Distinct roles of two ceramide synthases, CaLag1p and CaLac1p, in the morphogenesis of Candida...

Distinct roles of two ceramide synthases, CaLag1p andCaLac1p, in the morphogenesis of Candida albicansmmi_7961 728..745

Seon Ah Cheon,1† Jyotiranjan Bal,1

Yunkyoung Song,1‡ Hai-min Hwang,1 Ah Ruem Kim,1

Woo Kyu Kang,1 Hyun Ah Kang,2

Hans K. Hannibal-Bach,3 Jens Knudsen,3

Christer S. Ejsing3 and Jeong-Yoon Kim1*1Department of Microbiology and Molecular Biology,College of Bioscience and Biotechnology, ChungnamNational University, Daejeon 305-764, Korea.2Department of Life Science, Chung-Ang University,Seoul 156-756, Korea.3Department of Biochemistry and Molecular Biology,University of Southern Denmark, Campusvej 55, 5230Odense, Denmark.

Summary

Lag1p and Lac1p catalyse ceramide synthesis in Sac-charomyces cerevisiae. This study shows that Lag1family proteins are generally required for polarizedgrowth in hemiascomycetous yeast. However, in con-trast to S. cerevisiae where these proteins are func-tionally redundant, C. albicans Lag1p (CaLag1p) andLac1p (CaLac1p) are functionally distinct. Lack ofCaLag1p, but not CaLac1p, caused severe defects inthe growth and hyphal morphogenesis of C. albicans.Deletion of CaLAG1 decreased expression of thehypha-specific HWP1 and ECE1 genes. Moreover,overexpression of CaLAG1 induced pseudohyphalgrowth in this organism under non-hypha-inducingconditions, suggesting that CaLag1p is necessary forrelaying signals to induce hypha-specific gene expres-sion. Analysis of ceramide and sphingolipid composi-tion revealed that CaLag1p predominantly synthesizesceramides with C24:0/C26:0 fatty acid moieties, whichare involved in generating inositol-containing sphin-golipids, whereas CaLac1p produces ceramides withC18:0 fatty acid moieties, which are precursors forglucosylsphingolipids. Thus, our study demonstratesthat CaLag1p and CaLac1p have distinct substratespecificities and physiological roles in C. albicans.

Introduction

The establishment and maintenance of cell polarity areessential for morphogenesis and development in manybiological processes (Pruyne and Bretscher, 2000; Grebeet al., 2001). Cell polarity requires complex processesinvolving the spatial and temporal co-ordination of proteinlocalization and activation. In yeast, extensive studiesrelating to cell polarity have revealed the underlyingmechanisms responsible for mating projection formation,bud site selection and growth, and pseudohyphal orhyphal growth (Casamayor and Snyder, 2002; Berman,2006; Park and Bi, 2007; Slaughter et al., 2009). In par-ticular, investigation of morphogenesis in yeast and itsfilamentous forms has been critical to furthering ourunderstanding of eukaryotic cell differentiation and fungalpathogenesis.

Sphingolipids, which are critical for such fundamentaland diverse cellular functions as differentiation, migrationand apoptosis, comprise many types of lipid species char-acterized by a ceramide backbone structure (Dicksonet al., 2006; Cowart and Obeid, 2007). Sphingolipids playimportant structural roles in cell adhesion and the forma-tion of lipid rafts, which are assembled with apical proteinsand signal transduction molecules (Simons and Toomre,2000; Munro, 2003). Tight packing of the long, highlysaturated acyl chains of sphingolipids together withsterols (ergosterols in yeast) seems to provide lipid raftswith their signature property, namely their insolubility incertain non-ionic detergents and isolation as insolubledomains (Brown and London, 1998; Hoekstra et al.,2003). Thus, lipid rafts are thought to enable membranecompartmentalization.

Several studies have suggested that distinct popula-tions of lipid rafts consisting of different lipid componentsand/or proteins may coexist within cells, and that they canbe mobilized to different regions of the cell in response tostimuli (Madore et al., 1999; Bagnat and Simons, 2002a;Pike, 2003). Lipid rafts are involved in shmoo formation,polarized growth and morphogenesis in several yeastand filamentous fungal species, including Saccharomy-ces cerevisiae, Candida albicans, Schizosaccharomycespombe, Cryptococcus neoformans and Aspergillus nidu-lans (Bagnat et al., 2000; Wachtler et al., 2003; Nicholset al., 2004; Pearson et al., 2004). In fact, inhibition of lipid

Accepted 21 December, 2011. *For correspondence. E-mail [email protected]; Tel. (+82) 42 821 7469; Fax (+82) 42 822 7367. Presentaddresses: †Department of Life Science, Chung-Ang University,Seoul, Korea; ‡School of pharmacy, Sungkyunkwan University,Suwon, Gyeonggi-do, Korea.

Molecular Microbiology (2012) 83(4), 728–745 � doi:10.1111/j.1365-2958.2011.07961.xFirst published online 11 January 2012

© 2011 Blackwell Publishing Ltd

raft formation by specific drugs that block sphingolipid orergosterol biosynthesis leads to disruption of polarizedgrowth in these organisms (Hashida-Okado et al., 1996;Endo et al., 1997; Martin and Konopka, 2004).

