274

The structural features of sterols derived fromeukaryotes vary in plants, animals and fungi, asshown in Figure 2. The fungal sterol, ergosterol,differs from the animal sterol, cholesterol, by thepresence of unsaturations at C-7,8 in the ringstructure and at C-22 in the side chain and by thepresence of a methyl group at C-24 on the sidechain. The major plant sterols, sitosterol and stig-masterol, have ring structures identical to thatfound in cholesterol but have slight modificationsof the side chain. Common to all sterols is thesaturation at C-5,6 and the presence of the hy-droxyl group at C-3. The latter provides the onlyhydrophilic component of the molecule and allowsfor the proper orientation of the sterol molecule inthe membrane.

Much of the work in defining the role of sterol ineukaryotic membranes has been done using the


yeast model system. Ergosterol mutants have beenused to document the effects of sterol substitutionon membrane fluidity142 and membrane per-meability.20,122 Membrane sterol modification inyeast has also been shown to effect energy sourceutilization144 and the activity of membrane-boundATPase.46 In addition to providing these bulkfunctions for membranes, sterols have been impli-cated in providing a ‘sparking’ function52 involvedin the completion of the cell cycle. Unlike the bulkfunctions, which can be provided by a numberof sterols, the sparking function is conferred bythe presence of a specific sterol structure and isrequired in nanomolar quantities. Recent re-ports244,245 have indicated that the sparking func-tion may be related to the presence or absence ofheme. In addition, cloning and gene disruptionprocedures have contributed significantly indefining essential features of fungal membranesterol.143

S. cerevisiae has also served as a model systemfor defining aspects of sterol biosynthesis whichhave application for the development and evalu-ation of antifungal compounds. The sterol biosyn-thetic pathways or its end product are the targetsfor most of the antifungal compounds currentlyin use in agricultural settings and in combatinghuman infections. The polyene antifungals, such asnystatin and amphotericin B, function by bindingto membrane ergosterol and inducing cellular leak-age, resulting in cell death.30 Other groups ofantifungals, including the allylamines,217 morpho-lines15 and azoles,279 inhibit specific steps in ergo-sterol biosynthesis and will be discussed in moredetail later in this review. All of these compoundshave limitations as to their application to humaninfection or have seen the appearance of significantlevels of resistance in several pathogenicfungi.57,92,205,219,281,291 A recent review has out-lined factors which have led to this increasedresistance.293 In order to identify new possibletarget sites in the ergosterol pathway, molecularstudies of ergosterol biosynthesis have been em-ployed to identify essential steps in the pathwayagainst which antifungals might be developed.

Sterol biosynthesis: acetate to farnesylpyrophosphate

Sterol biosynthesis is a major metabolic commit-ment on the part of the cell and involves over 20distinct reactions. The pathway is initiated withacetyl-CoA, as depicted in Figure 3, which

Yeast 14, 1471–1510 (1998)

1483

Figure 2. The principal membrane sterols in fungi (ergosterol), animal cells (cholesterol), and higherplants (sitosterol and stigmasterol).

presents an abbreviated version of what is com-monly called the mevalonate or isoprenoid path-way. This portion of the pathway ends with theformation of farnesyl pyrophosphate, a pivotalintermediate which is the starting point for severalessential pathways. The syntheses of heme,287 quin-


ones196 and dolichols159 involve the participationof farnesyl components derived from this pathway.In addition, the farnesyl units and the relatedgeranyl and geranylgeranyl species are importantelements for the posttranslational modificationof proteins that require hydrophobic membrane

Yeast 14, 1471–1510 (1998)

1484 . .

phenomena. Subsequent work63 showed thatERG10 was induced by low levels of sterol andthat specific promoter regions regulate gene ex-pression. In a more recent report65 using theERG10 promoter fused to a reporter, additionalevidence was presented which suggests that regu-lation occurs at the transcriptional level and ismediated by pathway intermediates.

The conversion of acetoacetyl-CoA tohydroxymethylglutaryl-CoA (HMGCoA) by theERG13 gene product, HMGCoA synthase inyeast, deviates from that described in animal sys-tems in that the enzyme appears to be subject toregulatory control.230,265 The enzyme is a multimerpossibly comprised of two gene products, althoughonly one gene is found in the yeast database forthe production of this enzyme. The specificsof the regulatory mechanism involved remainuncharacterized.

The third enzyme in the pathway, HMGCoAreductase, is the most studied step in the meval-onate pathway. It is the major site of regulation incholesterol synthesis81 and the target for severalcompounds that are employed clinically ascholesterol-lowering agents. The yeast enzyme isalso subject to regulation by a number of factorsand conditions. Unlike humans, yeast has twocopies of the gene encoding HMGCoA reductase.This was discovered upon the disruption of one

213

Figure 3. The mevalonate pathway from acetyl-CoA to far-nesyl pyrophosphate. For a description of genes and geneproducts, see Table 3.
gene, HMG1, which resulted in a viable cell.
anchors for proper placement and function.Among such proteins are the ras proteins.44 Thus,farnesyl pyrophosphate leads to the synthesisof several critical end products, including sterols.For this reason, mutations in the pathway to thispoint are lethal, since multiple essential metabolicproducts cannot be synthesized.

The first step in the pathway, leading ultimatelyto the production of ergosterol, is the synthesis ofacetoacetyl-CoA from two molecules of acetyl-CoA, a reaction catalysed by acetoacetyl-CoAthiolase, encoded by the ERG10 gene.127,128 Yeastcontains two forms of the enzyme, one of which isfound in the mitochondrion, while the second islocated in the cytosol. This step in yeast, unlike thesituation in animal cells, is subject to regulation.Early studies described decreased enzyme activityupon the addition of exogenous sterol265 and in-creased activity resulting from ergosterol starva-tion.230 These studies, however, could not clearlydistinguish between transcriptional and trans-
lational mechanisms to explain the regulatory

This unanticipated result was explained by theidentification of a second gene, HMG2.22 Disrup-tion of both genes renders the cell non-viable, aswould be predicted. Yeast HMGCoA reductaseactivity shows feedback inhibition in the presenceof ergosterol18 and is subject to catabolite repres-sion.209 In addition, HMGCoA reductase activitywas found to be induced by unsaturated fattyacids29 and glucose.28 However, these studies wereconducted before the demonstration of two iso-forms of the enzyme. Thorsness et al.259 havefound that HMG1 expression was stimulated byheme while HMG2 expression was inhibited, againindicating a relationship between heme and sterolbiosynthesis.

Step 4 in the pathway is catalysed by mevalonatekinase, which is encoded by the ERG12 gene. Theyeast enzyme is subject to inhibition by certaindownstream intermediates. Farnesyl and geranylpyrophosphates exert an inhibitory influence onthe enzyme, while isopentenyl and dimethylallylpyrophosphates do not.66 Step 5 also involvesphosphorylation. The ERG8 gene encodes

Yeast 14, 1471–1510 (1998)

levels of enzyme activity and ergosterol produc-

1485

tion, indicating that this step may be rate-4,37

phosphomevalonate kinase,272 an enzyme which isnot subject to feedback inhibition by ergosterol.

Steps 6 and 7 of the pathway are largely unchar-acterized in terms of the properties of the enzymesinvolved. The ERG19 gene encodes the mevalonatepyrophosphate decarboxylase, which convertsmevalonate pyrophosphate to isopentenyl pyro-phosphate (IPP). Yeast erg19 mutants werefirst isolated in a broad screen which selectedfor temperature-sensitive, nystatin-resistant iso-lates.113,231 Among the many possibilities wereerg19 mutants, which were identified based onsterol intermediates that accumulated and enzymeassays37 specific for each possible step. The yeastERG19 gene has been cloned using sequence dataobtained from the human version of the gene.264

Overexpression of ERG19 has been shown to resultin a reduction of sterol accumulation,27 suggestingthat the enzyme is rate-limiting and that down-stream intermediates may accumulate and functionas feedback inhibitors at an early pathway step.The subsequent step converts IPP to dimethylallylpyrophosphate (DMAPP) through the action ofisopentenyl diphosphate isomerase, a product ofthe IDI1 gene. Although the IDI1 gene has beencloned, little is known about the properties of theenzyme and its regulation.3

The final step in the early portion of the path-way is the conversion of DMAPP to farnesyl andgeranyl pyrophosphates. This step is accomplishedby the action of farnesyl (geranyl) pyrophosphatesynthase, a product of the ERG20 gene. Theenzyme combines DMAPP and IPP to form ger-nyl pyrophosphate (GPP). The same enzyme isthen able to extend geranyl pyrophosphate bycombining it with a second IPP to form farnesylpyrophosphate (FPP). This brings the pathway tothe pivotal molecule, which is the substrate forseveral enzymes which initiate several metaboli-cally important pathways. Overexpression of theERG20 gene has been shown to result in increased

Figure 4. The ergosterol biosynthetic pathway from farnesylpyrophosphate to ergosterol. For a description of genes andgene products, see Table 3.

limiting.

Sterol biosynthesis in yeast: farnesylpyrophosphate to ergosterol

The conversion of farnesyl pyrophosphate to theend-product, sterol, represents an 11-step pathwaydedicated to ergosterol biosynthesis (Figure 4).Mutations in any step up to this point are lethalbecause of a failure to make multiple essential end


products. Mutations in the last eight steps of thispathway segment are lethal only if the intermediateformed cannot substitute functionally for ergo-sterol. Since yeasts require sterol, one would pre-dict that the first three steps (formation of squaleneto lanosterol) would be essential, since no sterolmolecule is synthesized to this point. This turnsout to be the case. In addition, one would predictthat regulatory phenomena specific to sterol levelswould be found in this portion of the pathway,

Yeast 14, 1471–1510 (1998)

1486 . .

Table 3. Genes involved of the biosynthesis of ergosterol in yeast.


ERG1 Squalene epoxidase 102, 103ERG2 Sterol C-8 isomerase 6, 8ERG3 Sterol C-5 desaturase 7, 149, 216ERG4 Sterol C-24 reductase 40, 133ERG5 Sterol C-22 desaturase 241ERG6 Sterol C-24 methyltransferase 17, 75, 177ERG7 Lanosterol synthase 50, 237ERG8 Phosphomevalonate kinase 272ERG9 Squalene synthase 108ERG10 Acetoacetyl-CoA thiolase 127, 128ERG11 Lanosterol C-14 demethylase 111, 258, 266ERG12 Mevalonate kinase 66ERG13 HMGCoA synthase 230, 265ERG19 Mevalonate pyrophosphate decarboxylase 113, 231ERG20 Farnesyl pyrophosphate synthase 4, 37ERG20 Geranyl pyrophosphate synthase 4, 37ERG24 Sterol C-14 reductase 133, 148, 158ERG25 Sterol C-4 methyloxidase 19, 74ERG26 Sterol C-3 dehydrogenase (decarboxylase) Gachotte et al. (pers. comm.)ERG(?)* Sterol C-3 keto reductaseHMG1, HMG2 HMGCoA reductase 81IDI1 Isopentenyl pyrophosphate isomerase 3

*The gene for the sterol C-3 keto reductase has yet to be isolated and characterized.

since no other products are formed from theintermediates. There are a few well-characterizedpoints of regulation, although the pathway re-mains largely unexplored in this regard.

Squalene synthase, a product of the ERG9 gene,combines two FPP molecules to form squalene. Asthe first enzyme in a linear pathway, this stepwould be expected to be an ideal place for regula-tion.108 The enzyme competes with other enzymesfor the FPP substrate and has been shown torespond to cellular sterol content in a mannersimilar to that shown by HMGCoA reductase.214

Studies of this type present specific limitations,since yeast synthesize sterol only under aerobicconditions and are unable to import sterol underthese conditions. Thus, sterol depletion in aerobiccells is possible only with the use of inhibitors orby using mutant strains. Using the latter approach,M’Baya et al.151 showed that early (pre-mevalonate) pathway erg mutants induced ERG9activity by 1.5 to 5-fold, suggesting that meval-onate may play a key role in the regulatory pro-cess. In the same study, excess sterol addedexogenously to anaerobically growing cells re-
sulted in the depression of squalene synthase

activity. These preliminary studies indicate thatthis branch enzyme is subject to regulation, butmuch remains to be done to define the controlmechanisms at the molecular level.

The ERG1 gene encodes squalene epoxidase(monooxygenase), which converts squalene to 2,3-oxidosqualene. This oxygen-requiring step102 pre-cludes sterol synthesis in anaerobic conditions,thus requiring the addition of exogenous sterol forgrowth. This step is inhibited by the allylamineclass217 of antifungals and by thiocarbamates.178

In fact, the ERG1 gene in yeast was cloned byusing a yeast library constructed from a strainthat was resistant to the allylamine, terbinafine.103

Employing the same conditions as described abovefor squalene synthase, squalene epoxidase activityincreased in all erg mutants grown aerobically,except erg11.151 The effects of excess sterol couldnot be determined under anaerobic conditions,since the enzyme requires oxygen.

The third step in the conversion of FPP toergosterol represents an impressive single-step con-version of 2,3-oxidosqualene to lanosterol, the firststerol molecule of the pathway. 2,3-Oxidosqualene
cyclase or lanosterol synthase, the product of the
Yeast 14, 1471–1510 (1998)

1487

ERG7 gene, performs several ring closures andbond cleavages in this very complex reaction.50

The ERG7 gene from yeast has been cloned by twolaboratories50,237 which should allow for charac-terization of this gene and the encoded enzyme.

The pathway has now reached the point where asterol molecule is present and the question to beasked from here to the end of the pathway iswhether the intermediate produced at each step isable to meet both the bulk and sparking require-ments and thus permit growth. One of the charac-teristics of this part of the pathway is that blocks inmost steps do not prevent subsequent enzymesfrom altering the improperly modified substrates.Thus, blocks lead to the accumulation of severalsterol structures that are not normal intermediatesof the pathway.

Mutants blocked in the terminal five steps of thepathway have been available for many years.17,177

They were isolated based on resistance to thepolyene nystatin, which shows less affinity forsterol intermediates than for ergosterol. The vi-ability of these mutants indicated that the sterolintermediates accumulated satisfied the sparkingfunction. However, as these were point mutations,trace and undetectable amounts of ergosterolmight be produced. Subsequent cloning and dis-ruption of each of the genes provided definitiveevidence that these genes are, indeed, non-essentialfor viability under normal growth conditions.

Lanosterol, or other sterols containing a C-14ámethyl group, cannot support growth of S. cerevi-siae except under very unusual circumstances.76

Thus, lanosterol C-14 demethylase, the product ofthe ERG11 gene, is an essential enzyme in sterolbiosynthesis. The enzyme is a cytochrome P-450enzyme and is the target of the azole antifungals,the most commonly employed family of drugs inhuman fungal infections.279 Yeast erg11 mutantshave been described258,266 but were found to beleaky and to contain a second leaky mutation inthe gene encoding the sterol C-5 desaturase(ERG3). Subsequent work21 using disrupted ver-sions of the ERG35 and ERG11111 genes confirmedthe essential nature of the ERG11 gene and theability of the accompanying ERG3 mutation tosuppress lethality of the erg11 phenotype. Thisdownstream suppression has been explained286 bythe accumulation of 14á-methyl-ergosta-8,24(28)-dien-3â,6á-diol in erg11 mutants. This unusualsterol is toxic and results from downstream metab-olism of C-14 methyl sterol intermediates thatcannot be completely desaturated at C-5. The diol


is not formed in cells that are missing a functionaldesaturase (erg3).

The lanosterol C-14 demethylase is one of thefew pathway reactions where regulation at themolecular level has been explored. Protein levels ofthe enzyme have been shown to respond to oxygenconcentration, carbon source and other growthconditions. Levels of ERG11 mRNA have beendemonstrated to increase during growth on glu-cose, in the presence of heme, and under condi-tions where oxygen is limited.277 In addition,promoter analysis identified two activating se-quences, one of which responds to the HAP1 geneproduct and one definite repressor element whichresponds to the ROX1 gene product. In a subse-quent report55 it has been shown that ERG11 isrepressed by the HAP1 gene product under lowheme conditions. Thus, the Hap1p transcriptionfactor has both positive and negative effects onERG11 expression, depending on the physiologicalconditions in the cell.

The ERG24 gene encodes the sterol C-14 re-ductase, which converts 4,4-dimethylcholesta-8,14,24-trienol to 4,4-dimethylcholesta-8,24-dienol. This enzyme and the sterol C-8 desaturase(ERG2) are the targets of the morpholine anti-fungals.15 Morpholine treatment leads to the pro-duction of ergosta-8,14-dienol (ignosterol), whichcannot support growth under normal conditions.Cloning and disruption133,148,158 of the ERG24gene indicated that it was essential, but conflictingevidence was provided as to whether growthinhibition was due to the accumulation ofnon-utilizable 8,14-dien sterols148 or the lack ofergosterol.158

Non-viability resulting from the elimination ofthe ERG24 gene product is also subject to suppres-sion in yeast. A mutant resistant to fenpropi-morph, a commonly employed morpholine, wasfound to produce ignosterol in the presence of thedrug.148 The mutation responsible for resistance,fen1, was able to allow growth with ignosterol.Recent reports have led to the identification of thefen1 mutation. SR 31747, an immunosuppressiveagent that also blocks yeast growth by inhibitingthe Ä8-Ä7 isomerase (ERG2p), has been used toselect for SR 31747-resistant mutants.239 The mu-tants fall into two categories and have been local-ized to FEN1 and SUR4. Mutations in FEN1 andSUR4 have pleiotropic effects in yeast and affectphospholipid synthesis, budding and other cellfunctions. Oh et al.194 have recently described twogenes, ELO2 and ELO3, that are involved in fatty

Yeast 14, 1471–1510 (1998)

1488 . .

acid elongation and sphingolipid synthesis. Se-quencing of ELO2 and ELO3 has indicated iden-tity to FEN1 and SUR4, respectively. A mutationin either of these genes was able to suppress theerg24 phenotype and permit growth. In addition,fen1 and sur4239 were shown to suppress the lethaleffects of an erg2 mutation which previously hadbeen reported to be non-lethal.8 These findingsindicate that sterol requirements for yeast mem-branes can vary depending on the presence orabsence of other membrane lipids.

The removal of the two methyl groups from theC-4 position remains the final step in yeast sterolbiosynthesis for which all the genes responsiblehave not been cloned. The process of demethyla-tion is very complex and has been described enzy-matically in cholesterol biosynthesis.74 The firststep employs the C-4 sterol methyloxidase (en-coded by the ERG25 gene) and results in theoxidation of the C-4á methyl group to a carboxylicacid. A second enzyme then removes the carboxy-lic acid, which results in the formation of a ketofunction at C-3. A third enzyme would reduce theC-3 keto function to the desired alcohol. TheERG25 gene, encoding C-4 sterol methyloxidase,has been isolated and cloned.19 As was the case forthe ERG11 and ERG24, the ERG25 gene wasshown to be essential for growth.

Subsequent studies indicated that the erg25 phe-notype was also subject to suppression, but by amechanism distinct from those observed in ERG11and ERG24 suppression.76 In this case, suppres-sion was due to the accumulation of two muta-tions. One was a mutation in the upstream ERG11gene, while the second was a leaky mutation ineither HEM2 or HEM4, genes in the heme bio-synthetic pathway. A curious observation in thetriple mutants (erg25, erg11, and hem2 or hem4)was that downstream conversions of lanosterolwere blocked and that the cell was now capable ofgrowing exclusively on lanosterol. This is the onlyreport of such a capability and has implicationsregarding the structural features of sterol that canbe tolerated in yeast.

The second gene in the C-4 demethylation reac-tion, ERG26, encoding a C-3 sterol dehydrogenase(decarboxylase), required to remove the C-4 car-boxylic acid groups, has recently been cloned anddisrupted (Gachotte et al., manuscript submitted).The ERG26 gene was reported to be essential anderg26 disruptants required both exogenous steroland a mutation in the heme pathway for viability.These results suggested that either the accumula-


tion of carboxylic acid sterol intermediates wastoxic to growing heme-competent yeast cells, orthat the heme mutation was required for sufficientuptake of exogenous sterol even under anaerobicconditions.

Zymosterol, the product of the C-4 demethyla-tion step, serves as the substrate for the sterol C-24methyltransferase. Yeast strains with mutations inthe encoding ERG6 gene have been available17,177

for a considerable period of time. This step hasbeen of particular interest, since it represents a stepnot found in cholesterol biosynthesis and might beprojected to provide some unique function inyeast. The yeast ERG6 gene has been cloned, andanalysis of a strain carrying an erg6 deletion/disruption indicated that it was not essential foraerobic growth. Mutants of erg6 were found tohave several deficiencies, including altered per-meability characteristics,20,122 the inability to uti-lize respiratory energy sources,144 reduced matingcapability75 and the inability to import tryp-tophan.75 Although survival is possible without afunctional ERG6 gene, the yeast is at a consider-able disadvantage, and the essentiality of a givengene may vary depending on changes in environ-mental conditions.197

As a target for the morpholine antifungals,15 thesterol C-8 isomerase was at one time postulated toprovide the sparking function. However, disrup-tion of the encoding ERG2 gene showed that it wasnot essential in yeast.6,8 Recent findings239 haveindicated that erg2 mutants are non-viable in anaerobic environment and that erg2 mutants can besuppressed by fen1/elo2 and sur4/elo3 mutations.Apparently, the strain in which the ERG2 disrup-tion was made8 carried an undetected fen1 or sur4mutation.

The next step in sterol biosynthesis is a desatu-ration which adds the C-5,6 double bond. This is ahighly conserved reaction in sterol biosynthesisand sterols from plant, animal and fungal sourceshave this feature. For this reason, the sterol C-5desaturase was hypothesized to be essential inproviding sparking sterol, and sterol-feeding ex-periments seemed to support the essential natureof this reaction.149,216 Subsequently, the encodingyeast ERG3 gene was cloned and disruption of thegene resulted in a strain that retained viabilityunder aerobic conditions.5 The resolution of thesecontradictory conclusions lies in considerationof other markers in the strains used.244 ERG3disruptions were done in a heme-competent strain,while feeding experiments were accomplished in a

Yeast 14, 1471–1510 (1998)

viability.

1489

heme-deficient background. The addition of hemeprecursor, allowing for heme synthesis in the lattersystem, permitted growth without ERG3 function.Therefore, ERG3 is essential only in heme-deficientcells and is thus required for respiratory growth.244

Further analysis245 has shown that heme, and notheme precursors prior to the heme biosynthesisblock, exhibits this characteristic, suggestingthat ergosterol, or sparking sterol and heme playimportant and interrelated roles in aerobic andanaerobic metabolism in yeast.

ERG3 is one of the few pathway genes in whichregulatory phenomena have been explored at themolecular level. Since the ERG3 gene has beenshown to be essential in some backgrounds244 andhas been implicated in the regulation of ergosterolbiosynthesis in the yeast strain GL7,187 it repre-sents a potentially important regulatory point inthe pathway. Two research groups have recentlyreported on regulation of ERG3.7,246 Using theERG3 promoter fused to the bacterial lacZ re-porter gene, both groups explored the activity ofthe promoter under a variety of conditions thateffect sterol biosynthesis. One study246 showed30–40 fold increases in ERG3 activity in strainsthat contained additional mutations in the ergos-terol pathway (erg5, erg6, and erg24) and wereunable to synthesize ergosterol. In addition, ERG3mRNA levels were significantly elevated in thesemutant strains. Increased mRNA levels were alsoobserved when sterol biosynthesis inhibitors wereused in place of the erg mutations. Deletion analy-sis of the ERG3 promoter identified a 22-bp regionas playing a major role in the sterol regulation oftranscription. The second study7 utilized a similarERG3 promoter–lacZ fusion to examine regula-tion of ERG3. Promoter activity was increased inseveral erg mutants (erg2, erg3, erg4, erg5 anderg6) as well as in cells that contain a null mutationin HMG1, the major yeast gene encoding HMG-CoA reductase. In addition, null mutations inboth genes (ARE1 and ARE2) responsible forsterol esterification resulted in decreased levels ofERG3 expression as well as a decrease in total cellsterol.

The final two steps of the pathway involve theintroduction of the double bond at C-22 and thereduction of the double bond at C-24(28). In bothcases, mutants produce sterol intermediates thatare close to the final structure and, thus, the cellshave characteristics very similar to those exhibitedby the wild type. The reduction at C-22 is accom-plished by the sterol C-22 desaturase, a product


of the ERG5 gene. This gene has been cloned,sequenced and disrupted and found to be non-essential for viability.241 The production of ergo-sterol results from the action of sterol C-24reductase, an enzyme encoded by the ERG4 gene.The ERG4 gene in yeast was cloned as YGL022and was shown to be non-essential for

40,133

Intracellular transport and location of sterol inyeast

The ultimate fate of sterol synthesized or im-ported by yeast is critical for the proper functionof the membranes of eukaryotes. Ergosterol is notpresent in equal amounts in all membranes andspecific preference is shown for the incorporationof ergosterol rather than sterol intermediates.Ergosterol is found in the highest concentrationsin the plasma membrane and in the secretoryvesicles.307 In contrast, the membrane ergosterolcontent in the microsomes, mitochondria andother intracellular membranes is significantlylower and the relative concentration of sterolintermediates is increased. Yeast differ from mosteukaryotes in that the mitochondrial ergosterol isconcentrated in the inner membrane rather than inthe outer membrane.274,307

The highest concentration of sterol found inyeast is in lipid particles which are comprisedprimarily of triacylglycerols and steryl esters.140,307

Of the latter, about half are ergosteryl esters, whilehalf are esters of sterol intermediates.307 Esterifi-cation of sterol takes place in the microsomes and,thus, the esters must be moved to the lipid particlesfor storage. Lipid particles were once thought to bestorage depots for sterols and fatty acids whichcould be retrieved during times when endogenoussterol synthesis does not occur.139 However, recentstudies have detected a significant level of sterolbiosynthetic enzymes in these structures. The C-24sterol methyltransferase (Erg6p) has been found inlipid particles307 at levels which suggest that mostof the methylation activity occurs at this location.More recently, squalene epoxidase (Erg1p) hasbeen detected in lipid particles and accounts for38% of the cellular squalene epoxidase protein, ascontrasted to the 62% of the enzyme found in theendoplasmic reticulum.138 Interestingly, the lipidparticle enzyme is not active but partial activitycan be generated when lipid particles are mixedwith microsomes devoid of enzyme, indicating thatthere is a factor in the endoplasmic reticulum that

Yeast 14, 1471–1510 (1998)

1490 . .

is necessary for squalene epoxidase activity. Thus,lipid particles play a more important role in whatis generally referred to as ‘sterol trafficking’ thanwas formerly assumed.

Sterol esterification in yeast has recently beenshown to be accomplished by the products of twodistinct genes.299 The enzymes are similar to theesterification enzymes found in human (acyl-coenzyme A: cholesterol acyl transferase orACAT). Deletions or disruptions of ARE1 andARE2 (ACAT-related enzymes) in yeast, individu-ally or together, do not result in a loss of viability.Deletion of ARE2 results in a 25% loss of sterolester levels, while elimination of ARE1 functionhas no effect on ester levels. The double deletionresults in a cell that forms no ester and exhibits anoverall decrease in sterol biosynthesis.299 The latterobservation suggests a regulatory role in sterolbiosynthesis for yeast ACATs.

The inverse of the ACAT function in yeast is thesteryl ester hydrolase function, which is necessaryfor the recycling of esterified sterol to free sterolfor incorporation into membrane. This enzymeactivity has been located primarily in the plasmamembrane and secretory vesicles.307 The process ofstored sterol mobilization requires movement ofsteryl esters from lipid particles to the site of esterhydrolysis and membrane synthesis. Growth in thepresence of the allylamine antifungal, terbinafine,an inhibitor of squalene epoxidase,217 requiresthat cells mobilize ergosterol from the steryl esterfraction. Experiments conducted under these con-ditions have shown that movement of esters fromthe lipid particles does not involve microtubulesand is sensitive to energy-supply disruption andthe inhibition of protein synthesis.139 The specificsof the mechanism involved remain undetermined.

inositol phosphorylceramide.

SPHINGOLIPIDS

Our knowledge of S. cerevisiae genes necessary forsphingolipid metabolism has increased many-foldsince the genome sequence was released in April1996. There have also been many advances inidentifying potential functions for sphingolipids inyeast (reviewed in ref. 60). Yet in spite of theseadvances, our understanding of sphingolipidmetabolism and function in yeast is in its infancy,and our level of understanding pales in compari-son to what is known about fungal glycerophos-pholipids and sterols. In this section of the review,we summarize current information about the genes


that govern sphingolipid metabolism and the littlethat is known about the location of the geneproducts. We will also attempt to identify areasthat seem ripe for future research.

Sphingolipids are characterized by the presenceof a sphingoid long-chain base that is amide-linkedto a fatty acid to form a ceramide. The type oflong-chain base and fatty acid differ considerablybetween organisms but are generally fairly uniformwithin an organism. For example, the long-chain base in S. cerevisiae is phytosphingosine(-erythro-2-amino-octadecane-1,3,4-triol). Phyto-sphingosine is also the primary long-chain basefound in most plant ceramides, with only traceamounts found in animal sphingolipids (reviewedin ref. 146). Phytosphingosine lacks the 4,5-doublebond found in sphingosine (-erythro-2-amino-trans-4-octadecene-1,3-diol), the predominantlong-chain base seen in mammalian sphingolipids,and has instead the hydroxyl on C-4 (Figure 5).Ceramides in S. cerevisiae contain a long fattyacid of 26 carbons while most, but not all, theceramides in animals contain shorter fattyacids.168

Ceramides are modified on the C-1 hydroxyl togive complex sphingolipids. Fungal sphingolipids,including those in S. cerevisiae, have inositol-phosphate attached to C-1 (Figure 5, see ref. 146).Animals are not known to make this modificationbut, instead, add glucose, galactose or phos-phocholine to yield glucosylceramide, galactosyl-ceramide and sphingomyelin, respectively.Glucosylceramide is further modified by additionof carbohydrates to give many hundreds of differ-ent types of complex sphingolipids.168 Plants areunique because they contain complex sphingo-lipids formed either from glucosylceramide or

60

Sphingolipid biosynthetic pathway: genes, enzymesand phenotypes

Sphingolipid synthesis begins with the conden-sation of serine and palmitoyl-CoA to yield thelong-chain base 3-ketodihydrosphingosine (Figure5). This reaction is catalysed by serine palmitoyl-transferase (SPT) (for a review, see ref. 169), apyridoxal phosphate-containing enzyme. Two es-sential genes, LCB1 and LCB2, are necessary forSPT activity and are thought to encode subunits ofthe enzyme.87,180,288 The growth defect producedby mutation of either LCB1 or LCB2 canbe circumvented by adding any one of several

Yeast 14, 1471–1510 (1998)

1491

Figure 5. The sphingolipid biosynthetic pathway in S. cerevisiae. Genes and the functions of their products are describedin more detail in Table 4. It is not known whether the physiological substrate for the Sur2 hydroxylase is dihydrosphin-gosine, as shown, or whether it is ceramide-containing dihydrospingosine (dihydroceramide, not shown), or whether bothcompounds are substrates 84, 85.

long-chain bases to the culture medium.203,289 Inthe absence of a long-chain base, the cells stopmaking sphingolipids and die within a few hours,even though protein, DNA and other lipid syn-thesis continues.203,289 It is not known how cellstake up the relatively hydrophobic long-chainbases from the culture medium. Other sphingolipidmetabolites, such as ceramides or long-chain basephosphates and the sphingolipids containing inosi-tol, are not taken up by cells and this property hasprevented many potentially interesting exper-iments from being performed. SPT is the targetof several potent natural inhibitors, includingsphingofungins,99,310 lipoxamycin,153 myriocin175

and viridofungins.154

In the second step in sphingolipid synthesis,3-ketodihydrosphingosine is reduced in a reactionutilizing NADPH204 to produce the long-chainbase dihydrosphingosine (sphinganine). The 3-ketosphinganine reductase is encoded by TSC10(YBR265w), as demonstrated by expressing thegene in E. coli.25


In the next reaction, a fatty acid (generally 26carbons long,147 but sometimes 24194) is linked byan amide bond to dihydrosphingosine to yielddihydroceramide. This reaction, catalysed by di-hydrosphingosine (sphinganine) N-acyltransferase(ceramide synthase), is poorly characterized sincethe enzyme has not been purified from any organ-ism, neither has any gene necessary for enzymeactivity been identified.

The dihydrosphingosine component of dihy-droceramide is hydroxylated at C-4 to yield aceramide (sometimes referred to as phytoceramide)containing the long-chain base generally referredto as phytosphingosine.

Although the pathway diagrammed in Figure 5shows dihydrosphingosine as the substrate for C-4hydroxylation by the Sur2p/Syr2p, it is still notclear if dihydroceramide or both compounds aresubstrates for hydroxylation at C-4. A rapid accu-mulation of free phytosphingosine following heatshock, which precedes an increase in the level ofceramide, suggests that free dihydrosphingosine

Yeast 14, 1471–1510 (1998)

1492 . .

can be hydroxylated prior to incorporation intodihydroceramide, at least under these experimentalconditions.61 What we do know is that the SUR2/SYR2 gene is necessary for hydroxylation of thelong-chain base at C-4.84,85

The predicted Sur2 protein shows similarity todesaturase/hydroxylase enzymes characterized bya motif containing eight histidines grouped intothree clusters. The clusters may be part of thecatalytic site, known to contain oxo-diiron, Fe-O-Fe.232 The SUR2/SYR2 gene is not essential forvegetative growth but a deletion mutant has theinteresting property of increased resistance to theantifungal compound syringomycin E.84 It is un-clear why hydroxylation of the long-chain basecomponent of sphingolipids sensitizes cells tosyringomycin E. Sensitivity would not seem toprovide a selective advantage to cells in the wild, sothere must be some other reason why most fungihydroxylate C-4 of their long-chain bases.