Acyl-CoA-dependent ceramide synthases encoded bythe LAG1 and LAC1 genes catalyse the synthesis of(phyto/dihydro)ceramide, a key building block of complexsphingolipids in S. cerevisiae (Guillas et al., 2001; Schor-ling et al., 2001). Single deletion of either LAG1 or LAC1does not lead to a remarkable growth defect due to func-tional redundancy of these genes. Double deletion of LAG1and LAC1, however, causes severe growth defects or celldeath depending on the genetic background of the strain(Jiang et al., 1998; Barz and Walter, 1999). Lack of cera-mide synthase results in a reduced rate of GPI-anchoredprotein transport to the Golgi and a dramatic change insphingolipid level, including accumulation of long chainbases, phytosphingosine and dihydrosphingosine, anddepletion of dihydroceramide and complex sphingolipids(Barz and Walter, 1999; Guillas et al., 2001; Schorlinget al., 2001). Despite these studies, whether and howceramide synthases are involved in filamentous growthin S. cerevisiae remains largely unknown. In this study,we demonstrate that ceramide synthases are generallyrequired for filamentous growth in hemiascomycetousyeast. In particular, two ceramide synthases in C. albicans,CaLag1p and CaLac1p, synthesize different types of cera-mides. Finally, our data show that CaLag1p contributesto the synthesis of inositol-containing sphingolipids thatare required for cell growth and morphogenesis, whileCaLac1p is essential for the synthesis of glucosylsphin-golipids that play a minor role in morphogenesis.

Results

Ceramide synthases are required for polarized growth inhemiascomycetous yeast

To investigate whether LAG1 family genes are involvedin the hyphal morphogenesis of C. albicans, we deletedCaLAG1 (orf19.3249) and CaLAC1 (orf19.7354) (http://www.candidagenome.org/) from the CAI4 strain usingthe CaURA3-dpl200 cassette. Growth of the homozy-gous Calag1D/Calag1D mutant (Calag1D2-1 in Table 1,referred to as Calag1D unless otherwise indicated) wasseverely impaired. However, the homozygous Calac1D/Calac1D mutant (Calac1D2-1 in Table 1, referred to asCalac1D unless otherwise indicated) grew as well asthe wild-type strain. In various hypha-inducing media,Calag1D was unable to form hyphae; most of the mutantcells remained as clumps, and a fraction of the cellsformed short germ tubes or pseudohyphae-like shapes(Fig. 1A). On the contrary, Calac1D retained the ability toform hyphae in liquid hypha-inducing media; however,

the hyphae appeared to be curved and short in length,especially in Lee’s and Spider media (Fig. 1A). Bothmutants formed smooth colonies on solid hypha-inducing media (Fig. S1). These results suggest thatCaLag1p is required for normal cell growth and hyphalmorphogenesis of C. albicans, while CaLac1p plays aminor role in hyphal growth.

Next, we investigated whether the LAG1 family genesare required for filamentous growth of the S1278b geneticbackground of S. cerevisiae, which is inducible by nitrogenlimitation in diploid strains or by butanol in haploid strains.Single deletion of ScLAG1 or ScLAC1 had no effect onfilamentous growth of diploid strains on solid nitrogen-limiting SLAD medium or haploid strains in liquid butanol-based induction medium (Fig. 1B). However, deletion ofboth ScLAG1 and ScLAC1 in haploid strains preventedS. cerevisiae from growing as a filamentous form inbutanol-based induction medium. Expression of eithergene in the Sclag1D Sclac1D double mutant restoredthe filamentous growth phenotype induced by butanol(Fig. 1B). Thus, our data indicate that ScLAG1 andScLAC1 are functionally redundant for filamentous growthof S. cerevisiae and that either gene alone is sufficient.These results, together with the established roles of cera-mide synthases in hyphal growth of C. albicans and Yar-rowia lipolytica (Fig. S2), suggest that ceramide synthasesmay be generally required for the polarized growth ofhemiascomycetous yeast species.

Functional similarity of the Lag1 family proteins

Phylogenetic analysis of the Lag1 protein family fromS. cerevisiae, C. albicans, Y. lipolytica, and the filamen-tous fungi A. nidulans demonstrated that these proteinscan be divided into two groups (Fig. S3A). In addition,sequence comparison of the 52-amino-acid Lag1 motif,which is essential for the ceramide synthase activity(Jiang et al., 1998; Spassieva et al., 2006), also hints thatScLag1p, ScLac1p, CaLag1p, YlLag1p and A. nidulansLagA may constitute one group while CaLac1p, YlLac1pand A. nidulans BarA comprise another group (Fig. S3B).To test this possibility, we examined which genes couldrescue the growth defect of the Sclag1D Sclac1D mutant insynthetic medium and induce filamentous growth of themutant in butanol-based induction medium. Althoughthe Sclag1D Sclac1D mutant is able to grow in syn-thetic medium supplemented with an excess amount oftryptophan (200 mg ml-1) (Fig. S3C), it fails to grow in syn-thetic medium containing a low amount of tryptophan(20 mg ml-1) probably because the mutant is Trp auxotrophand lacks the high-affinity tryptophan permease, Tat2p,targeted to the plasma membrane through the organiza-tion of lipid rafts (Umebayashi and Nakano, 2003). TheSclag1D Sclac1D mutant carrying a low-copy TRP1 vector