Ceramide is converted to inositol phosphoryl-ceramide (IPC), the first of three so-called complexsphingolipids, each of which contains inositolphosphate. The inositol phosphate is transferredfrom phosphatidylinositol to the C-1 OH groupof ceramide (Figure 5). This is a very commonmodification to C-1 of ceramide in fungi146 andis catalysed by phosphatidylinositol:ceramidephosphoinositol transferase (IPC synthase), amembrane-bound enzyme.23 The AUR1 gene en-codes IPC synthase or a subunit of the enzyme.181

IPC synthase activity is inhibited by the antifungalagents aureobasidin A181 and khafrefungin,155

which are potent inhibitors with an IC50 of lessthan 1 n.155,181 IPC synthase is a promising targetfor development of antifungal drugs because thisenzyme is not found in mammals and its inhibitionleads to fungal cell death. More efficacious anti-fungal drugs are needed for treatment of patientstaking immunosuppressive drugs and in those withAIDS.

IPC is mannosylated to yield mannose-inositol-P-ceramide (MIPC). This reaction requires twogenes, SUR124 and CSG2.303 IPC accumulates inthe absence of either gene. The Sur1 protein isprobably a mannosyltransferase since it contains aregion of 93 amino acids, which is similar to twoyeast á-1,6-mannosyltransferases, Och1p185 andHoc1p.186 The function of the Csg2 protein is notobvious. It has an EF-Ca+ +-binding domain and9–10 predicted transmembrane domains.26,257 Asdiscussed below, it is likely that IPC is delivered tothe Golgi apparatus with the inositol–phosphate


head group facing the cytosol. It is then flippedacross the membrane so that it can be manno-sylated in the lumen of the Golgi. Perhaps the Csg2protein does the flipping.

Strains mutated in CSG2 have proved useful inidentifying genes involved in sphingolipid metab-olism. Such strains do not grow in the presence of100 m Ca2+ and make only one species of IPCand no MIPC or M(IP)2C.303 Seven classes ofsuppressors of calcium-sensitive growth, referredto as SCS genes, have been identified includingSCS1/LCB2 and SCS7/FAH1 (Table 4). The SCSsuppressor genes seem to partially restore synthesisof some complex sphingolipids or adjust the con-centration of different species of IPC so that cellgrowth is restored. A recent variation of the sup-pressor selection procedure has uncovered over adozen new SCS complementation groups (T.Dunn and T. Beeler, personal communication).Identifying the function of all of the SCS genesshould provide new insights into sphingolipidmetabolism and function.

The final and most abundant sphingolipid,M(IP)2C, is formed by transfer of inositol phos-phate from phosphatidylinositol onto MIPC (Fig-ure 5), a reaction very similar to the one requiringthe Aur1 protein. This similarity suggested thatopen-reading frame YDR072c, renamed IPT1,62

whose predicted protein was similar to the Aur1protein, might be necessary for synthesis ofM(IP)2C. This prediction proved correct, since anipt1 deletion mutant lacked M(IP)2C synthase ac-tivity and made no detectable M(IP)2C. The de-letion mutant had an increased level of MIPC.62

M(IP)2C normally accounts for about 75% of thesphingolipids in wild-type S. cerevisiae cells, withthe other 25% being divided between IPC andMIPC.247 It appears that S. cerevisiae is able tosense its total sphingolipid content and adjust thelevel of MIPC to compensate for the absence ofM(IP)2C.62,136

Under laboratory conditions, the ipt1 deletionmutant grows at a normal rate in complex ordefined medium. Phenotypically it shows a slightsensitivity to a high concentration of calcium.62 Itseems unlikely that S. cerevisiae cells would retainM(IP)2C if it were not providing some selectiveadvantage. A clue about M(IP)2C function comesfrom analysis of a genetically uncharacterized mu-tant strain that is unable to make M(IP)2C. Itshowed increased resistance to the polyene anti-biotic nystatin.136 Nystatin is thought to kill cellsby interacting with sterols, with the complexes

Yeast 14, 1471–1510 (1998)

1493

Table 4. Genes for S. cerevisiae sphingolipid metabolism.


AUR1 Synthesis of IPC, possible IPC synthase or a subunit of the enzyme 181CSG2 Addition of mannose to IPC, function unknown 303DPL1 Breakdown of long-chain base phosphates, long-chain base–phosphate

lyase218

ELO2/FEN1 Fatty acid elongase (synthesis of C-24 fatty acids) 194ELO3/SUR4 Fatty acid elongase (conversion of C-24 to C-26 fatty acids) 194FAH1/SCS7 Hydroxylation of the C-26 fatty acid in ceramide 85, 174IPT1/SYR4 Synthesis of M(IP)2C, possible M(IP)2C synthase 62, 136LCB1 Synthesis of long-chain bases, subunit of serine palmitoyltransferase 32LCB2/SCS1 Synthesis of long-chain bases, catalytic subunit of serine

palmitoyltransferase180, 303

LCB3/YSR2 Dephosphorylation of long-chain base phosphates, long-chainbase-1-phosphate phosphatase

156, 157, 208

LCB4 Long-chain base kinase 182LCB5 Long-chain base kinase 182SUR1/CSG1 Necessary for á-mannosylation of IPC 24SUR2/SYR2 Hydroxylation of sphinganine or sphinganine-containing

dihydroceramides at the C-4 position to yield phytoceramide84, 85

TSC10 3-Ketosphinganine reductase 25YSR3/LBP2 Long-chain base-1-phosphate phosphatase 156, 157

forming pores in the plasma membrane.119 Leberet al.136 suggest that M(IP)2C, in addition toergosterol, is necessary for nystatin action onmembranes and that the two types of lipids mayexist as microdomains within the plasma mem-brane. Experimentally, it needs to be shown thatM(IP)2C lipids have a propensity to interact withergosterol and to do so better than MIPC and IPC.

The complex sphingolipids of S. cerevisiae, IPC,MIPC and M(IP)2C, are mixtures, not uniquemolecular species, which differ in chain length andthe extent of hydroxylation of both the long-chainbase and the fatty acid.146 These differences arelikely to be physiologically important but support-ing data are meagre. It is known that cells grown at25)C primarily contain C-18 phytosphingosine,whereas when grown at 37)C they contain anincreased amount of C-20 phytosphingosine.61 Themechanism underlying this change is not entirelyclear, but it may be partly a consequence ofincreased C-18 fatty acid synthesis over C-16 syn-thesis, known to occur during a heat shock.195

In addition, cells grown anaerobically have nohydroxyl groups on the fatty acid component ofceramide (R. L. Lester, unpublished data).

Finally, one area of sphingolipid metabolismthat has recently received attention is the forma-


tion and breakdown of phosphorylated long-chainbases, dihydrosphingosine-1-phosphate (DHS-1-P)and phytosphingosine-1-phosphate (PHS-1-P). In-terest in these compounds stems from work ontissue-culture cells suggesting that the mammalianlong-chain base phosphate, sphingosine-1-phosphate (SPP), is a signalling molecule. Depend-ing on the cell type, it is thought to regulategrowth, motility or programmed cell death(reviewed in refs 207, 249, 250, 309).

Until recently, the existence of DHS-1-P andPHS-1-P in S. cerevisiae has been indirect andbased upon the existence of mutations that sup-press the choline or ethanolamine requirement of astrain mutated in cho1 and lacking phosphatidyl-serine synthase activity.11 Presumably the suppres-sor enabled cells to derive ethanolamine from ametabolite, the most likely one being the phos-phoethanolamine produced from breakdown ofDHS-1-P and PHS-1-P (Figure 5). Direct evidencefor the existence of both DHS-1-P157,182 and PHS-1-P182 in S. cerevisiae cells has been presented.

Progress in identifying the genes for synthesisand degradation of long-chain base phosphateshas been rapid. DPS-1-P and PHS-1-P are de-graded in S. cerevisiae by a long-chain base phos-phate lyase activity encoded by DPL1.218 It was

Yeast 14, 1471–1510 (1998)

1494 . .

not clear if DHS-1-P and PHS-1-P could also bedegraded by dephosphorylation until two phos-phatase genes, LCB3/YSR2156,157,208 and YSR3/LBP2156,157 were uncovered. It now seems clearthat either the lyase or phosphatase pathway candegrade long-chain base phosphates: the physio-logical function and regulation of each pathwayremain to be determined. Mammals have a homo-logue of the DPL1 lyase,305 but no homologue ofthe phosphatases has been identified yet. Finally,two S. cerevisiae genes, LCB4 and LCB5, encod-ing all of the detectable long-chain base kinaseactivity in log phase cells, have been identified.182

They appear to have mammalian homologuesbased upon searches of expressed sequence tagdatabases.

The genes known to be necessary for metab-olism of sphingolipids are summarized in Table 4.A major task is to characterize the enzymes bio-chemically. The challenge of purifying and bio-chemically characterizing these enzymes cannot beover-emphasized, considering that no enzyme inyeast sphingolipid metabolism has been purified.Such biochemical studies are essential if we areeven to begin to understand how sphingolipidmetabolism is regulated and how it is coordinatedwith other lipid metabolism and with cellularphysiology. While at least one gene has beenidentified for nearly all steps in yeast sphingolipidmetabolism (Figure 5 and Table 4), there areprobably more genes to be identified, either onesencoding subunits of multimeric enzymes or onesencoding regulatory molecules.

Cellular location, sites of synthesis and functionsof sphingolipids

Unlike proteins, which can usually be localizedin cells by immunomicroscopy, localizing lipidscan be a challenge, particularly since antibodiesspecific for individual types of yeast lipids, amongthem sphingolipids, are not available. Generally,the location of sphingolipids has been accom-plished by isolating subcellular fractions andanalysing them biochemically. Using these ap-proaches, it appears that IPC, MIPC and M(IP)2Care located primarily in the plasma membrane ofS. cerevisiae.94,199 It remains to be determinedwhether they are present in both the inner andouter leaflet of the plasma membrane or if there isan asymmetric distribution, as there is for manytypes of lipids present in the plasma membrane ofhigher eukaryotes. Sphingolipids are detected in


other locations in S. cerevisiae cells, especially IPC,which is found in the Golgi as well as in thevacuole. Trace amounts of sphingolipids are foundin mitochondria but none has been detectedin lipid particles, and nuclei have not beenexamined.94

Some yeast proteins are anchored to membranesby ceramide in an uncharacterized process thatinvolves replacing a glycosylphosphatidylinositolanchor with a ceramide anchor. This remodellingseems to be done in the Golgi apparatus.49,212,240

Recently, C-26 fatty acids have been detected inthe nucleus, although it is not known if they arefree or incorporated into sphingolipids, glycero-lipids or some novel lipid(s) species.227 These C-26fatty acids have been hypothesized to play a role instabilizing the sharp curvature in the membraneoccurring at nuclear pore complexes. C-26 fattyacids have also been found in the sn-2 position ofthe glycerol moiety of glycosylphosphatidyl-inositol-anchored proteins. Other potential rolesfor very long-chain fatty acids in yeasts and otherorganisms have been suggested.226

The endoplasmic reticulum (ER) is the site forde novo long-chain base synthesis in all organismsthat have been examined and also where all thesteps up to, and including, ceramide formationoccur. Some ceramide is converted to IPC in S.cerevisiae before transport to the Golgi apparatus,followed by further synthesis of IPC and the othercomplex sphingolipids (Figure 5).206 In S. cerevi-siae, most of the ceramide and IPC are believed tobe transported to the Golgi apparatus and then tothe plasma membrane by secretory vesicles.100,206

However, it would not be surprising if alternativeforms of transport are found.

Nothing is known about the site(s) of synthesisof DHS-1-P and PHS-1-P by the Lcb4 and Lcb5kinases. A portion of both lipid kinases is found inthe soluble, and in the 100 000#g membranefraction, of cells.182 It is presumed that at leastsome fraction of one of them is in the endoplasmicreticulum, since this is where DHS and PHS aresynthesized and where substrates are most likely tobe located. It is not known whether DHS or PHSexist in other organelles, where they could serve assubstrates for LCB kinase, or whether some long-chain bases are in the cytoplasm and thus availablefor phosphorylation. Localizing the enzymes forDHS-1-P and PHS-1-P metabolism, Lcb3, Lcb4p,Lcb5p, Lbp2p and Dpl1p will be importantfor determining whether DHS-1-P and PHS-1-Pare regulatory molecules, as is currently

Yeast 14, 1471–1510 (1998)

1495

suspected,135,156,182 or if they are simply inter-mediates in the pathway that directs sphingo-lipid metabolites into aminoglycerophospholipidsynthesis (Figure 5).

Long-chain base phosphates are found in alleukaryotes that have been examined, making itlikely that they provide a function(s) with survivalvalue. This function is not obvious, however, sincemutants lacking Lcb4 and Lcb5 kinase activity,and unable to make any detectable DHS-1-P orPHS-1-P, grow normally in complex or syntheticmedium.182 Other data suggest that long-chainbase phosphates are important for resistance toheat shock in stationary phase cells.135,156 Whatneeds to be done to verify the role of DHS-1-P andPHS-1-P in mediating resistance to heat stress instationary phase is to demonstrate that the level ofthese compounds changes during heat treatment.

Finally, complex mammalian sphingolipids,such as sphingomyelin, are turned over in mosttypes of cells, either as a part of normal cellularphysiology or in response to extracellular signals.The breakdown products (such as ceramide, sphin-gosine and SPP) are believed to regulate multiplecellular processes, including growth regulation,differentiation and apoptosis, to name but afew.88,207,249,250 In contrast to mammals, it appearsthat the majority of S. cerevisiae sphingolipids arestable and are not turned over in haploid cellsduring vegetative growth.290 Thus, the productionof potential sphingolipid signalling molecules inS. cerevisiae, including DHS, PHS, DHS-1-P,PHS-1-P and ceramide,61,107,290 are most likelyderived from de novo synthesis, not from theturnover of preformed sphingolipids.

Sphingolipids are involved in other cellular pro-cesses in S. cerevisiae (reviewed in ref. 60). Theyhave been shown to be necessary for resistance to37)C, low pH and osmotic stress,200 and for sur-vival in stationary phase (R. Dickson, unpublishedresults). The exact role of sphingolipids in thesestress responses remains to be elucidated. Heatshock has been shown to cause a rapid andtransient increase in dihydrosphingosine andphytosphingosine, and a stable increase in cera-mide, which suggests that one or all of thesemolecules are second messengers during the heatstress response.61,107 Potential processes signalledby one or more of these compounds are trehaloseaccumulation and induction of transcription ofTPS2, encoding a subunit of trehalose synthase.61

Calcium homeostasis or components in calcium-mediated signalling pathways may be regulated by


sphingolipids or vice versa,26,56,193,303 but themechanisms await characterization. It has beensuggested that ceramide regulates cell cycle arrestat G1 via a pathway that utilizes a ceramide-activated protein phosphatase encoded by SIT4,TPD3 and CYC55.188 However, these experimentsare based upon the use of ceramide-containingN-linked acetate (C-2 ceramide) and it is question-able if this compound really mimics the behaviourof authentic ceramide, with its very hydrophobicC-26 fatty acid. In fact, one published reportfound that C-2 ceramide did not arrest growth.69 Ifceramide is regulating the cell cycle, its concen-tration should change in a cell-cycle-dependentmanner.

Finally, ceramide synthesis is known to benecessary for trafficking of secretory vesicles fromthe endoplasmic reticulum to the Golgi appara-tus.100,243,256 It remains to be determined whatceramide is doing in vesicle trafficking.

GENERAL CONCLUSIONS AND FUTUREPERSPECTIVES

Sequencing of the yeast genome has opened newavenues of research for yeast lipidologists. Hom-ology searches and the development of rapidtechniques to construct deletion mutants havefacilitated analysis of a large number of genes fortheir possible involvement in yeast lipid metab-bolism. As a consequence of these developments, anumber of new genes involved in lipid metabolismhave been discovered during the last few years. Ithas become evident that some of the lipid syntheticenzymes exist as isoenzymes, such as Psd1p andPsd2p, Are1p and Are2p, or acyltransferasesinvolved in PA biosynthesis. Minor enzymaticactivities that were ignored in the past were thuspinpointed and the responsible genes and geneproducts identified.

Great progress has been made in identifyingS. cerevisiae genes necessary for sphingolipidmetabolism. At the current rate of progress, itappears likely that S. cerevisiae will be the firsteukaryotic organism for which all genes necessaryfor sphingolipid metabolism are identified. Thisinformation will provide a unique opportunity tobiochemically characterize sphingolipid metabolicenzymes and to identify, by both luck and design,new and unimagined functions for sphingolipids.

Besides the discovery of structural genes,some mechanisms for regulating lipid biosynthetic

Yeast 14, 1471–1510 (1998)

1496 . .

pathways have been unravelled. The most promi-nent regulatory pathway for yeast phospholipidformation is transcriptional control by inositol.This regulatory circuit forms the basis for cross-pathway regulation (for reviews, see refs 35, 82,198).

Other levels of regulating lipid metabolism andlipid assembly into yeast membranes also deservemention. Since lipid synthesis occurs in specificorganelles, the physiological state of the yeast cellcan greatly influence lipid synthesis. The mostprominent example of such phenomena is thecontrol of mitochondrial development by oxygen.Not only are CL synthesis and the heme requiredfor certain steps of sterol biosynthesis dependentupon mitochondrial development, but so is theenergy state of a yeast cell, since it can changedramatically during anaerobiosis or as a conse-quence of mitochondrial dysfunction. Since moststeps of lipid biosynthesis are directly or indirectlydependent upon the availability of energy, suchchanges affect lipid metabolism. As anotherexample, sphingolipid synthesis is linked to proteinsecretion, because steps of sphingolipid formationoccur in organelles along the secretory pathway,and intermediates and products are transported totheir destination(s) by the protein secretory ma-chinery. Thus, lipid transport is not only requiredfor lipid targeting and lipid assembly into cellularmembranes, but also to link lipid biosyntheticpathways, in which steps are distributed amongvarious organelles. Finally, the interplay of theendoplasmic reticulum and mitochondria duringsynthesis of aminoglycerophospholipids, and theinterplay of the endoplasmic reticulum with lipidparticles during sterol synthesis or the depositionof triacylglycerols and steryl esters, are also exam-ples of processes that regulate lipid metabolism.

Future research efforts need to be directed to-wards understanding how lipid synthesis and turn-over are integrated with other metabolic pathwaysand cellular processes during all aspects of S.cerevisiae cell biology. In the case of phospho-lipids, understanding mechanisms of targetingamong organelles will be a significant focus area.Identification of components involved in theseprocesses will open new aspects of organelle bio-genesis from the viewpoint of the lipidologist.Improved analytical methods will allow us todetect less abundant species and phospholipidsthat may play important roles in signalling pro-cesses or membrane integrity. New levels of regu-lation of phospholipid biosynthesis and turnover,


like the one described for SEC14, will be of greatinterest.

Remodelling and degradation of lipids may playan important role in the maintenance of a specificmembrane lipid composition and in intracellularsignalling. Distinct phospholipid molecules areproduced, for example, by phospholipase A2-catalysed deacylation in the sn-2 position of exist-ing phospholipids, followed by reacylation of thelysophospholipid with a coenzyme-A-activatedfatty acid. Deacylation and reacylation of glycero-phospholipids appear to be very efficient,284 butcellular functions that depend on this energeticallyexpensive process have yet to be identified.

Phospholipases of S. cerevisiae are only poorlycharacterized. Phospholipase B (PLB) activitiescatalysing the complete deacylation of glycero-phospholipids have been identified in the plasmamembrane and the periplasmic space ofyeast.141,294 The physiological function and rel-evance of the two forms of the enzyme are notclear. Interestingly, disruption of the PLB1 genecompletely abolishes PLB activity in vitro, but hasno effect on cell viability and lipid compositionand only slightly effects release of deacylationproducts into the culture medium.141 Thus, S.cerevisiae is likely to have additional genes en-coding PLB activities that have escaped detection.

The S. cerevisiae PLC gene, encoding a phos-pholipase C (PLC), is not essential. Cells lackingPlc1p, however, grow poorly on fermentablecarbon sources, are unable to grow on non-fermentable carbon sources or at elevated tem-peratures, and are extremely sensitive tohyperosmotic stress.73 In yeast, PLC-dependentinositolphospholipid turnover functions to main-tain cell morphology, chromosome segregation,cytokinesis and the integrity of the plasma mem-brane, and may be required at several or all stagesof the cell cycle.300

Phospholipase D (PLD) cleavage releases thesecond messenger phosphatidic acid. Only recentlywas the yeast gene encoding Pld1p identified.160,285

Surprisingly, PLD1 is identical to SPO14, whichencodes a factor essential for sporulation. The roleof PLD activity in spore formation, its site ofaction and the nature of the target lipid(s) have notyet been established.

Similar to phospholipid metabolism, the sterolbiosynthetic pathway contains several steps thatare subject to regulation. Most of these are un-explored and the availability of the genes for moststeps makes this an imperative area for research in

Yeast 14, 1471–1510 (1998)

1497

the near future. In addition, we are beginning tounravel some of the elements of the overall schemefor targeting sterol to specific cellular membranesystems, selection of sterol for esterification orutilization, storage and recovery of sterol, andmovement of various sterol forms throughout thecell. All of these trafficking phenomena have im-plications for the regulation of sterol biosynthesisas well. Finally, there remains the interest in yeaststerol metabolism and physiology as a model forthe same events that occur in mammalian systems.For example, the yeast Ncr1 protein has beenfound to be 42% identical to the human Niemann-Pick product, which seems to function in steroltransport or sensing.254 These would seem tobe areas of great future interest, as we noware reaching the end of the discovery andcharacterization of sterol biosynthetic genesand related aspects concerned with essentiality andsuppression.

In the field of yeast sphingolipid research, thechallenge for the future will be to purify andbiochemically characterize the enzymes, most ofwhich are membrane-bound, and establish theircellular location(s) in intact, not disrupted, cells.Compared to glycerophospholipids and sterols, weknow very little about the function of sphingo-lipids and even less about how their synthesis iscontrolled. Identifying new functions for the polarand hydrophobic components of sphingolipids andunderstanding how de novo synthesis is regulatedare two areas that could greatly benefit frominsightful hypotheses and clever experimental de-signs. Unlike mammalian cells, where complexsphingolipids are normally turned over,168 there isno detectable turnover of complex sphingolipids(IPC, MIPC and M(IP)2C) in haploid yeastcells.290 This may represent a fundamental differ-ence in sphingolipid metabolism between fungi andcomplex eukaryotes. It is possible that smallamounts of complex sphingolipids are turned overin S. cerevisiae cells, but improved analytical tech-niques are needed to identify and to quantify suchchanges. Sphingolipid metabolites produced byturnover have the potential to be important regu-latory molecules, as seems to be the case in highereukaryotes.

In conclusion, using a combination of cell biol-ogy, molecular biology and biochemical tech-niques, future experiments in yeast are likely toreveal new roles for lipids as structural elements inmembranes. They should also identify new roles oflipids as modulators of enzymes and mediators of


cellular processes, such as sporulation, mating,stress resistance and protein assembly into mem-branes. This experimental background will also bethe basis for extended comparative studies oflipids and lipid metabolism in yeast and highereukaryotes.

ACKNOWLEDGEMENTS

The authors are grateful to F. Paltauf and R.Schneiter for critically reading the manuscript. Wethank Matthew Kennedy for preparing Figures 2,3 and 4. Research in G.D.’s laboratory wasfinancially supported by the Fonds zur Forderungder wissenschaftlichen Forschung in O} sterreich(projects 11491 and 12076), the EUROFANproject BIO4-CT95-0080, and the AustrianMinistry of Science and Transportation (project950080). R.C.D.’s research has been supported inpart by grant GM43102 from the United StatesPublic Health Service and by grant EHR-9108764from the National Science Foundation. Researchin the laboratories of M.B. and N.D.L. has beensupported by NIH grant RO1 A538598 to M.B.and a Johnson & Johnson Focused Giving Awardto M.B. and N.D.L.

REFERENCES

1. Achleitner, G., Zweytick, D., Trotter, P., Voelker,D. and Daum, G. (1995). Synthesis and intra-cellular transport of aminoglycerophospholipidsin permeabilized cells of the yeast, S. cerevisiae.J. Biol. Chem. 270, 29836–29842.

2. Al-Feel, W., Chirala, S. S. and Wakil, S. J. (1992).Cloning of the yeast FAS3 gene and primarystructure of yeast acetyl-CoA carboxylase. Proc.Natl Acad. Sci. USA 89, 4534–4538.

3. Anderson, M. S., Muehlbacher, M., Street, I. P.,Proffitt, J. and Poulter, C. D. (1989). Isopentenyldiphosphate:dimethylallyl diphosphate isomerase:an improved purification of the enzyme and isola-tion of the gene from Saccharomyces cerevisiae.J. Biol. Chem. 264, 19169–19175.

4. Anderson, M. S., Yarger, J. G., Burck, C. L. andPoulter, C. D. (1989). Farnesyl diphosphate syn-thase molecular cloning, sequence and expressionof an essential gene from Saccharomyces cerevisiae.J. Biol. Chem. 264, 19176–19184.

5. Arthington, B. A., Bennett, L. G., Skatrud, P. L.,Guynn, C. J., Barbuch, R. J., Ulbright, C. E. andBard, M. (1991). Cloning, disruption and sequenceof the gene encoding yeast C-5 desaturase. Gene102, 39–44.

6. Arthington, B. A., Hoskins, J., Skatrud, P. L. and
Bard, M. (1991). Nucleotide sequence of the gene
Yeast 14, 1471–1510 (1998)

1498 . .

encoding yeast sterol C-8 isomerase. Gene 107,173–174.

7. Arthington-Skaggs, B. A., Crowell, D. N., Yang,H., Sturley, S. L. and Bard, M. (1996). Positive andnegative regulation of a sterol biosynthetic gene(ERG3) in the post-squalene portion of the yeastergosterol pathway. FEBS Lett. 392, 161–165.

8. Ashman, W. H., Barbuch, R. J., Ulbright, C. E.,Jarrett, H. W. and Bard, M. (1991). Cloning anddisruption of the yeast C-8 isomerase gene. Lipids26, 628–632.

9. Athenstaedt, K. and Daum, G. (1997). Biosynthe-sis of phosphatidic acid in lipid particles and endo-plasmic reticulum of Saccharomyces cerevisiae.J. Bacteriol. 179, 7611–7616.

10. Atkinson, K. (1983). In Thomson, W. W., Madd,J. B. and Gibbs, M. (Eds) Biosynthesis and Functionof Plant Lipids, American Society of Plant Physi-ology Monograph, Waverly Press, Baltimore, MD,pp. 229–249.

11. Atkinson, K. D. (1984). Saccharomyces cerevisiaerecessive suppressor that circumvents phosphati-dylserine deficiency. Genetics 108, 533–543.

12. Atkinson, K. D., Jensen, B., Kolat, A. I., Storm,E. M., Henry, S. A. and Fogel, S. (1980). Yeastmutants auxotrophic for choline or ethanolamine.J. Bacteriol. 141, 558–564.

13. Bailis, A. M., Lopes, J. M., Kohlwein, S. D. andHenry, S. A. (1992). Cis and trans regulatoryelements required for regulation of the CHO1 geneof Saccharomyces cerevisiae. Nucl. Acids Res. 20,1411–1418.

14. Bailis, A. M., Poole, M. A., Carman, G. M. andHenry, S. A. (1987). The membrane-associatedenzyme phosphatidylserine synthase is regulated atthe level of mRNA abundance. Mol. Cell. Biol. 7,167–176.

15. Baloch, R. I. and Mercer, E. I. (1987). Inhibition ofsterol 8-7 isomerase and 14-reductase by fenpropi-morph, tridemorph and fenpropidin in cell-freesystems from Saccharomyces cerevisiae. Phyto-chemistry 26, 663–668.

16. Bankaitis, V. A., Malehorn, D. E., Emr, S. E. andGreene, R. (1989). The Saccharomyces cerevisiaeSEC14 gene encodes a cytosolic factor that isrequired for transport of secretory proteins fromthe yeast Golgi complex. J. Cell Biol. 108, 1271–1281.

17. Bard, M. (1972). Biochemical and genetic aspectsof nystatin resistance in Saccharomyces cerevisiae.J. Bacteriol. 11, 649–657.

18. Bard, M. and Downing, J. F. (1981). Genetic andbiochemical aspects of yeast sterol regulation in-volving 3-hydroxy-3-methylglutaryl coenzyme Areductase. J. Gen. Microbiol. 125, 415–420.

19. Bard, M., Bruner, D. A., Pierson, C. A., Lees,N. D., Biermann, B., Frye, L., Koegel, C. and
Barbuch, R. (1996). Cloning and characterization

of ERG25, the Saccharomyces cerevisiae gene en-coding C-4 sterol methyl oxidase. Proc. Natl Acad.Sci. USA 93, 186–190.

20. Bard, M., Lees, N. D., Burrows, L. S. andKleinhans, F. W. (1978). Differences in crystalviolet uptake and cation induced death amongyeast sterol mutants. J. Bacteriol. 135, 1146–1148.

21. Bard, M., Lees, N. D., Turi, T., Craft, D., Cofrin,L., Barbuch, R., Koegel, C. and Loper, J. C.(1993). Sterol biosynthesis and viability of erg11(cytochrome P450 lanosterol demethylase) muta-tions in Saccharomyces cerevisiae and Candidaalbicans. Lipids 28, 963–967.

22. Basson, M. E., Thorsness, M. and Rine, J. (1986).Saccharomyces cerevisiae contains two functionalgenes encoding 3-hydroxy-3-methylglutaryl-co-enzyme A reductase. Proc. Natl Acad. Sci. USA 83,5563–5567.

23. Becker, G. W. and Lester, R. L. (1980). Biosyn-thesis of phosphoinositol-containing sphingo-lipids from phosphatidylinositol by a membranepreparation from Saccharomyces cerevisiae.J. Bacteriol. 142, 747–754.

24. Beeler, T. J., Fu, D., Rivera, J., Monaghan, E.,Gable, K. and Dunn, T. M. (1997). SUR1 (CSG1/BCL21), a gene necessary for growth of Saccharo-myces cerevisiae in the presence of high Ca+ +

concentrations at 37)C, is required for mannosyla-tion of inositolphosphorylceramide. Mol. Gen.Genet. 255, 570–579.

25. Beeler, T., Bacikova, D., Gable, K., Hopkins, L.,Johnson, C., Slife, H. and Dunn, T. (1998). TheSaccharomyces cerevisiae TSC10/YBR265w geneencoding 3-ketosphinganine reductase is identifiedin a screen for temperature sensitive suppressors ofthe Ca-sensitive csg2 mutant. J. Biol. Chem. (inpress).

26. Beeler, T., Gable, K., Zhao, C. and Dunn, T.(1994). A novel protein, CSG2p, is required forCa2+ regulation in Saccharomyces cerevisiae.J. Biol. Chem. 269, 7279–7284.

27. Berges, T., Guyonnet, D. and Karst, F. (1997). TheSaccharomyces cerevisiae mevalonate diphosphatedecarboxylase is essential for viability and a singleleu-to-pro mutation in a conserved sequence leadsto thermosensitivity. J. Bacteriol. 179, 4664–4670.

28. Berndt, J., Boll, M. and Gaumet, R. (1973). Regu-lation of sterol biosynthesis in yeast: inductionof 3-hydroxy-3-methylglutaryl CoA reductase byglucose. Biochem. Biophys. Res. Commun. 51, 843–848.

29. Boll, M., Lowel, M. and Berndt, J. (1980). Effect ofunsaturated fatty acids on sterol biosynthesis inyeast. Biochim. Biophys. Acta 620, 429–439.

30. Brajtburg, J., Powderly, W. G., Kobayashi, G. S.and Medoff, G. (1990). Amphotericin B: currentunderstanding of mechanisms of action. Anti-
microb. Agents Chemother. 34, 183–188.
Yeast 14, 1471–1510 (1998)

1499

31. Brody, S., Oh, C., Hoja, U. and Schweizer, E.(1997). Mitochondrial acyl carrier protein is in-volved in lipoic acid synthesis in Saccharomycescerevisiae. FEBS Lett. 408, 217–220.

32. Buede, R., Rinker-Schaffer, C., Pinto, W. J.,Lester, R. L. and Dickson, R. C. (1991). Cloningand characterization of LCB1, a Saccharomycesgene required for biosynthesis of the long-chainbase component of sphingolipids. J. Bacteriol. 173,4325–4332.

33. Camici, O. and Corazzi, L. (1997). Phosphatidyl-serine translocation into brain mitochondria: in-volvement of a fusogenic protein associated withmitochondrial membranes. Mol. Cell. Biochem.175, 71–80.

34. Carman, G. M. (1997). Phosphatidate phosphatasesand diacylglycerol pyrophosphate phosphatasesin Saccharomyces cerevisiae and Escherichia coli.Biochim. Biophys. Acta 1348, 45–55.

35. Carman, G. M. and Zeimetz, G. M. (1996). Regu-lation of phospholipid biosynthesis in the yeastSaccharomyces cerevisiae. J. Biol. Chem. 271,13293–13296.

36. Caro, L. H. P., Tettelin, H., Vossen, J. H., Ram,A. F. J., van den Ende, H. and Klis, F. M. (1997).In silicio identification of glycosyl-phosphatidyl-inositol-anchored plasma-membrane and cell wallproteins of Saccharomyces cerevisiae. Yeast 13,1477–1489.

37. Chambon, C., Ladeveze, V., Servouse, M.,Blanchard, L., Javeiot, C., Vladescu, B. and Karst,F. (1991). Sterol pathway in yeast. Identificationand properties of mutant strains defective inmevalonate diphosphate decarboxylase and far-nesyl diphosphate synthase. Lipids 26, 633–636.

38. Chang, S. C., Heacock, P. N., Clancey, C. J. andDowhan, W. (1998). The PEL1 gene (renamedPGS1) encodes the phosphatidylglycerophosphatesynthase of Saccharomyces cerevisiae. J. Biol.Chem. 273, 9829–9836.

39. Chang, S. C., Heacock, P. N., Mileykovskaya, E.,Voelker, D. R. and Dowhan, W. (1998). Isolationand characterization of the gene (CLS1) encodingcardiolipin synthase of Saccharomyces cerevisiae.J. Biol. Chem. 273, 14933–14941.