Distinct roles of CaLag1p and CaLac1p in C. albicans 729

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 83, 728–745

expressing ScLAC1 (pRS314-ScLAC1) was transformedwith a low-copy URA3 vector expressing each LAG1family gene and grown on synthetic medium containing20 mg ml-1 tryptophan and 5-fluroanthranilic acid (5-FAA).Since 5-FAA is toxic to Trp+ cells, cells growing on themedium are thought to express a functional homologue ofScLAG1 and ScLAC1. Figure 2A shows that CaLAG1,YlLAG1 and lagA rescued the Sclag1D Sclac1D mutantunlike CaLAC1, YlLAC1 and barA. Furthermore, butanol-induced filamentous growth of Sclag1D Sclac1D was alsorecovered by expression of CaLAG1, YlLAG1 and lagA(Fig. 2B), but not CaLAC1, YlLAC1 and barA (data notshown). These results suggest that ScLag1p, ScLac1p,CaLag1p, YlLag1p and A. nidulans LagA are similarfunctionally.

We also investigated whether CaLac1p, YlLac1p andBarA constitute a distinct group with functional homology.To address this question, we examined the capability ofCaLAC1 and barA to rescue the morphological defectof the Yllac1D mutant (Fig. S2). Each gene was cloneddownstream of the constitutive Y. lipolytica TEF promoterin an ARS-based vector and then expressed in Yllac1D.Expression of barA resulted in hyphal growth of Yllac1D(Fig. 2C), while CaLAC1 expression could not rescuethis defect. Moreover, expression of ScLAG1, ScLAC1,CaLAG1, YlLAG1 and lagA did not restore filamentousgrowth in Yllac1D (data not shown). These results suggestthat YlLAC1 and A. nidulans barA are functional homo-logues in terms of hypha formation. However, it remainsto be determined why CaLAC1 was not sufficient for

Table 1. Strains used in this study.

Strain Parental strain Genotype Source

C. albicansSC5314 Clinical isolate Gillum et al. (1984)CAI4 SC5314 ura3::imm434/ura3::imm434 Fonzi and Irwin (1993)Calag1D1-1 CAI4 As CAI4, but CaLAG1/Calag1::dpl200-URA3-dpl200 This studyCalag1D1-2 Calag1D1-1 As CAI4, but CaLAG1/Calag1::dpl200 This studyCalag1D2-1 Calag1D1-2 As CAI4, but Calag1::dpl200/Calag1::dpl200-URA3-dpl200 This studyCalag1D2-2 Calag1D2-1 As CAI4, but Calag1::dpl200/Calag1::dpl200 This studyCalag1D3-1 Calag1D2-2 As CAI4, but Calag1::dpl200/Calag1::CaLAG1-dpl200-URA3-dpl200 This studyCalac1D1-1 CAI4 As CAI4, but CaLAC1/Calac1::dpl200-URA3-dpl200 This studyCalac1D1-2 Calac1D1-1 As CAI4, but CaLAC1/Calac1::dpl200 This studyCalac1D2-1 Calac1D1-2 As CAI4, but Calac1::dpl200/Calac1::dpl200-URA3-dpl200 This studyCalac1D2-2 Calac1D2-1 As CAI4, but Calac1::dpl200/Calac1::dpl200 This studyCalac1D3-1 Calac1D2-2 As CAI4, but Calac1::dpl200/Calac1::CaLAC1-dpl200-URA3-dpl200 This studyCAI4-PE CAI4 As CAI4, but EFG1/efg1::pPCK1-3HA-EFG1-URA3 This studyCalag1DPE Calag1D2-2 As CAI4, but Calag1::dpl200/Calag1::dpl200

EFG1/efg1::pPCK1-3HA-EFG1-URA3This study

CAI4-PG CAI4 As CAI4, but CaLAG1/Calag1::pPCK1-3HA-CaLAG1-URA3 This studyCalag1DPG Calag1D2-2 As CAI4, but Calag1::dpl200/Calag1::pPCK1-3HA-CaLAG1-URA3 This studyCalag1DPC Calag1D2-2 As CAI4, but Calag1::dpl200/Calag1::dpl200

CaLAC1/Calac1::pPCK1-CaLAC1-URA3This study

Calag1DAC Calag1D2-2 As CAI4, but Calag1::dpl200/Calag1::dpl200CaLAC1/Calac1::pADH1-CaLAC1-URA3

This study

S. cerevisiae10560-2B MATa ura3-52 his3::hisG leu2::hisG Liu et al. (1993)10560-5B MATa ura3-52 trp1::hisG leu2::hisG Liu et al. (1993)10560-2B/5B 10560-2B & 10560-5B MATa/a ura3-52/ura3-52 leu2::hisG/leu2::hisG his3::hisG/HIS

trp1::hisG/TRP1This study

Sclag1D2B 10560-2B As 10560-2B, but lag1::tc190-URA3-tc190 This studySclag1D5B 10560-5B As 10560-5B, but lag1::tc190-URA3-tc190 This studySclac1D2B 10560-2B As 10560-2B, but lac1::LEU2 This studySclac1D5B 10560-5B As 10560-5B, but lac1::LEU2 This studySclag1D25B Sclag1D2B & Sclag1D5B As 10560-2B/5B, but lag1::tc190-URA3-tc190/lag1::tc190-URA3-tc190 This studySclac1D25B Sclac1D2B & Sclac1D5B As 10560-2B/5B, but lac1::LEU2/lac1::LEU2 This studySclac1DtU Sclac1D2B As 10560-2B, but lac1::LEU2 trp1::tc190-URA3-tc190 This studySclac1Dt Sclac1DtU As 10560-2B, but lac1::LEU2 trp1::tc190 This studySGC97 Sclac1Dt As 10560-2B, but lac1::LEU2 trp1::tc190 lag1::HIS3 pRS314-ScLAC1 This studySGC98 SGC97 As 10560-2B, but lac1::LEU2 trp1::tc190 lag1::HIS3 pRENC-ScLAG1 This studySGC99 SGC97 As 10560-2B, but lac1::LEU2 trp1::tc190 lag1::HIS3 pRENC-ScLAC1 This study