40. Chen, W., Capieaux, E., Balzi, E. and Goffeau, A.(1991). The YGL022 gene encodes a putative trans-port protein. Yeast 7, 305–308.

41. Chirala, S. S., Kuziora, M. A., Spector, D. M. andWakil, S. J. (1987). Complementation of mutationsand nucleotide sequence of FAS1 gene encodingbeta subunit of yeast fatty acid synthase. J. Biol.Chem. 262, 4231–4240.

42. Christiansen, K. (1978). Triacylglycerol synthesisin lipid particles from baker’s yeast (Saccharomycescerevisiae). Biochim. Biophys. Acta 530, 78–90.

43. Clancey, C. J., Chang, S.-C. and Dowhan, W.
(1993). Cloning of a gene (PDS1) encoding phos-

phatidylserine decarboxylase from Saccharomycescerevisiae by complementation of an Escherichiacoli mutant. J. Biol. Chem. 268, 24580–24590.

44. Clark, S. (1992). Protein isoprenylation andmethylation at carboxy-terminal cysteine residues.Annu. Rev. Biochem. 61, 355–386.

45. Cleves, A. E., McGee, T. P., Whitters, E. A.,Champion, K. M., Aitken, J. R., Dowhan, W.,Goebl, M. and Bankaitis, V. A. (1991). Mutationsin the CDP–choline pathway for phospholipid bio-synthesis bypass the requirement for an essentialphospholipid transfer protein. Cell 64, 789–800.

46. Cobon, G. S. and Haslam, J. M. (1973). The effectof altered sterol composition on the temperaturedependence of yeast mitochondrial ATPase. Bio-chem. Biophys. Res. Commun. 52, 320–326.

47. Conzelmann, A., Fankhauser, C. and Desponds,C. (1990). Myoinositol gets incorporated into nu-merous membrane glycoproteins of Saccharomycescerevisiae; incorporation is dependent on phospho-mannomutase (sec53). EMBO J. 9, 653–661.

48. Conzelmann, A., Fankhauser, C., Puoti, A. andDesponds, C. (1991). Biosynthesis of glycophospho-inositol anchors in Saccharomyces cerevisiae. CellBiol. Int. Rep. 15, 863–873.

49. Conzelmann, A., Puoti, A., Lester, R. L. andDesponds, C. (1992). Two different types of lipidmoieties are present in glycophosphoinositol-anchored membrane proteins of Saccharomycescerevisiae. EMBO J. 11, 457–466.

50. Corey, E. J., Matsuda, S. P. T. and Bartel, B.(1994). Molecular cloning, characterization andoverexpression of the Saccharomyces cerevisiaegene encoding lanosterol synthase. Proc. NatlAcad. Sci. USA 91, 2211–2215.

51. Cutler, N. S., Heitman, J. and Cardenas, M. E.(1997). STT4 is an essential phosphatidylinositol4-kinase that is a target of wortmannin in Saccha-romyces cerevisiae. J. Biol. Chem. 272, 27671–27677.

52. Dahl, C., Biemann, H. P. and Dahl, J. (1987). Aprotein kinase antigenically related to pp60 srcpossibly involved in yeast cell cycle control: posi-tive in vivo regulation of sterol. Proc. Natl Acad.Sci. USA 84, 4012–4016.

53. Daum, G. and Paltauf, F. (1980). Triacylglycerolsas fatty acid donors for membrane phospholipidbiosynthesis in yeast. Monatsh. Chemie 111, 355–363.

54. Daum, G. and Vance, J. (1997). Import of lipidsinto mitochondria. Prog. Lipid Res. 36, 103–130.

55. Defranoux, N., Gaisne, M. and Verdiere, J. (1994).Functional analysis of the zinc cluster domain ofthe CYP1 (HAP1) complex regulator in heme-sufficient and heme-deficient yeast cells. Mol. Gen.Genet. 242, 699–707.

56. DeMesquita, J. F., Paschoalin, V. M. F. and
Panek, A. D. (1997). Modulation of trehalase
Yeast 14, 1471–1510 (1998)

1500 . .

activity in Saccharomyces cerevisiae by an intrinsicprotein. Biochim. Biophys. Acta 1334, 233–239.

57. Denning, D. W., Venkateswarlu, K., Oakley,K. L., Anderson, M. J., Manning, N. J., Stevens,D. A., Warnock, D. W. and Kelly, S. L. (1997).Itraconazole resistance in Aspergillus fumigatus.Antimicrob. Agents Chemother. 41, 1364–1368.

58. Desrivieres, S., Cooke, F. T., Parker, P. J. andHall, M. N. (1998). MSS4, a phosphatidylinositol-4-phosphate 5-kinase required for organization ofthe actin cytoskeleton in Saccharomyces cerevisiae.J. Biol. Chem. 273, 15787–15793.

59. DeWald, D. B., Wurmser, A. E. and Emr, S. D.(1997). Regulation of the Saccharomyces cerevisiaeVps34p phosphatidylinositol 3-kinase. Biochem.Soc. Trans. 25, 1141–1146.

60. Dickson, R. C. (1998). Sphingolipid functions inSaccharomyces cerevisiae: comparison to mam-mals. Annu. Rev. Biochem. 67, 27–48.

61. Dickson, R. C., Nagiec, E. E., Skrzypek, M.,Tillman, P., Wells, G. B. and Lester, R. L. (1997).Sphingolipids are potential heat stress signals inSaccharomyces. J. Biol. Chem. 272, 30196–30200.

62. Dickson, R. C., Nagiec, E. E., Wells, G. B.,Nagiec, M. M. and Lester, R. L. (1997). Synthesisof mannose-(inositol-P)2-ceramide, the majorsphingolipid in Saccharomyces cerevisiae, requiresthe IPT1(YDR072c) gene. J. Biol. Chem. 272,29620–29625.

63. Dimster-Denk, D. and Rine, J. (1996). Transcrip-tional regulation of a sterol-biosynthetic enzyme bysterol levels in Saccharomyces cerevisiae. Mol. Cell.Biol. 16, 3981–3989.

64. Dittrich, F., Zajonc, D., Huhne, K., Hoja, U.,Ekici, A., Greiner, E., Klein, H., Hofmann, J.,Bessoule, J. J., Sperling, P. and Schweizer, E.(1998). Fatty acid elongation in yeast—biochemical characteristics of the enzyme systemand isolation of elongation-defective mutants. Eur.J. Biochem. 252, 477–485.

65. Dixon, G., Scanlon, D., Cooper, S. and Broad, P.(1997). A reporter gene assay for fungal sterolbiosynthesis inhibitors. J. Steroid Biochem. Mol.Biol. 62, 165–171.

66. Dorsey, J. K. and Porter, J. W. (1973). Inhibitionof mevalonate kinase by geranyl and farnesylpyrophosphates. J. Biol. Chem. 243, 4667–4670.

67. Dove, S. K., Cooke, F. T., Douglas, M. R., Sayers,L. G., Parker, P. J. and Michell, R. H. (1997).Osmotic stress activates phosphatidylinositol 3,5-bisphosphate synthesis. Nature 390, 187–192.

68. Dowhan, W. (1997). CDP-diacylglycerol synthaseof microorganisms. Biochim. Biophys. Acta 1348,157–165.

69. Ella, K. M., Qi, C., Dolan, J. W., Thompson, R. P.and Meier, K. E. (1997). Characterization of asphingomyelinase activity in Saccharomyces cerevi-
siae. Arch. Biochem. Biophys. 340, 101–110.

70. Fischl, A. S. and Carman, G. M. (1983).Phosphatidylinositol biosynthesis in Saccharo-myces cerevisiae: purification and properties ofmicrosome-associated phosphatidylinositol syn-thase. J. Bacteriol. 154, 304–311.

71. Fischl, A. S., Homann, M. J. and Carman, G. M.(1986). Phosphatidylinositol synthase from Saccha-romyces cerevisiae. Reconstitution, characteriza-tion, and regulation of activity. J. Biol. Chem. 261,3178–3183.

72. Flanagan, C. A., Schnieders, E. A., Emerick,A. W., Kunisawa, R., Admon, A. and Thorner,J. (1993). Phosphatidylinositol 4-kinase: genestructure and requirement for yeast cell viability.Science 262, 1444–1448.

73. Flick, J. S. and Thorner, J. (1993). Genetic andbiochemical characterization of a phosphatidyl-inositol-specific phospholipase C in Saccharomycescerevisiae. Mol. Cell. Biol. 13, 5861–5876.

74. Fukushima, H., Grinstead, G. F. and Gaylor, J. L.(1981). Total enzymatic synthesis of cholesterolfrom lanosterol. J. Biol. Chem. 256, 4822–4826.

75. Gaber, R. F., Copple, D. M., Kennedy, B. K.,Vidal, M. and Bard, M. (1989). The yeast geneERG6 is required for normal membrane functionbut is not essential for biosynthesis of the cell-cycle-sparking sterol. Mol. Cell. Biol. 9, 3447–3456.

76. Gachotte, D., Pierson, C. A., Lees, N. D.,Barbuch, R., Koegel, C. and Bard, M. (1997). Ayeast sterol auxotroph (erg25) is rescued byaddition of azole antifungals and reduced levelsof heme. Proc. Natl Acad. Sci. USA 94, 11173–11178.

77. Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F.and Daum, G. (1995). Characterization of a micro-somal subfraction associated with mitochondriaof the yeast, Saccharomyces cerevisiae. Biochim.Biophys. Acta 1234, 214–220.

78. Garcia-Bustos, J. F., Marini, F., Stevenson, I.,Frei, C. and Hall, M. N. (1994). PIK1, an essentialphosphatidylinositol 4-kinase associated with theyeast nucleus. EMBO J. 13, 2352–2361.

79. Gaynor, P. M., Hubbell, S., Schmidt, A. J., Lina,R. A., Minskoff, S. A. and Greenberg, M. L.(1991). Regulation of phosphatidylglycerolphos-phate synthase in Saccharomyces cerevisiae byfactors affecting mitochondrial development.J. Bacteriol. 173, 6124–6131.

80. Gnamusch, E., Kalaus, C., Hrastnik, C., Paltauf,F. and Daum, G. (1992). Transport of phospho-lipids between subcellular membranes of wild-type yeast cells and of the phosphatidylinositoltransfer protein-deficient strain Saccharomycescerevisiae sec14. Biochim. Biophys. Acta 1111, 120–126.

81. Goldstein, J. L. and Brown, M. S. (1990). Regu-lation of the mevalonate pathway. Nature 343,
425–430.
Yeast 14, 1471–1510 (1998)

1501

82. Greenberg, M. L. and Lopes, J. M. (1996). Geneticregulation of phospholipid biosynthesis in Saccha-romyces cerevisiae. Microbiol. Rev. 60, 1–20.

83. Greenberg, M. L., Hubbell, S. and Lam, C. (1988).Inositol regulates phosphatidylglycerolphosphatesynthase expression in Saccharomyces cerevisiae.Mol. Cell. Biol. 8, 4773–4779.

84. Grilley, M. M., Stock, S. D., Dickson, R. C.,Lester, R. L. and Takemoto, J. Y. (1998). Syringo-mycin action gene SYR2 is essential for sphingo-lipid 4-hydroxylation in Saccharomyces cerevisiae.J. Biol. Chem. 273, 11062–11068.

85. Haak, D., Gable, K., Beeler, T. and Dunn, T.(1997). Hydroxylation of Saccharomyces cerevisiaeceramides requires Sur2p and Scs7p. J. Biol. Chem.272, 29704–29710.

86. Hamada, K., Fukuchi, S., Arisawa, M., Baba, M.and Kitada, K. (1998). Screening for glycosylphos-phatidylinositol (GPI)-dependent cell wall proteinsin Saccharomyces cerevisiae. Mol. Gen. Genet. 258,53–59.

87. Hanada, K., Hara, T., Nishijima, M., Kuge, O.,Dickson, R. C. and Nagiec, M. M. (1997). Amammalian homolog of the yeast LCB1 encodesa component of serine palmitoyltransferase, theenzyme catalyzing the first step in sphingolipidsynthesis. J. Biol. Chem. 272, 32108–32114.

88. Hannun, Y. A. (1996). Functions of ceramide incoordinating cellular responses to stress. Science274, 1855–1859.

89. Harington, A., Herbert, C. J., Tung, B., Getz, G. S.and Slonimski, P. P. (1993). Identification of a newnuclear gene (CEM1) encoding a protein homolo-gous to a beta-keto-acyl synthase which is essentialfor mitochondrial respiration in Saccharomycescerevisiae. Mol. Microbiol. 9, 545–555.

90. Harington, A., Schwarz, E., Slonimski, P. P. andHerbert, C. J. (1994). Subcellular relocalization ofa long-chain fatty acid CoA ligase by a suppressormutation alleviates a respiration deficiency inSaccharomyces cerevisiae. EMBO J. 13, 5531–5538.

91. Hasslacher, M., Ivessa, A. S., Paltauf, F. andKohlwein, S. D. (1993). Acetyl-CoA carboxylasefrom yeast is an essential enzyme and is regulatedby factors that control phospholipid metabolism.J. Biol. Chem. 268, 10946–10952.

92. Hebeka, E. K. and Solotovsky, M. (1965). Devel-opment of resistance to polyene antibiotics inCandida albicans. J. Bacteriol. 89, 1533–1539.

93. Hechtberger, P. and Daum, G. (1995). Intracellulartransport of inositol-containing sphingolipids inthe yeast, Saccharomyces cerevisiae. FEBS Lett.367, 201–204.

94. Hechtberger, P., Zinser, E., Saf, R., Hummel, K.,Paltauf, F. and Daum, G. (1994). Characterization,
quantification and subcellular localization of

inositol-containing sphingolipids of the yeastSaccharomyces cerevisiae. Eur. J. Biochem. 225,641–649.

95. Herman, P. K. and Emr, S. D. (1990). Characteri-zation of VPS34, a gene required for vacuolarprotein sorting and vacuole segregation in Saccha-romyces cerevisiae. Mol. Cell. Biol. 10, 6742–6754.

96. Hjelmstad, R. H. and Bell, R. M. (1987). Mutantsof Saccharomyces cerevisiae defective in sn-1,2-diacylglycerol cholinephosphotransferase: isola-tion, characterization and cloning of the CPT1gene. J. Biol. Chem. 262, 3909–3917.

97. Hjelmstad, R. H. and Bell, R. M. (1991). sn-1,2-diacylglycerol choline- and ethanolaminephospho-transferases in Saccharomyces cerevisiae. Mixedmicellar analysis of the CPT1 and EPT1 geneproducts. J. Biol. Chem. 266, 4357–4365.

98. Homma, K., Terui, S., Minemura, M., Qadota, H.,Anraku, Y., Kanaho, Y. and Ohya, Y. (1998).Phosphatidylinositol-4-phosphate 5-kinase local-ized on the plasma membrane is essential for yeastcell morphogenesis. J. Biol. Chem. 273, 15779–15786.

99. Horn, W. S., Smith, J. L., Bills, G. F., Raghoobar,S. L., Helms, G. L., Kurtz, M. B., Marrinan, J. A.,Frommer, B. R., Thornton, R. A. and Mandala,S. M. (1992). Sphingofungins E and F: novel serinepalmitoyltransferase inhibitors from Paecilomycesvariotii. J. Antibiot. (Tokyo) 45, 1692–1696.

100. Horvath, A., Suetterlin, C., Manning-Krieg, U.,Movva, N. R. and Riezman, H. (1994). Ceramidesynthesis enhances transport of GPI-anchored pro-teins to the Golgi apparatus in yeast. EMBO J. 13,3687–3695.

101. Hosaka, K., Kodaki, T. and Yamashita, S. (1989).Cloning and characterization of the yeast CKI geneencoding choline kinase and its expression inEscherichia coli. J. Biol. Chem. 264, 2053–2059.

102. Jahnke, L. and Klein, H. P. (1983). Oxygen re-quirement for formation and activity of thesqualene epoxidase in Saccharomyces cerevisiae.J. Bacteriol. 155, 488–492.

103. Jandrositz, G., Turnowsky, F. and Hogenauer, G.(1991). The gene encoding squalene epoxidase fromSaccharomyces cerevisiae: cloning and characteri-zation. Gene 107, 155–160.

104. Janitor, M. and Subik, J. (1993). Molecular cloningof the PEL1 gene of Saccharomyces cerevisiae thatis essential for the viability of petite mutants. Curr.Genet. 24, 307–312.

105. Janitor, M., Jarosch, E., Schweyen, R. J. andSubik, J. (1995). Molecular characterization of thePEL1 gene encoding a putative phosphatidylserinesynthase. Yeast 11, 1223–1231.

106. Janitor, M., Obernauerova, M., Kohlwein, S. D.and Subik, J. (1996). The pel1 mutant of Saccharo-myces cerevisiae is deficient in cardiolipin and does
not survive the disruption of the CHO1 gene
Yeast 14, 1471–1510 (1998)

1502 . .

encoding phosphatidylserine synthase. FEMSMicobiol. Lett. 140, 43–47.

107. Jenkins, G. M., Richards, A., Wahl, T., Mao, C.,Obeid, L. and Hannun, Y (1997). Involvement ofyeast sphingolipids in the heat stress response inSaccharomyces cerevisiae. J. Biol. Chem. 252,32566–32572.