Y. lipolyticaCX39-74B MATB trp1 ATCC 32339UL2 CX39-74B MATB trp1 leu2::mini-TRP1 Cheon et al. (2003)UL3 UL2 MATB trp1 leu2::trp1 This studyYllac1D UL3 MATB trp1 leu2::trp1Yllac1::mini-TRP1 This study

730 S. A. Cheon et al. �


rescuing the defective filamentous growth of the Yllac1Dmutant.

CaLag1p and CaLac1p possess distinct cellularfunctions in C. albicans

To further our knowledge of the differential roles ofCaLag1p and CaLac1p in C. albicans hyphal morphogen-esis, we investigated whether lipid rafts are polarized in

Calag1D and Calac1D. Staining these strains with thesterol-binding dye filipin demonstrated that the sterol-richdomains did not localize to sites of polarized growth inCalag1D. However, these domains were found in budand growing tips of Calac1D similar to wild type (Fig. 3A).This result indicates that sphingolipids synthesized byCaLag1p are crucial for lipid raft polarizations, which isthought to contribute to C. albicans hyphal morphogen-esis (Martin and Konopka, 2004).

Fig. 1. Ceramide synthase is required for cell polarity in hemiascomycetous yeast.A. C. albicans wild-type (SC5314) and mutant strains CaLAG1/Calag1D (Calag1D1-1), Calag1D/Calag1D (Calag1D2-1),Calag1D/Calag1D[CaLAG1] (Calag1D3-1), CaLAC1/Calac1D (Calac1D1-1), Calac1D/Calac1D (Calac1D2-1), Calac1D/Calac1D[CaLAC1](Calac1D3-1) grown overnight in liquid YPD medium were transferred to each hypha-inducing medium and incubated at 37°C for 3 h.B. S. cerevisiae diploid strains (upper panel): wild type (10560-2B/5B), Sclag1D/Sclag1D (Sclag1D25B) and Sclac1D/Sclac1D (Sclac1D25B)were grown at 30°C for 5 days on SLAD medium. Haploid strains (lower panel): wild type (10560-2B), Sclag1D (Sclag1D2B), Sclac1D(Sclac1D2B), Sclag1D Sclac1D[pRENC-ScLAG1] (SGC98) and Sclag1D Sclac1D[pRENC-ScLAC1] (SGC99) grown overnight in liquid YPDmedium were inoculated into liquid YPD or YPGR (YP + galactose and raffinose) media with or without 1% (v/v) butanol and cultured at 30°Cfor 8 h. The ScLAG1 and ScLAC1 genes in the pRENC plasmids were expressed from the GAL1 promoter.Scale bar, 5 mm.



Fig. 2. Functional similarity of LAG1 familygenes.A. CaLAG1, YlLAG1 and AnlagA rescue thegrowth defect of the Sclag1D Sclac1D doublemutant. A Sclag1D Sclac1D double mutantcarrying a low-copy TRP1 vector expressingScLAC1 (SGC97, MATa ura3-52 his3::hisGleu2::hisG Sclac1::LEU2 trp1::tc190Sclag1::HIS3 pRS314-ScLAC1) wasco-transformed with a low-copy URA3 vectorexpressing each LAG1 family gene controlledby the GAL1 promoter. Transformants growingon synthetic complete medium withoutleucine, histidine, tryptophan and uracil werestreaked on repression (glucose) or inductionmedium (galactose) that lacks leucine,histidine and uracil, but contains 0.4 g l-1

5-FAA.B. CaLAG1, YlLAG1 and AnlagA restore thepseudohyphal growth of the Sclag1D Sclac1Ddouble mutant. Sclag1D Sclac1D mutant cellscarrying GAL1 promoter-driven CaLAG1,YlLAG1 or AnlagA were inoculated into YPDor YPGR (YP + galactose and raffinose)medium with or without 1% (v/v) butanol andcultured at 30°C for 8 h. The ratio of length towidth was measured to clearly distinguishpseudohyphal cells from yeast-form cells. Theaverage length-to-width ratios for yeast-formand pseudohyphal cells (n > 100 for eachsample) were about 1.5 and larger than 2.5respectively. See Fig. S3D for details.C. Morphological defect of the Yllac1D mutantis restored by A. nidulans barA. Yllac1Dmutants carrying a control vector (pINATX1),YlLAC1, CaLAC1 or AnbarA were grownunder hypha-inducing conditions.Scale bar, 5 mm.