108. Jennings, S. M., Tsay, Y. H., Fisch, T. M. andRobinson, G. W. (1991). Molecular cloning andcharacterization of the yeast gene for squalenesynthetase. Proc. Natl Acad. Sci. USA 88, 6038–6042.

109. Jiang, F., Rizavi, H. S. and Greenberg, M. L.(1997). Cardiolipin is not essential for the growthof Saccharomyces cerevisiae on fermentable or non-fermentable carbon sources. Mol. Microbiol. 26,481–491.

110. Johnston, J. M. and Paltauf, F. (1970). Lipidmetabolism in inositol-deficient yeast, Saccharo-myces carlsbergensis. II. Incorporation of labeledprecursors into lipids by whole cells and activitiesof some enzymes involved in lipid formation.Biochim. Biophys. Acta 218, 431–440.

111. Kalb, V. K., Woods, C. W., Turi, T. G., Dey,C. R., Sutter, T. R. and Loper, J. (1987). Primarystructure of the P450 lanosterol demethylasegene from Saccharomyces cerevisiae. DNA 6, 529–537.

112. Kanipes, M. I. and Henry, S. A. (1997). Thephospholipid methyltransferases in yeast. Biochim.Biophys. Acta 1348, 134–141.

113. Karst, F. and Lacroute, F. (1977). Ergosterol bio-synthesis in Saccharomyces cerevisiae. Mol. Gen.Genet. 154, 269–277.

114. Kearns, B. G., McGee, T. P., Mayinger, P.,Gedvilaite, A., Phillips, S. E., Kagiwada, S. andBankaitis, V. A. (1997). Essential role for diacyl-glycerol in protein transport from the yeast Golgicomplex. Nature 387, 101–105.

115. Kelley, M. J. and Carman, G. M. (1987). Purifica-tion and characterization of CDP-diacylglycerolsynthase from Saccharomyces cerevisiae. J. Biol.Chem. 262, 14563–14570.

116. Kelley, M. J., Bailis, A. M., Henry, S. A. andCarman, G. M. (1988). Regulation of phospholipidbiosynthesis in Saccharomyces cerevisiae by inosi-tol. J. Biol. Chem. 263, 18078–18085.

117. Kennedy, E. P. and Weis, S. B. (1956). The func-tion of cytidine coenzymes in the biosynthesis ofphospholipids. J. Biol. Chem. 222, 193–214.

118. Kent, C. (1997). CTP:phosphocholine cytidylyl-transferase. Biochim. Biophys. Acta 1348, 79–90.

119. Kerridge, D. (1986). Mode of action of clinicallyimportant antifungal drugs. Adv. Microb. Physiol.27, 1–72.

120. Kim, K. H., Voelker, D. R., Flocco, M. T. and
Carman, G. M. (1998). Expression, purification

and characterization of choline kinase, productof the CKI gene from Saccharomyces cerevisiae.J. Biol. Chem. 273, 6844–6852.

121. Kiyono, K., Kiura, K., Kushima, Y., Hikiji, T.,Fukushima, M., Shibuya, I. and Ohta, A. (1987).Primary structure and product characterization ofthe Saccharomyces cerevisiae CHO1 gene that en-codes phosphatidylserine synthase. J. Biochem.102, 1089–1100.

122. Kleinhans, F. W., Lees, N. D., Bard, M., Haak,R. A. and Woods, R. A. (1979). ESR determi-nation of membrane permeability in a yeast sterolmutant. Chem. Phys. Lipids 23, 143–154.

123. Kodaki, T. and Yamashita, S. (1987). Yeastphosphatidylethanolamine methylation pathway:cloning and characterization of two distinctmethyltransferase genes. J. Biol. Chem. 262, 15428–15435.

124. Kodaki, T. and Yamashita, S. (1989). Characteri-zation of the methyltransferases in the yeast phos-phatidylethanolamine methylation pathway byselective gene disruption. Eur. J. Biochem. 185,243–251.

125. Kodaki, T., Hosaka, K., Nikawa, J. andYamashita, S. (1991). Identification of the up-stream activation sequences responsible for theexpression and regulation of the PEL1 and PEM2genes encoding the enzymes of the phosphatidyl-ethanolamine methylation pathway in Saccharo-myces cerevisiae. J. Biochem. 109, 276–287.

126. Kohlwein, S. D., Daum, G., Schneiter, R. andPaltauf, F. (1996). Phospholipids: synthesis, sort-ing, subcellular traffic—the yeast approach. TrendsCell Biol. 6, 260–266.

127. Kornblatt, J. A. and Rudney, H. (1971). Twoforms of acetoacetyl coenzyme A thiolase in yeastI. Separation and properties. J. Biol. Chem. 246,4417–4423.

128. Kornblatt, J. and Rudney, H. (1971). Two formsof acetoacetyl coenzyme A thiolase in yeast II.Intracellular location and relationship to growth.J. Biol. Chem. 246, 4424–4430.

129. Kuchler, K., Daum, G. and Paltauf, F. (1986).Subcellular and submitochondrial localization ofphospholipid-synthesizing enzymes in Saccharo-myces cerevisiae. J. Bacteriol. 165, 901–910.

130. Kuge, O. and Nishijima, M. (1997). Phosphatidyl-serine synthase I and II of mammalian cells. Bio-chim. Biophys. Acta 1348, 151–156.

131. Lai, K. and McGraw, P. (1994). Dual control ofinositol transport in Saccharomyces cerevisiae byirreversible inactivation of permease and regulationof permease synthesis by INO2, INO4 and OPI1.J. Biol. Chem. 269, 2245–2251.

132. Lai, K., Bolognese, C. P., Swift S. and McGraw, P.(1995). Regulation of inositol transport in Saccha-
romyces cerevisiae involves inositol-induced
Yeast 14, 1471–1510 (1998)

1503

changes in permease stability and endocyticdegradation in the vacuole. J. Biol. Chem. 270,2525–2534.

133. Lai, M. H., Bard, M., Pierson, C. A., Alexander,J. F., Goebl, M., Carter, G. T. and Kirsch, D. T.(1994). The identification of a gene family in theSaccharomyces cerevisiae ergosterol biosynthesispathway. Gene 40, 41–49.

134. Lamping, E., Kohlwein, S. D., Henry, S. A. andPaltauf, F. (1991). Coordinate regulation of phos-phatidylserine decarboxylation in Saccharomycescerevisiae. J. Bacteriol. 173, 6432–6437.

135. Lanterman, M. M. and Saba, J. D. (1998). Char-acterization of sphingosine kinase (SK) activity inSaccharomyces cerevisiae and isolation of SK-deficient mutants. Biochemistry 332, 525–531.

136. Leber, A., Fischer, P., Schneiter, R., Kohlwein,S. D. and Daum, G. (1997). The yeast mic2 mutantis defective in the formation of mannosyl-diinositolphosphorylceramide. FEBS Lett. 411,211–214.

137. Leber, A., Hrastnik, C. and Daum, G. (1995).Phospholipid-synthesizing enzymes in Golgi mem-branes of the yeast Saccharomyces cerevisiae. FEBSLett. 377, 271–274.

138. Leber, R., Landl, K., Zinser, E., Ahorn, H., Spok,A., Kohlwein, S. D., Turnowsky, F. and Daum, G.(1998). Dual localization of squalene epoxidase,Erg1p, in yeast reflects a relationship betweenendoplasmic reticulum and lipid particles. Mol.Biol. Cell 9, 375–386.

139. Leber, R., Zinser, E., Hrastnik, C., Paltauf, F. andDaum, G. (1995). Export of steryl esters from lipidparticles and release of free sterols in the yeastSaccharomyces cerevisiae. Biochim. Biophys. Acta1234, 119–126.

140. Leber, R., Zinser, E., Zellnig, G., Paltauf, F.and Daum, G. (1994). Characterization of lipidparticles of the yeast, Saccharomyces cerevisiae.Yeast 10, 1421–1428.

141. Lee, K. S., Patton, J. L., Fido, M., Hines, L. K.,Kohlwein, S. D., Paltauf, F., Henry, S. A. andLevin, D. E. (1994). The Saccharomyces cerevisiaePLB1 gene encodes a protein required for lyso-phospholipase and phospholipase B activity.J. Biol. Chem. 269, 19725–19730.

142. Lees, N. D., Bard, M., Kemple, M. D., Haak,R. A. and Kleinhans, F. W. (1979). ESR determi-nation of membrane order parameter in yeaststerol mutants. Biochim. Biophys. Acta 553, 469–475.

143. Lees, N. D., Bard, M. and Kirsch, D. R. (1997).Biochemistry and molecular biology of sterol syn-thesis in Saccharomyces cerevisiae. In Parish, E. J.and Nes, W. D. (Eds), Biochemistry and Function ofSterols. CRC Press, Boca Raton, FL, pp. 85–99.

144. Lees, N. D., Lofton, S. L., Woods, R. A. and Bard,
M. (1980). The effects of varied energy source

and detergent on the growth of sterol mutants ofSaccharomyces cerevisiae. J. Gen. Microbiol. 118,209–214.

145. Leidich, S. D., Drapp, D. A. and Orlean, P. (1994).A conditionally lethal yeast mutant blocked at thefirst step in glycosyl phosphatidylinositol anchorsynthesis. J. Biol. Chem. 269, 10193–10196.

146. Lester, R. L. and Dickson, R. C. (1993). Sphingo-lipids with inositol phosphate-containing headgroups. Adv. Lipid Res. 26, 253–272.

147. Lester, R. L., Wells, G. B., Oxford, G. andDickson, R. C. (1993). Mutant strains of Saccha-romyces cerevisiae lacking sphingolipids synthesizenovel inositol glycerophospholipids that mimicsphingolipid structures. J. Biol. Chem. 268, 845–856.

148. Lorenz, R. T. and Parks, L. W. (1992). Cloning,sequencing and disruption of the gene encodingsterol C-14 reductase in Saccharomyces cerevisiae.DNA Cell Biol. 11, 685–692.

149. Lorenz, R. T., Casey, W. M. and Parks, L. W.(1989). Structural discrimination in the sparkingfunction of sterols in the yeast Saccharomycescerevisiae. J. Bacteriol. 171, 6169–6173.

150. Lorenz, R. T., Rodriguez, R. J., Lewis, T. A. andParks, L. W. (1986). Characteristics of sterol up-take in Saccharomyces cerevisiae. J. Bacteriol. 167,981–985.

151. M’Baya, B., Fegueur, M., Servouse, M. and Karst,F. (1989). Regulation of squalene synthase andsqualene epoxidase activities in Saccharomycescerevisiae. Lipids 24, 1020–1023.

152. Majumder, A. L., Johnson, M. D. and Henry, S. A.(1997). 1L-myo-inositol-1-phosphate synthase. Bio-chim. Biophys. Acta 1348, 245–256.

153. Mandala, S. M., Frommer, B. R., Thornton, R. A.,Kurtz, M. B., Young, M., Cabello, M. A.,Genilloud, O., Leisch, J. M., Smith, J. L. andHorn, W. S. (1994). Inhibition of serine palmitoyl-transferase activity by lipoxamycin. J. Antibiot.(Tokyo) 47, 376–379.

154. Mandala, S. M., Thornton, R. A., Frommer, B. R.,Dreikorn, S. and Kurtz, M. B. (1997). Viridiofun-gins, novel inhibitors of sphingolipid synthesis.J. Antibiot. (Tokyo) 50, 339–343.

155. Mandala, S. M., Thornton, R. A., Rosenbach, M.,Milligan, J., Garcia-Calvo, M., Bull, H. G. andKurtz, M. B. (1997). Khafrefungin, a novel inhibi-tor of sphingolipid synthesis. J. Biol. Chem. 272,32709–32714.

156. Mandala, S. M., Thornton, R., Tu, Z., Kurtz,M. B., Nickels, J., Broach, J., Menzeleev, R. andSpiegel, S. (1998). Sphingoid base 1-phosphatephosphatase: a key regulator of sphingolipidmetabolism and stress response. Proc. Natl Acad.Sci. USA 95, 150–155.

157. Mao, C., Wadleight, M., Jenkins, G. M., Hannun,
Y. A. and Obeid, L. M. (1997). Identification
Yeast 14, 1471–1510 (1998)

1504 . .

and characterization of Saccharomyces cerevi-siae dihydrosphingosine-1-phosphate phosphatase.J. Biol. Chem. 272, 28690–28694.

158. Marcireau, D., Guyonnet, D. and Karst, F. (1992).Construction and growth properties of a yeaststrain defective in sterol 14-reductase. Curr. Genet.22, 267–272.

159. Matsuoka, S., Sagami, H., Kurisaki, A. and Ogura,K. (1991). Viable product specificity of microsomaldehydrolichyl diphosphate synthase from rat liver.J. Biol. Chem. 266, 3464–3468.

160. Mayr, J. A., Kohlwein, S. D. and Paltauf, F.(1996). Identification of a novel, Ca2+-dependentphospholipase D with preference for phosphatidyl-serine and phosphatidylethanolamine in Saccharo-myces cerevisiae. FEBS Lett. 393, 236–240.

161. McGee, T. P., Skinner, H. B. and Bankaitis, V. A.(1994). Functional redundancy of the CDP-ethanolamine and CDP-choline pathway enzymesin phospholipid synthesis: ethanolamine-dependenteffects on steady state membrane phospholipidcomposition in Saccharomyces cerevisiae. J. Bac-teriol. 176, 6861–6868.

162. McGraw, P. and Henry, S. A. (1989). Mutations inthe Saccharomyces cerevisiae opi3 gene: effects onphospholipid methylation, growth and cross-pathway regulation of inositol synthesis. Genetics122, 317–330.

163. McMaster, C. R. and Bell, R. M. (1994). Phos-phatidylcholine biosynthesis in Saccharomyces cer-evisiae. Regulatory insight from studies employingnull and chimeric sn-1,2-diacylglycerol choline- andethanolaminephosphotransferases. J. Biol. Chem.269, 28010–28016.

164. McMaster, C. R. and Bell, R. M. (1994). Phos-phatidylcholine biosynthesis via the CDP-cholinepathway in Saccharomyces cerevisiae. Multiplemechanisms of regulation. J. Biol. Chem. 269,14776–14783.

165. McMaster, C. R. and Bell, R. M. (1997). CDP-ethanolamine: 1,2-diacylglycerol ethanolamine-phosphotransferase. Biochim. Biophys. Acta 1348,117–123.

166. McMaster, C. R. and Bell, R. M. (1997). CDP-choline: 1,2-diacylglycerol cholinephosphotrans-ferase. Biochim. Biophys. Acta 1348, 100–110.

167. McMaster, C. R., Morash, S. C. and Bell, R. M.(1996). Phospholipid and cation activation of chi-meric choline/ethanolamine phosphotransferases.Biochem. J. 313, 729–735.

168. Merrill, A. H. and Sweeley, C. C. (1996). In D. E.Vance and J. E. Vance (Eds), Biochemistry of lipids,lipoproteins and membranes. Elsevier Science, NewYork, pp. 309–339.

169. Merrill A. H. Jr. and Jones, D. D. (1990). Anupdate of the enzymology and regulation ofsphingomyelin metabolism. Biochim. Biophys. Acta
1044, 1–12.

170. Min-Seok, R., Kawamata, Y., Nakamura, H.,Ohta, A. and Takagi, M. (1996). Isolation andcharacterization of ECT1 gene encoding CTP-phosphoethanolamine cytidylyltransferase fromSaccharomyces cerevisiae. J. Biochem. 120, 1040–1047.

171. Minskoff, S. A. and Greenberg, M. L. (1997).Phosphatidylglycerolphosphate synthase fromyeast. Biochim. Biophys. Acta 1348, 187–191.

172. Minskoff, S. A., Racenis, P. V., Granger, J.,Larkins, L., Hajra, A. K. and Greenberg, M. L.(1994). Regulation of phosphatidic acid bio-synthetic enzymes in Saccharomyces cerevisiae.J. Lipid Res. 35, 2254–2262.

173. Mitchell, A. G. and Martin, C. E. (1995). A novelcytochrome b5-like domain is linked to the car-boxyl terminus of the Saccharomyces cerevisiaedelta-9 fatty acid desaturase. J. Biol. Chem. 270,29766–29772.

174. Mitchell, A. G. and Martin, C. E. (1997). Fah1p, aSaccharomyces cerevisiae cytochrome b5 fusionprotein and its Arabidopsis thaliana homolog thatlacks the cytochrome b5 domain both function inthe alpha-hydroxylation of sphingolipid-associatedvery long chain fatty acids. J. Biol. Chem. 272,28281–28288.

175. Miyake, Y., Kozutsumi, Y., Nakamura, S., Fujita,T. and Kawasaki, T. (1995). Serine palmitoyltrans-ferase is the primary target of a sphingosine-likeimmunosuppressant, ISP-1/myriocin. Biochem.Biophys. Res. Commun. 211, 396–403.

176. Mohamed, A. H., Chirala, S. S., Mody, N. H.,Huang, W. Y. and Wakil, S. J. (1988). Primarystructure of the multifunctional alpha subunit pro-tein of yeast fatty acid synthase derived from FAS2gene sequence. J. Biol. Chem. 263, 12315–12325.

177. Molzhan, S. W. and Woods, R. A. (1972). Polyeneresistance and the isolation of sterol mutants inSaccharomyces cerevisiae. J. Gen. Microbiol 72,339–348.

178. Monk, J. P. and Brogden, R. N. (1991). Naftifine.A review of its antimicrobial activity and therapeu-tic use in superficial dermatomycoses. Drugs 42,659–672.

179. Morash, S. C., McMaster, C. R., Helmstad, R. H.and Bell, R. M. (1994). Studies employing Saccha-romyces cerevisiae cpt1 and ept1 null mutants im-plicate the CPT1 gene in coordinate regulation ofphospholipid biosynthesis. J. Biol. Chem. 269,28769–28776.

180. Nagiec, M. M., Baltisberger, J. A., Wells, G. B.,Lester, R. L. and Dickson, R. (1994). The LCB2gene of Saccharomyces and the related LCB1 geneencode subunits of serine palmitoyltransferase, theinitial enzyme in sphingolipid synthesis. Proc. NatlAcad. Sci USA 91, 7899–7902.

181. Nagiec, M. M., Nagiec, E. E., Baltisberger, J. A.,
Wells, G. B., Lester, R. L. and Dickson, R. C.
Yeast 14, 1471–1510 (1998)

1505

(1997). Sphingolipid synthesis as a target for anti-fungal drugs—complementation of the inositolphosphorylceramide synthase defect in strain ofSaccharomyces cerevisiae by the AUR1 gene.J. Biol. Chem. 272, 9809–9817.

182. Nagiec, M. M., Skrzypek, M., Nagiec, E. E.,Lester, R. L. and Dickson, R. C. (1998). The LCB4(YOR171c) and LCB5 (YLR60w) genes of Saccha-romyces encode sphingolipid long chain basekinases. J. Biol. Chem. 273, 19437–19442.

183. Nagiec, M. M., Wells, G. B., Lester, R. L. andDickson, R. C. (1993). A suppressor gene thatenables Saccharomyces cerevisiae to grow withoutmaking sphingolipids encodes a protein that re-sembles an Escherichia coli fatty acyltransferase.J. Biol. Chem. 268, 22156–22163.

184. Nakashima, A., Hosaka, K. and Nikawa, J. (1997).Cloning of a human cDNA for CTP-phospho-ethanolamine cytidylyltransferase by complement-ation in vivo of a yeast mutant. J. Biol. Chem. 272,9567–9572.

185. Nakayama, K., Nagasu, T., Shimma, Y.,Kuromitsu, J. and Jigami, Y. (1992). OCH1 en-codes a novel membrane-bound mannosyltrans-ferase: outer chain elongation of asparagine-linkedoligosaccharides. EMBO J. 11, 2511–2519.

186. Neiman, A. M., Mhaiskar, V., Manus, V.,Galibert, F. and Dean, N. (1997). Saccharomycescerevisiae HOC1, a suppressor of pkc1, encodesa putative glycosyltransferase. Genetics 145, 637–645.

187. Nes, W. R. and Dhanuka, J. C. (1988). Inhibitionof sterol synthesis by Ä5-sterols in a sterolauxotroph of yeast defective in oxidosqualenecyclase and cytochrome P-450. J. Biol. Chem. 263,11844–11850.

188. Nickels, J. T. and Broach, J. R. (1996). Aceramide-activated protein phosphatase mediatesceramide-induced G1 arrest of Saccharomycescerevisiae. Genes. Dev. 10, 382–394.

189. Nikawa, J.-I. and Yamashita, S. (1997). Phosphati-dylinositol synthase from yeast. Biochim. Biophys.Acta 1348, 173–178.

190. Nikawa, J.-I., Kodaki, T. and Yamashita, S.(1987). Primary structure and disruption of thephosphatidylinositol synthase gene of Saccharomy-ces cerevisiae. J. Biol. Chem. 262, 4876–4881.

191. Nikawa, J.-I., Nagumo, T. and Yamashita, S.(1982). myo-Inositol transport in Saccharomycescerevisiae. J. Bacteriol. 150, 441–446.

192. Nikawa, J.-I., Tsukagoshi, Y. and Yamashita, S.(1991). Isolation and characterization of two dis-tinct myo-inositol transporter genes of Saccharo-myces cerevisiae. J. Biol. Chem. 266, 11184–11191.

193. Nwaka, S., Kopp, M. and Holzer, H. (1995).Expression and function of the trehalase genesNTH1 and YBR0106 in Saccharomyces cerevisiae.
J. Biol. Chem. 270, 10193–10198.

194. Oh, C.-S., Toke, D. A., Mandala, S. and Martin,C. E. (1997). ELO2 and ELO3, homologues of theSaccharomyces cerevisiae ELO1 gene, function infatty acid elongation and are required for sphin-golipid formation. J. Biol. Chem. 272, 17376–17384.

195. Okuyama, H., Saito, M., Joshi, V. C., Gunsberg, S.and Wakil, S. J. (1979). Regulation by temperatureof the chain length of fatty acids in yeast. J. Biol.Chem. 254, 12281–12284.

196. Olson, R. E. and Rudney, H. (1983). Biosynthesisof ubiquinone. Vitam. Horm. 40, 1–43.

197. Palermo, L. M., Leak, F. W., Tove, S. and Parks,L. W. (1997). Assessment of the essentiality of ERGgenes late in ergosterol biosynthesis in Saccharo-myces cerevisiae. Curr. Genet. 32, 93–99.

198. Paltauf, F., Kohlwein, S. D. and Henry, S. A.(1992). Regulation and Compartmentalization ofLipid Synthesis in Yeast. In The Molecular andCellular Biology of the Yeast Saccharomyces: GeneExpression. Cold Spring Harbor Laboratory Press,New York, pp. 415–500.

199. Patton, J. L. and Lester, R. L. (1991). The phos-phoinositol sphingolipids of Saccharomyces cerevi-siae are highly localized in the plasma membrane.J. Bacteriol. 173, 3101–3108.

200. Patton, J. L., Srinivasan, B., Dickson, R. C. andLester, R. L. (1992). Phenotypes of sphingolipid-dependent strains of Saccharomyces cerevisiae.J. Bacteriol. 174, 7180–7184.

201. Patton-Vogt, J. L., Griac, P., Sreenivas, A., Bruno,V., Dowd, S., Swede, M. J. and Henry, S. A.(1997). Role of the yeast phosphatidylinositol/phosphatidylcholine transfer protein (Sec14p) inphosphatidylcholine turnover and INO1 regula-tion. J. Biol. Chem. 272, 20873–20883.

202. Payne, W. E. and Fitzgerald-Hayes, M. (1993). Amutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharo-myces cerevisiae, causes aberrant mitoticchromosome segregation. Mol. Cell. Biol. 13, 4351–4364.

203. Pinto, W. J., Srinivasan, B., Shepherd, S., Schmidt,A., Dickson, R. C. and Lester, R. L. (1992).Sphingolipid long-chain base auxotrophs ofSaccharomyces cerevisiae: genetics, physiology anda method for their selection. J. Bacteriol. 174,2565–2574.

204. Pinto, W. J., Wells, G. W. and Lester, R. L. (1992).Characterization of enzymatic synthesis ofsphingolipid long chain bases in Saccharomycescerevisiae: mutant strains exhibiting long-chainauxotrophy are deficient in serine palmitoyltrans-ferase activity. J. Bacteriol. 174, 2575–2581.

205. Powderly, W. G., Kobayashi, G. S., Herzig, G. P.and Medoff, G. (1988). Amphotericin B-resistantyeast infection in severely immunocompromised
patients. Am. J. Med. 84, 826–832.
Yeast 14, 1471–1510 (1998)

1506 . .

206. Puoti, A., Desponds, C. and Conzelmann, A.(1991). Biosynthesis of mannosylinositolphos-phoceramide in Saccharomyces cerevisiae is depen-dent on genes controlling the flow of secretoryvesicles from the endoplasmic reticulum to theGolgi. J. Cell Biol. 113, 515–525.

207. Pyne, S., Tolan, D. G., Conway, A. M. and Pyne,N. (1997). Sphingolipids as differential regulatorsof cellular signaling processes. Biochem. Soc.Trans. 25, 549–556.

208. Qie, L. X., Nagiec, M. M., Baltisberger, J. A.,Lester, R. L. and Dickson, R. (1997). Identificationof a Saccharomyces gene, LCB3, necessary forincorporation of exogenous long chain bases intosphingolipids. J. Biol. Chem. 272, 16110–16117.

209. Quain, D. E. and Haslam, J. M. (1979). Yeast3-hydroxy-3-methylglutaryl-CoA reductase: an en-zyme committed to catabolite derepression beforeexhaustion of fermentable substrate. J. Gen. Micro-biol. 111, 343–351.

210. Racenis, P. V., Lai, J. L., Das, A. K., Mullick,P. C., Hajra, A. K. and Greenberg, M. L. (1992).The acyldihydroxyacetone phosphate pathway en-zymes for glycerolipid biosynthesis are present inthe yeast Saccharomyces cerevisiae. J. Bacteriol.174, 5702–5710.

211. Rakowska, M., Zborowski, J. and Corazzi, L.(1994). A fusogenic protein from rat microsomalmembranes: partial purification and reconstitutioninto liposomes. Membr. Biol. 142, 35–42.

212. Reggiori, F., Canivenc-Gansel, E. andConzelmann, A. (1997). Lipid remodeling leads tothe introduction and exchange of defined ceramideson GPI proteins in the ER and Golgi of Saccharo-myces cerevisiae. EMBO J. 16, 3506–3518.

213. Rine, J., Hansen, W., Hardeman, E. and Davis,R. W. (1983). Targeted selection of recombinantclones through gene dosage effects. Proc. NatlAcad. Sci USA 80, 6750–6754.

214. Robinson, G. W., Tsay, Y. H., Kienzle, B. K.,Smith-Monroy, C. A. and Bishop, R. W. (1993).Conservation between human and fungal squalenesynthetases: similarities in structure, function andregulation. Mol. Cell. Biol. 13, 2706–2717.

215. Robinson, K. S., Lai, K., Cannon, T. A. andMcGraw, P. (1996). Inositol transport in Saccha-romyces cerevisiae is regulated by transcriptionaland degradative endocytic mechanisms during thegrowth cycle that are distinct from inositol-inducedregulation. Mol. Biol. Cell 7, 81–89.

216. Rodriguez, R. J. and Parks, L. W. (1983). Struc-tural and physiological features of sterols necessaryto satisfy bulk membrane and sparking require-ments in yeast sterol auxotrophs. Arch. Biochem.Biophys. 225, 861–871.

217. Ryder, N. S. (1991). Squalene epoxidase as atarget for the allylamines. Biochem. Soc. Trans. 19,
774–777.

218. Saba, D. J., Nara, F., Bielawska, A., Garrett, S.and Hannun, Y. A. (1997). The BST1 gene ofSaccharomyces cerevisiae is the sphingosine-1-phosphate lyase. J. Biol. Chem. 272, 26087–26090.

219. Sanglard, D., Kuchler, K., Ischer, F., Pagani, J. L.,Monod, M. and Bille, J. (1995). Mechanisms ofresistance to azole antifungal agents in Candidaalbicans isolates from AIDS patients involvespecific multidrug transporters. Antimicrob. AgentsChemother. 39, 2378–2386.

220. Schjerling, C. K., Hummel, R., Hansen, J. K.,Borsting, C., Mikkelsen, J. M., Kristiansen, K. andKnudsen, J. (1996). Disruption of the gene encod-ing the acyl-CoA-binding protein (ACB1) perturbsacyl-CoA metabolism in Saccharomyces cerevisiae.J. Biol. Chem. 271, 22514–22521.

221. Schlame, M. and Greenberg, M. L. (1997). Cardio-lipin synthase from yeast. Biochim. Biophys. Acta1348, 201–206.

222. Schlossman, D. M. and Bell, R. M. (1978). Glyc-erolipid biosynthesis in Saccharomyces cerevisiae:sn-glycerol-3-phosphate and dihydroxyacetonephosphate acyltransferase activities. J. Bacteriol.133, 1368–1376.

223. Schmidt, A., Bickle, M., Beck, T. and Hall, M. N.(1997). The yeast phosphatidylinositol kinasehomolog TOR2 activates RHO1 and RHO2 via theexchange factor ROM2. Cell 88, 531–542.

224. Schneider, R., Brors, B., Burger, F., Camrath, S.and Weiss, H. (1997). Two genes of the putativemitochondrial fatty acid synthase in the genome ofSaccharomyces cerevisiae. Curr. Genet. 32, 384–388.

225. Schneider, R., Massow, M., Lisowsky, T. andWeiss, H. (1995). Different respiratory-defectivephenotypes of Neurospora crassa and Saccharo-myces cerevisiae after inactivation of the geneencoding the mitochondrial acyl carrier protein.Curr. Genet. 29, 10–17.

226. Schneiter, R. and Kohlwein, S. D. (1997). Or-ganelle structure, function and inheritance in yeast:a role for fatty acid synthesis? Cell 88, 431–434.

227. Schneiter, R., Hitomi, M., Ivessa, A. S., Fasch,E.-V., Kohlwein, S. D. and Tartakoff, A. M.(1996). A yeast acetyl-CoA carboxylase mutantlinks very long-chain fatty acid synthesis to struc-ture and function of the nuclear membrane/porecomplex. Mol. Cell. Biol. 16, 7161–7172.

228. Schweizer, E. and Bolling, H. (1970). A Saccharo-myces cerevisiae mutant defective in saturated fattyacid biosynthesis. Proc. Natl Acad. Sci. USA 67,660–666.

229. Schweizer, M., Lebert, C., Holtke, J., Roberts,L. M. and Schweizer E. (1984). Molecular cloningof the yeast fatty acid synthetase genes, FAS1 andFAS2: illustrating the structure of the FAS1 clustergene by transcript mapping and transformation
studies. Mol. Gen. Genet. 194, 457–465.
Yeast 14, 1471–1510 (1998)

1507

230. Servouse, M. and Karst, F. (1986). Regulation ofearly enzymes of ergosterol biosynthesis in yeast.Biochem. J. 240, 541–547.

231. Servouse, M., Mons, N., Baillargeat, J. L. andKarst, F. (1984). Isolation and characterization ofyeast mutants blocked in mevalonic acid forma-tion. Biochem. Biophys. Res. Commun. 123, 424–430.

232. Shanklin, J., Achim, C., Schmidt, H., Fox, B. G.and Munck, E. (1997). Mossbauer studies ofalkane omega-hydroxylase: evidence for a diironcluster in an integral-membrane enzyme. Proc. NatlAcad. Sci. USA 94, 2981–2986.

233. Shen, H. and Dowhan, W. (1996). Reduction ofCDP-diacylglycerol synthase activity results in theexcretion of inositol by Saccharomyces cerevisiae.J. Biol. Chem. 271, 29043–29048.

234. Shen, H. and Dowhan, W. (1997). Regulationof phospholipid biosynthetic enzyme activity bythe level of CDP-diacylglycerol synthase activity.J. Biol. Chem. 272, 11215–11220.

235. Shen, H. and Dowhan, W. (1998). Regulation ofphosphatidylglycerophosphate synthase levels inSaccharomyces cerevisiae. J. Biol. Chem. 273,11638–11642.

236. Shen, H., Heacock, P. N., Clancey, C. J. andDowhan, W. (1996). The CDS1 gene encodingCDP-diacylglycerol synthase in Saccharomycescerevisiae is essential for cell growth. J. Biol. Chem.271, 789–795.

237. Shi, Z., Buntel, C. J. and Griffin, J. H. (1994).Isolation of the gene encoding 2,3-oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae.Proc. Natl Acad. Sci. USA 91, 7370–7374.

238. Shiao, Y. J., Balcerzak, B. and Vance, J. E. (1998).A mitochondrial membrane protein is requiredfor translocation of phosphatidylserine frommitochondria-associated membranes to mitochon-dria. Biochem. J. 331, 217–223.

239. 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). The immuno-suppressant SR 31747 blocks cell proliferation byinhibiting a steroid isomerase in Saccharomycescerevisiae. Mol. Cell. Biol. 16, 2719–2727.

240. Sipos, G., Reggiori, F., Vionnet, C. andConzelmann, A. (1997). Alternative lipid re-modeling pathways for glycosylphosphatidylinosi-tol membrane anchors in Saccharomyces cerevisiae.EMBO J. 16, 3494–3505.

241. Skaggs, B. A., Alexander, J. F., Pierson, C. A.,Schweitzer, K. S., Chun, K. T., Koegel, C.,Barbuch, R. and Bard, M. (1996). Cloning andcharacterization of the Saccharomyces cerevisiaeC-22 sterol desaturase gene encoding a secondcytochrome P-450 involved in ergosterol bio-
synthesis. Gene 169, 105–109.

242. Skinner, H. B., McGee, T. P., McMaster,C. R., Fry, M. R., Bell, R. M. and Bankaitis,V. A. (1995). The Saccharomyces cerevisiaephosphatidylinositol-transfer protein effects aligand-dependent inhibition of choline–phosphatecytidylyltransferase activity. Proc. Natl Acad. Sci.USA 92, 112–116.