Previous studies suggest that reduced ceramidesynthesis severely impairs vacuole morphogenesis andthat the Sclag1D Sclac1D strain cannot form definedvacuoles (Faergeman et al., 2004). Thus, we analysedvacuolar morphology in the Calag1D and Calac1Dmutants. Our data demonstrate that Calag1D and

Calac1D mutant cells rapidly took up the fluorescentlipophilic dye FM4-64, resulting in staining of endocyticvesicles (Fig. 3B). However, while Calac1D producedvacuoles similar to wild-type cells, Calag1D displayed avery diffuse staining pattern with some vacuoles exhib-iting an aberrant, fragmented morphology (Fig. 3B).

Fig. 3. CaLag1p, but not CaLac1p, is required for polarization of lipid rafts and normal vacuole morphogenesis.A. Inability of the Calag1D mutant to polarize sterol-enriched domains to a budding or hyphal tip. Cells were grown at 30°C in YPD or at 37°Cin YPD + serum (10%) for 3 h, stained for 10 min with filipin (10 mg ml-1) and then analysed immediately by UV-fluorescence microscopy.B. The Calag1D mutant forms aberrant, fragmented vacuoles. The enlarged inset area shows the vacuole morphology in more detail. Cellsgrown in YPD medium to exponential phase at 30°C were treated with the vacuolar dye FM4-64 for 20 min and chased for 0, 30 and 120 minin YNB medium.C. The Calag1D mutant is multinucleate. Nuclei were visualized by DAPI staining.Scale bar, 5 mm.



These data indicate that CaLag1p is required for normalvacuolar morphology but not endocytosis. Therefore,CaLag1p, but not CaLac1p, contributes to hyphal growthin C. albicans by synthesizing specific ceramides/sphingolipids that are associated with proper vacuoleintegration and function.

Previously, we reported that morphologically defec-tive C. albicans mutants displayed a multinucleatephenotype (Song et al., 2008). Interestingly, we foundthat the Calag1D mutant, but not Calac1D, also pro-duced multinucleate yeast cells (Fig. 3C), further con-firming that CaLag1p plays a cellular role distinct fromCaLac1p.

Overexpression of CaLAG1 induces hyphal growth inC. albicans under non-hyphal growth conditions

To determine whether the inability of Calag1D to formhyphae is due to lack of structural membrane compo-nents or disruption of signalling pathways that mediatehyphal growth, we analysed the mRNA level of twohypha-specific genes, HWP1 and ECE1, under hypha-inducing conditions. Expression of these genes inCalag1D decreased substantially compared with wildtype, suggesting that CaLag1p contributes to C. albicanshyphal morphogenesis by affecting, at least partially,transcriptional activation of hypha-specific genes(Fig. 4A). Since Efg1p, a transcription factor regulated bythe cAMP/PKA signalling pathway, modulates HWP1 andECE1 expression, we proceeded to examine whetherthis pathway also mediates the effects of sphingolipidssynthesized by CaLag1p on hypha formation. Overex-pression of Efg1p from pPCK1-3HAEFG1 integrated intothe EFG1 locus partially restored hyphal growth ofCalag1D, even though lipid rafts were not polarized inthis mutant (Fig. 4B).

Since CaLag1p is necessary for mediating hypha-specific gene expression, we reasoned that overexpres-sion of this protein could induce hyphal growth ofC. albicans even under non-hyphal growth conditions.To evaluate this possibility, we integrated the pPCK1-3HACaLAG1 vector into the CaLAG1 locus of wild-typeand Calag1D mutant strains. Intriguingly, activation ofthe CaPCK1 promoter under non-hypha-inducing condi-tions promoted filamentous growth of cells carrying thepPCK1-3HACaLAG1 gene (Fig. 4C). This result impliesthat CaLAG1 expression should be regulated to ensurea proper morphology for C. albicans growth and survival.However, Calcofluor white staining revealed that a largenumber of the filamentous cells were pseudohyphae, nottrue hyphae (Fig. 4D). Thus, it is probable that, althoughCaLag1p overexpression stimulates signalling eventsthat mediate polarized growth in C. albicans, it is notsufficient for true hypha formation.

CaLag1p and CaLac1p synthesize ceramides forinositol-containing sphingolipids and glucosylceramidesrespectively

Our data establish that CaLag1p and CaLac1p play distinctcellular roles in C. albicans, especially with respect tohyphal growth. Thus, we proceeded to investigate whatdetermines the functional difference between theseceramide synthases. To assess whether CaLag1p andCaLac1p possess unique substrate specificities, leading tothe synthesis of different ceramide species, we analysedthe composition of ceramides, inositol-containing sphin-golipids and hexosylceramides in wild-type, Calag1D andCalac1D strains grown in non-hypha- or hypha-inducingmedium. This experiment revealed that the types andquantities of ceramides were not drastically differentbetween the cells grown under the two culture conditions(Fig. 5A–F and Fig. S4A–F), except for the relativeincrease of IPC, MIPC and M(IP)2C 44:0;4 species over42:0;4 species, along with about twofold relative decreasein total sphingolipids, in hypha-inducing medium. Ingeneral, CaLag1p synthesizes phytoceramides that areprecursors for inositol-containing sphingolipids, whileCaLac1p is responsible for producing dihydroceramidesthat constitute hexosylceramides (Fig. 5A–F). From thelipidomic data, we deduced that CaLag1p uses C24:0-CoAand C26:0-CoA primarily to synthesize ceramide species.Moreover, these data revealed that CaLac1p uses C18:0-CoA and C24:0-CoA as major and minor substratesrespectively. The fact that CaLag1p and CaLac1p pro-duced a variety of ceramide species, including odd-numbered ceramides, indicates that their substratespecificities are not very strict (Fig. 5A). On the contrary,the substrate specificity of inositol phosphorylceramide(IPC) synthase, IPC mannosyl transferase and inositol-phosphotransferase in the following steps may contributeto the synthesis of the final specific products, such asM(IP)2C 42:0;4 and 44:0;4, in the wild-type strain (Fig. 5D).The Calag1D mutant was capable of synthesize M(IP)2C36:0;3, 36:0;4, 42:0;3 and 42:0;4 (Fig. 5D), demonstratingthat ceramide species produced by CaLac1p in theabsence of CaLag1p can be used as substrates of IPCsynthase to supply inositol-containing sphingolipids,although at levels that are insufficient for supporting normalcell function.

These results also indicate that hexosylceramides wereabsent in Calac1D and that hexosylceramide (37:2;3) wasformed as the almost exclusive product in the wild-typeand Calag1D strains (Fig. 5F). Because a methyl groupis attached at the C9 position of the long chain base ofceramides in C. albicans (Oura and Kajiwara, 2010) and4-desaturation and C4-hydroxylation of the long-chainbase are mutually exclusive, hexosylceramide (37:2;3)was identified as the methylated diunsaturated hexosylce-



ramide (d18:2/18:0(2-OH)). To confirm that CaLac1p isrequired for hexosylceramide synthesis and that thehexosylceramide is glucosylceramide, we analysed hexo-sylceramides using thin-layer chromatography (TLC).Figure 5G shows that glucosylceramide is absent in the

Calac1D mutant, suggesting that CaLac1p is the ceramidesynthase responsible for glucosylceramide synthesis inC. albicans.

However, our data demonstrate that the amount ofglucosylceramide present was reduced substantially in

Fig. 4. Expression of the hypha-specific HWP1 and ECE1 genes is reduced dramatically in the Calag1D mutant.A. Expression of the HWP1 and ECE1 genes is reduced dramatically in Calag1D/Calag1D (Calag1D2-1). Total RNA was extracted from cellsincubated in YPD medium at 30°C and YPD + serum (10%) at 37°C for 1, 3 or 5 h.B. Overexpression of CaEFG1 partially rescues hyphal growth of Calag1D. The CaEFG1 gene was controlled by the PCK1 promoter. Eachstrain grown overnight in repressing medium (S4D) was transferred to inducing medium (SCAA) and then incubated for 8 h at 30°C.C. Overexpression of CaLAG1 induces hyphal growth of C. albicans. The endogenous CaLAG1 gene was replaced with a CaLAG1 genecontrolled by the PCK1 promoter in wild type (CAI4-PG) and Calag1D/Calag1D (Calag1DPG). Each strain grown overnight in repressingmedium (S4D) was transferred to inducing medium (SCAA) and then incubated at 30°C.D. Quantification of three cellular forms (yeast, pseudohyphae and true hyphae) of CaLAG1-overexpressing C. albicans cells. To distinguishbetween pseudohyphae and true hyphae, cells grown at 30°C for 8 h in SCAA were stained with Calcofluor white (n = 200 for each strain).Scale bar, 5 mm.



Fig. 5. CaLag1p and CaLac1p synthesize ceramides for inositol-containing sphingolipids and glucosylceramides respectively.A–D. Composition of ceramides and inositol-containing sphingolipids.E. Relative amounts of IPC, MIPC and M(IP)2C to the phospholipids (PI + PS + PA + LIPC + IPC + MIPC + M(IP)2C) in each strain.F. Composition of hexosylceramides. Lipid extracts from wild type (SC5314), Calag1D/Calag1D mutant (Calag1D2-1), Calac1D/Calac1D mutant(Calac1D2-1) strains grown in YPD plus serum medium were analysed by mass spectrometry. No glucosylceramide species were detected inthe Calac1D strain. Mass spectra were recorded on the LTQ Orbitrap XL with a target mass resolution of 100 000 at m/z 700. This massresolution allows the baseline separation of two molecular ions differing by only 0.014 Da. The high mass resolution and accuracy allowedidentification of the odd-numbered ceramide species. For example, the mass difference between Cer 43:0;4 (m/z 696.6512) and Cer 42:1;5(m/z 696.6148) was 0.0364 atomic mass unit, which was enough to identify each species. Cer 36:0;3 = Cer(t18:0/18:0 or d18:0/18:0(2-OH));Cer 36:0;4 = Cer(t18:0/18:0(2-OH)); Cer 42:0;3 = Cer(d18:0/24:0(2-OH) or t18:0/24:0); Cer 42:0;4 = Cer(t18:0/24:0(2-OH)); Cer44:0;4 = Cer(t18:0/26:0(2-OH)). Error bar indicates standard deviation (n = 3, technical repeats).G. TLC analysis of glucosylceramide extracted from the C. albicans wild-type SC5314, Calag1D/Calag1D (Calag1D2-1) mutant andCalac1D/Calac1D (Calac1D2-1) mutant strains under yeast and hypha induction conditions. Glucosylceramide from soybean was used as astandard. Structural differences in the fatty acid and sphingoid base residues render the mobilities of glucosylceramides from yeast and plantsare slightly different under the conditions used in this study.H. Analysis of CaLAG1 and CaLAC1 mRNAs from wild-type, Calag1D/Calag1D (Calag1D2-1) and Calac1D/Calac1D (Calac1D2-1) strains byqRT-PCR.