243. Skrzypek, M., Lester, R. L. and Dickson, R. C.(1997). Suppressor gene analysis reveals an essen-tial role for sphingolipids in transport of glyco-sylphosphatidylinositol-anchored proteins in Sac-charomyces cerevisiae. J. Bacteriol. 179, 1513–1520.

244. Smith, S. J. and Parks, L. (1993). The ERG3 genein Saccharomyces cerevisiae is required for theutilization of respiratory substrates and in heme-deficient cells. Yeast 9, 1177–1187.

245. Smith, S. J. and Parks, L. W. (1997). Requirementof heme to replace the sparking sterol functionin the yeast Saccharomyces cerevisiae. Biochim.Biophys. Acta 1345, 71–76.

246. Smith, S. J., Crowley, J. H. and Parks, L. W.(1996). Transcriptional regulation by ergosterol inthe yeast Saccharomyces cerevisiae. Mol. Cell. Biol.16, 5427–5432.

247. Smith, S. W. and Lester, R. L. (1974). Inositolphosphorylceramide, a novel substance and a chiefmember of a major group of yeast sphingolipidscontaining single inositol phosphate. J. Biol. Chem.249, 3395–3405.

248. Sperka-Gottlieb, C. D. M., Fasch, E.-V., Kuchler,K., Bailis, A. M., Henry, S. A., Paltauf, F. andKohlwein, S. D. (1990). The hydrophilic and acidicN-terminus of the integral membrane enzymephosphatidylserine synthase is required for efficientmembrane insertion. Yeast 6, 331–343.

249. Spiegel, S. and Merrill, A. H. Jr. (1996). Sphingo-lipid metabolism and cell growth regulation.FASEB J. 10, 1388–1397.

250. Spiegel, S., Foster, D. and Kolesnick, R. N. (1996).Signal transduction through lipid second messen-gers. Curr. Opin. Cell Biol. 8, 159–167.

251. Sreenivas, A., Patton-Vogt, J. L., Bruno, V., Griac,P. and Henry, S. A. (1998) A role of phospholipaseD (Pld1p) in growth, secretion and regulation ofmembrane lipid synthesis in yeast. J. Biol. Chem.273, 16635–16638.

252. Stack, J. H. and Emr, S. D. (1994). Vps34prequired for yeast vacuolar protein sorting is amultiple specificity kinase that exhibits bothprotein kinase and phosphatidylinositol-specific PI3-kinase activities. J. Biol. Chem. 269, 31552–31562.

253. Stewart, L. C. and Yaffe, M. P. (1991). A role forunsaturated fatty acids in mitochondrial movementand inheritance. J. Cell Biol. 115, 1249–1257.

254. Sturley, S. (1998). A molecular approach to under-standing human sterol metabolism using yeast
genetics. Curr. Opin. Lipidol. 9, 85–91.
Yeast 14, 1471–1510 (1998)

1508 . .

255. Summers, E. F., Letts, V. A., McGraw, P. andHenry, S. A. (1988). Saccharomyces cerevisiae cho2mutants are deficient in phospholipid methylationand cross-pathway regulation of inositol synthesis.Genetics 120, 909–922.

256. Sutterlin, C., Doering, T. L., Schimmoeller, F.,Schroeder, S. and Riezman, H. (1997). Specificrequirements for the ER to Golgi transport ofGPI-anchored proteins in yeast. J. Cell Sci. 110,2703–2714.

257. Tanida, I., Takita, Y., Hasegawa, A., Ohya, Y. andAnraku, Y. (1996). Yeast Cls2p/Csg2p localized onthe endoplasmic reticulum membrane regulates anon-exchangeable intracellular Ca2+ pool coopera-tively with calcineurin. FEBS Lett. 379, 38–42.

258. Taylor, F. R., Rodriguez, R. J. and Parks, L. W.(1983). Requirement for a second sterol biosyn-thetic mutation for viability of a sterol C-14demethylation defect in Saccharomyces cerevisiae.J. Bacteriol. 155, 64–68.

259. Thorsness, M., Schafer, W., D’Ari, L. and Rine, J.(1989). Positive and negative transcriptional con-trol by heme of genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharo-myces cerevisiae. Mol. Cell. Biol. 9, 5702–5712.

260. Tillman, T. S. and Bell, R. M. (1986). Mutants ofSaccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase. J. Biol. Chem. 261,9144–9149.

261. Toke, D. A. and Martin, C. E. (1996). Isolationand characterization of a gene affecting fatty acidelongation in Saccharomyces cerevisiae. J. Biol.Chem. 271, 18413–18422.

262. Toke, D. A., Bennett, W. L., Dillon, D. A., Wu,W. I., Chen, X., Ostrander, D. B., Oshiro, J.,Cremesti, A., Voelker, D. R., Fischl, A. S. andCarman, G. M. (1998). Isolation and characteriza-tion of the Saccharomyces cerevisiae DPP1 geneencoding diacylglycerol pyrophosphate phos-phatase. J. Biol. Chem. 273, 3278–3284.

263. Toke, D. A., Bennett, W. L., Oshiro, J., Wu, W. I.,Voelker, D. R. and Carman, G. M. (1998). Isola-tion and characterization of the Saccharomycescerevisiae LPP1 gene encoding a Mg2+-independent phosphatidate phosphatase. J. Biol.Chem. 273, 14331–14338.

264. Toth, M. J. and Huwyler, L. (1996). Molecularcloning and expression of the cDNAs encodinghuman and yeast mevalonate pyrophosphate de-carboxylase. J. Biol. Chem. 271, 7895–7898.

265. Trocha, P. J. and Sprinson, D. B. (1976). Locationand regulation of early enzymes of sterol biosyn-thesis in yeast. Arch. Biochem. Biophys. 174, 45–51.

266. Trocha, P. J., Jasne, S. J. and Sprinson, D. B.(1977). Yeast mutants blocked in removing themethyl group of lanosterol at C-14. Separation ofsterols by high-pressure liquid chromatography.
Biochemistry 16, 4721–4726.

267. Trotter P. J. and Voelker D. R. (1994). Lipidtransport processes in eukaryotic cells. Biochim.Biophys. Acta 1213, 241–262.

268. Trotter, P. J. and Voelker, D. R. (1995). Identifi-cation of a non-mitochondrial phosphatidylserinedecarboxylase activity (PSD2) in the yeast Saccha-romyces cerevisiae. J. Biol. Chem. 270, 6062–6070.

269. Trotter, P. J., Pedretti, J. and Voelker, D. R.(1993). Phosphatidylserine decarboxylase fromSaccharomyces cerevisiae. Isolation of mutants,cloning of the gene and creation of a null allele.J. Biol. Chem. 268, 21416–21424.

270. Trotter, P. J., Pedretti, J., Yates, R. and Voelker,D. R. (1995). Phosphatidylserine decarboxylase 2of Saccharomyces cerevisiae. Cloning and mappingof the gene, heterologous expression and creationof the null allele. J. Biol. Chem. 270, 6071–6080.

271. Trotter, P. J., Wu, W. I., Pedretti, J., Yates, R. andVoelker, D. R. (1998). A genetic screen for amino-phospholipid transport mutants identifies the phos-phatidylinositol 4-kinase, Stt4p, as an essentialcomponent in phosphatidylserine metabolism.J. Biol. Chem. 273, 13189–13196.

272. Tsay, Y. H. and Robinson, G. W. (1991). Cloningand characterization of ERG8, an essential gene ofSaccharomyces cerevisiae encoding phosphomeval-onate kinase. Mol. Cell. Biol. 11, 620–631.

273. Tsukagoshi, Y., Nikawa, J., Hosaka, K. andYamashita, S. (1991). Expression in Escherichiacoli of the Saccharomyces cerevisiae CCT geneencoding cholinephosphate cytidylyltransferase.J. Bacteriol. 173, 2134–2136.

274. Tuller, G. and Daum, G. (1995). Import of sterolsinto mitochondria of the yeast Saccharomycescerevisiae. FEBS Lett. 372, 29–32.

275. Tuller, G., Achleitner, G., Gaigg, B., Krasser, A.,Kainersdorfer, E., Kohlwein, S. D., Perktold, A.,Zellnig, G. and Daum, G. (1998). Translocation ofphospholipids between the endoplasmic reticulumand mitochondria in yeast. Lipid and Protein Traf-fic, NATO ASI Series Vol. H106, pp. 333–342.

276. Tuller, G., Hrastnik, C., Achleitner, G.,Schiefthaler, U., Klein, F. and Daum, G. (1998).YDL142c encodes cardiolipin synthase (Clslp) andis non-essential for aerobic growth of Saccharo-myces cerevisiae. FEBS Lett. 421, 15–18.

277. Turi, T. G. and Loper, J. C. (1992). Multipleregulatory elements control expression of the geneencoding the Saccharomyces cerevisiae cytochromeP450 lanosterol 14á-demethylase (ERG11). J. Biol.Chem. 267, 2046–2056.

278. Van den Bosch, H. and de Vet, E. C. J. M. (1997).Alkyl-dihydroxyacetone-phosphate synthase. Bio-chim. Biophys. Acta 1348, 35–44.

279. Van den Bossche, H., Lauwers, W., Willemsens,G., Marichal, P., Cornelissen, F. and Cools, W.
(1984). Molecular basis for antimycotic and
Yeast 14, 1471–1510 (1998)

1509

antibacterial activity of N-substituted imidazolesand triazoles: The inhibition of isoprenoid biosyn-thesis. Pestic. Sci. 15, 188–198.

280. Van Meer, G. (1993). Transport and sorting ofmembrane lipids. Curr. Opin. Cell Biol. 5, 661–673.

281. Venkateswarlu, K., Taylor, M., Manning, N. J.,Rinaldi, M. G. and Kelly, S. (1997). Fluconazoletolerance in clinical isolates of Cryptococcus neo-formans. Antimicrob. Agents Chemother. 41, 748–751.

282. Voelker, D. R. (1991). Organelle biosynthesis andintracellular lipid transport in eukaryotes. Micro-biol. Rev. 55, 543–560.

283. Voelker, D. R. (1997). Phosphatidylserine decar-boxylase. Biochim. Biophys. Acta 1348, 236–244.

284. Wagner, S. and Paltauf, F. (1994). Generation ofglycerophospholipid molecular species in the yeastSaccharomyces cerevisiae. Fatty acid pattern ofphospholipid classes and selective acyl turnover atsb-1 and sn-2 positions. Yeast 10, 1429–1437.

285. Waksman, M., Eli, Y., Liscovitch, M. and Gerst,J. E. (1996). Identification and characterization ofa gene encoding phospholipase D activity in yeast.J. Biol. Chem. 271, 2361–2364.

286. Watson, P. E., Rose, M. E., Ellis, S. W., England,H. and Kelly, S. L. (1989). Defective sterol C5-6desaturation and azole resistance: a new hypothesisfor the mode of action of azole antifungals. Bio-chem. Biophys. Res. Commun. 164, 1170–1175.

287. Weinstein, J. D., Branchaud, R., Beale, S. I.,Bement, W. J. and Sinclair, P. R. (1986). Biosyn-thesis of the farnesyl moiety of heme á fromexogenous mevalonic acid by cultured chick livercells. Arch. Biochem. Biophys. 245, 44–50.

288. Weiss, B. and Stoffel, W. (1997). Human andmurine serine-palmitoyl-CoA transferase—cloning,expression and characterization of the key enzymein sphingolipid synthesis. Eur. J. Biochem. 249,239–247.

289. Wells, G. B. and Lester, R. L. (1983). The isolationand characterization of a mutant strain of Saccha-romyces cerevisiae that requires a long-chain basefor growth and for synthesis of phosphosphingo-lipids. J. Biol. Chem. 258, 10200–10203.

290. Wells, G. B., Dickson, R. C. and Lester, R. L.(1998). Heat-induced elevation of ceramide inSaccharomyces cerevisiae via de novo synthesis.J. Biol. Chem. 273, 7235–7243.

291. Wheat, J., Marichal, P., Van den Bossche H., LeMonte, A. and Connolly, P. (1997). Hypothesison the mechanism of resistance to fluconazolein Histoplasma capsulatum. Antimicrob. AgentsChemother. 41, 410–414.

292. White, M. J., Lopes, J. M. and Henry, S. A. (1991).Inositol metabolism in yeast. Adv. Microb. Physiol.32, 2–47.

293. White, T. C., Marr, K. A. and Bowden, R. A.
(1998). Clinical, cellular and molecular factors that

contribute to antifungal drug resistance. Clin.Microbiol. Rev. 11, 382–402.

294. Witt, W., Schweingruber, M. E. and Mertsching,A. (1984). Phospholipase B from the plasma mem-brane of Saccharomyces cerevisiae. Separation oftwo forms with carbohydrate content. Biochim.Biophys. Acta 795, 108–116.

295. Wu, W. I., Liu, Y., Riedel, B., Wissing, J. B.,Fischl, A. S. and Carman, G. M. (1996). Purifica-tion and characterization of diacylglycerol pyro-phosphate phosphatase from Saccharomycescerevisiae. J. Biol. Chem. 271, 1868–1876.

296. Yamashita, M. and Hosaka, K. (1997). Cholinekinase from yeast. Biochim. Biophys. Acta 1348,63–69.

297. Yamashita, S. and Nikawa, J.-I. (1997). Phosphati-dylserine synthase from yeast. Biochim. Biophys.Acta 1348, 228–235.

298. Yamashita, S., Oshima, A., Nikawa, J.-I. andHosaka, K. (1982). Regulation of the phos-phatidylethanolamine methylation pathway inSaccharomyces cerevisiae. Eur. J. Biochem. 128,589–595.

299. Yang, H., Bard, M., Bruner, D. A., Gleeson, A.,Deckelbaum, R. J., Aljinovic, G., Pohl, T. M.,Rothstein, R. and Sturley, S. L. (1996). Sterolesterification in yeast: a two-gene process. Science272, 1353–1356.

300. Yoko-o, T., Kato, H., Matsui Y., Takenawa, T.and Toh-e, A. (1995). Isolation and characteriza-tion of temperature-sensitive plc1 mutants of theyeast Saccharomyces cerevisiae. Mol. Gen. Genet.247, 148–156.

301. Yoko-o, T., Matsui, Y., Yagisawa, H., Nojima, H.,Uno, I. and Toh-e, A. (1993). The putativephosphoinositide-specific phospholipase C gene,PLC1, of the yeast Saccharomyces cerevisiae isimportant for cell growth. Proc. Natl Acad. Sci.USA 90, 1804–1808.

302. Yoshida, S., Ohya, Y., Goebl, M., Nakano, A. andAnraku, Y. (1994). A novel gene, STT4, encodes aphosphatidylinositol 4-kinase in the PKC1 proteinkinase pathway of Saccharomyces cerevisiae.J. Biol. Chem. 269, 1166–1172.

303. Zhao, C., Beeler, T. and Dunn, T. (1994). Suppres-sors of the Ca2+-sensitive yeast mutant (csg2)identify genes involved in sphingolipid biosynthe-sis. Cloning and characterization of SCS1, a generequired for serine palmitoyltransferase activity.J. Biol. Chem. 269, 21480–21488.

304. Zhao, M., Schlame, M., Rua, D. and Greenberg,M. L. (1998). Cardiolipin synthase is associatedwith a large complex in yeast mitochondria. J. Biol.Chem. 273, 2402–2408.

305. Zhou, J. H. and Saba, J. (1998). Identification ofthe first mammalian sphingosine phosphate lyasegene and its functional expression in yeast. Bio-
chem. Biophys. Res. Commun. 242, 502–507.
Yeast 14, 1471–1510 (1998)

1510 . .

306. Zinser, E. and Daum, G. (1995). Isolation andbiochemical characterization of organelles fromthe yeast, Saccharomyces cerevisiae. Yeast 11, 493–536.

307. Zinser, E., Paltauf, F. and Daum, G. (1993). Sterolcomposition of yeast organellar membranes andsubcellular distribution of enzymes involved insterol metabolism. J. Bacteriol. 175, 2853–2858.

308. Zinser, E., Sperka-Gottlieb, C. D. M., Fasch,E.-V., Kohlwein, S. D., Paltauf, F. and Daum, G.(1991). Phospholipid synthesis and lipid compos-


ition of subcellular membranes in the unicellulareukaryote Saccharomyces cerevisiae. J. Bacteriol.173, 2026–2034.

309. Zu Heringdorf, D. M., van Koppen, C. J. andJakobs, K. H. (1997). Molecular diversity ofsphingolipid signaling. FEBS Lett. 410, 34–38.

310. Zweerink, M. M., Edison, A. M., Wells, G. B.,Pinto, W. and Lester, R. L. (1992). Characteriza-tion of a novel, potent and specific inhibitor ofserine palmitoyltransferase. J. Biol. Chem. 267,25032–25038.

Yeast 14, 1471–1510 (1998)

Biochemistry, Cell Biology and Molecular Biology of Lipids ... · PDF fileyeast Yeast 14,...

Documents

Transcript of Biochemistry, Cell Biology and Molecular Biology of Lipids ... · PDF fileyeast Yeast 14,...