Calag1D compared with the wild-type strain (SC5314). Toexamine whether this reduction was due to decreasedCaLAC1 expression, we analysed the level of CaLAG1 andCaLAC1 mRNA using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). We found that theexpression of CaLAC1 and CaLAG1 was downregulated inCalag1D and Calac1D respectively (Fig. 5H). These resultssupport our TLC analysis. However, the mechanism under-lying how and when CaLag1p and CaLac1p regulate eachother remains to be elucidated.

Overexpression of CaLAC1 partially suppresses thedefective phenotypes of the Calag1D mutant

Our analysis revealed that CaLac1p can synthesizeinositol-containing sphingolipids (i.e. 36:0;3, 36:0;4,42:0;3, 42:0;4) in Calag1D, although at levels that areinsufficient for supporting normal cell function (Fig. 5A–E).Therefore, despite the distinct cellular functions ofCaLag1p and CaLac1p, CaLAC1 overexpression couldsuppress the defective phenotypes observed in Calag1D.To investigate this hypothesis, we constructed a Calag1Dstrain that overexpresses CaLAC1 by replacing one copyof the wild-type CaLAC1 gene with pPCK1-CaLAC1 whoseexpression is controlled by the inducible CaPCK1 pro-moter. Growth of the strain in synthetic casamino acid(SCAA) medium induced the CaPCK1 promoter, therebycausing CaLac1p overexpression. This strain grew betterin this medium than the parental Calag1D mutant did. In

contrast, culturing the cells in synthetic complete (SC-URAS) medium repressed the CaPCK1 promoter and thestrain grew slowly, similar to Calag1D (Fig. 6A and Fig. S5).This result indicates that overexpression of CaLAC1 canpartially complement the growth defect of the Calag1Dmutant.

Next, we determined whether CaLAC1 overexpressioncould rescue the defective hyphal morphogenesis ofCalag1D. To this end, we created a new strain carryingpADH1-CaLAC1, which expresses CaLAC1 constitutivelyfrom the ADH1 promoter.As shown in Fig. 6B, the Calag1Dstrain carrying pADH1-CaLAC1 formed hyphae in hypha-inducing serum medium, although the hyphal lengthobserved was much shorter than that seen in wild-typecells. These results indicate that, while the endogenouslevel of CaLac1p is not enough to suppress the absence ofCaLag1p, overexpression of CaLAC1 can partially rescuethe defective phenotypes of the Calag1D mutant.

Discussion

Lipid rafts, membrane microdomains enriched in sphin-golipids and ergosterol, polarize to the shmoo tip inpheromone-induced S. cerevisiae cells and to the hyphaltip in C. albicans and A. nidulans (Bagnat and Simons,2002b; Martin and Konopka, 2004; Van et al., 2004; Liet al., 2006). Nonetheless, direct involvement of the Lag1family of ceramide synthases, which play critical roles insphingolipid synthesis, in the filamentous growth of yeast

Fig. 6. Overexpression of CaLAC1 partially suppresses the defective phenotypes of the Calag1D mutant.A. Overexpression of CaLAC1 partially restores the growth defect of the Calag1D mutant. Wild type (SC5314), Calag1D/Calag1D (Calag1D2-1)and Calag1D/Calag1D (Calag1DPC), which carries the CaLAC1 gene (pPCK1-CaLAC1) regulated by the inducible PCK1 promoter, weregrown overnight in YPD medium, inoculated into liquid PCK1-repressing (SC-URAS) or -inducing (SCAA) medium, and then measured fortheir growth at 30°C.B. Overexpression of CaLAC1 partially rescues the defective hyphal growth of the Calag1D mutant. Wild type (SC5314), Calag1D/Calag1D(Calag1D2-1) and Calag1D/Calag1D (Calag1DAC), which carries the CaLAC1 gene (pADH1-CaLAC1) expressed from the constitutive ADH1promoter, were grown in 10% serum medium for 3 h.



remains poorly understood. Our study began after a screenrevealed the requirement of Y. lipolytica Lac1 protein(YlLac1p) in hyphal growth in this organism (Fig. S2). Ourresults showed that Lag1 family proteins play an essentialrole in filamentous growth in S. cerevisiae and C. albicans.Thus, this study indicates that Lag1 family proteins aregenerally required for filamentous growth in hemiascomy-cetous yeast. More importantly, we demonstrate thattwo ceramide synthases in C. albicans, CaLag1p andCaLac1p, produce distinct ceramides of differing fatty acidchain length, suggesting that these enzymes have distinctin vivo functions.

Phylogenetic analysis indicates that ceramide syn-thases of hemiascomycetous yeast and filamentous fungican be divided into two categories, namely one thatincludes S. cerevisiae LAG1 and LAC1, C. albicans LAG1,Y. lipolytica LAG1 and A. nidulans lagA, and the other thatincludes C. albicans LAC1, Y. lipolytica LAC1 and A. nidu-lans barA (Fig. S3A and B). Studies have established thatthe six mammalian Lag1 homologues possess specificsubstrate specificities to different fatty acyl-CoAs (Guillaset al., 2003; Riebeling et al., 2003). The two groups offungal ceramide synthases are expected to also possessdifferent substrate specificities and in vivo functions (Liet al., 2006; Rittenour et al., 2011; Ternes et al., 2011). Thefirst group of yeast and fungi ceramide synthases generallyparticipates in cell growth. In the case of Y. lipolytica LAG1,we failed to obtain a Yllag1D mutant, which indicates thatYlLAG1 may be an essential gene. However, the secondgroup is not necessary for cell growth. We found thatall members belonging to each group, except CaLAC1,were functionally complementary (Fig. 2A–C). It remainsunclear why CaLAC1 could not rescue the defective phe-notypes of Yllac1D despite the fact that CaLAC1 andYlLAC1 could be categorized into the same phylogeneticgroup. It is possible that differences in substrate specificitydo not allow for functional swapping between YlLac1p andCaLac1p. Such a scenario is consistent with the observa-tions that YlLac1p uses C16 fatty acids for glucosylceram-ide synthesis (J. Bal and J.-Y. Kim, unpublished), whileCaLac1p favours C18 fatty acids (Fig. 5F). Analysis of thesubstrate specificities of the ceramide synthases will likelyprovide more clues as to why YlLac1p cannot be function-ally replaced by CaLac1p.

Sphinganine level is thought to be a regulator of cellpolarity development in HepG2 cells. Increased sphinga-nine resulting from inhibition of dihydroceramide synthaseby fumonisin B1 dramatically perturbed cell polarity devel-opment. In contrast, decreased sphinganine caused byinhibition of serine palmitoyltransferase with L-cycloserinestimulated cell polarity development (Van et al., 2004). Totest whether sphinganine accumulation caused the loss ofcell polarity in Calag1D, we treated the mutant cells withmyriocin, a specific inhibitor of serine palmitoyltransferase

that decreases sphinganine level. Our result demonstratesthat treatment with varying concentrations of myriocin didnot rescue the defective hyphal growth of the Calag1Dmutant (Fig. S6A), indicating that sphinganine accumula-tion may not be involved in the loss of cell polarity in theCalag1D mutant. We also tested whether exogenous addi-tion of phytosphingosine (PHS), dihydrosphingosine(DHS) or sphingosine (SPH) can block hyphal growth inC. albicans. However, as shown in Fig. S6B, PHS, DHS orSPH did not affect the hyphal growth of C. albicans inserum medium, which confirms that sphinganine accumu-lation is not associated with the defective hyphal growth ofthe Calag1D mutant.

Lipid rafts may contribute to the localization of specificproteins, such as polarity landmark and signalling proteins,which are critical for cell polarity (Fischer et al., 2008).Thus, failure to polarize lipid rafts due to sphingolipiddeficiency most likely causes disruption of special mem-brane structures involved in polarized growth and plat-forms on which multi-protein signalling complexes arebased. As such, it is necessary to uncover which sphin-golipid species play a major role in lipid raft polarization.Our study shows that Calag1D, which lacks the complexinositol-containing sphingolipids, IPC, MIPC and M(IP)2Cwith C24:0/C26:0 fatty acid moieties, is defective in hyphalgrowth. This result suggests that the synthesis of sphin-golipids composed of C24:0/C26:0 fatty acid moieties iscritical for the lipid raft polarization and signalling platformformation required for C. albicans morphogenesis. Otherstudies support the importance of sphingolipids with verylong chain fatty acids in microdomain-mediated signaltransduction in mammalian cells (Iwabuchi et al., 2010;Ohno et al., 2010). However, why fatty acid length insphingolipids is important for this signalling functionremains to be elucidated.

Our data show that expression of hypha-specific HWP1and ECE1 genes was low in Calag1D compared with wildtype (Fig. 4A). Overexpression of Efg1p, a transcriptionfactor involved in the cAMP/PKA signalling pathway, par-tially recovered hyphal growth in this mutant (Fig. 4B).Moreover, overexpression of CaLag1p induced filamen-tous growth of C. albicans without any external stimulirequired for the morphogenetic switch (Fig. 4C and D).Altogether, these results suggest that sphingolipids con-taining C24:0/C26:0 fatty acid moieties or intermediates inthe sphingolipid biosynthesis pathway may participate inregulating the expression of genes involved in C. albicanshyphal growth. This may be the first report demonstratinga direct link between sphingolipid biosynthesis andtranscription regulation in C. albicans. Nevertheless,previous studies have suggested a possible involvementof sphingolipids in gene expression regulation throughactin cytoskeleton dynamics. Disruption of sphingolipidbiosynthesis eliminates the signalling of Mss4p,



phosphatidylinositol-4-phosphate 5-kinase, which controlsCdc42p to regulate actin cytoskeleton assembly in S. cer-evisiae (Kobayashi et al., 2005; Yakir-Tamang and Gerst,2009). Inhibition of IPC synthesis by Aureobasidin (AbA)resulted in a lack of actin cables, but not cortical actinpatches, in S. cerevisiae (Endo et al., 1997), which issimilar with our result that cortical actin patches polarize tobudding cells in Calag1D as in wild type and Calac1D(Fig. S7). In addition, expression of HWP1 is coupled toactin cytoskeletal dynamics in C. albicans (Wolyniak andSundstrom, 2007). Therefore, we speculate that sphin-golipids with C24:0/C26:0 fatty acid moieties may berequired for the polarized localization of a factor(s) control-ling actin polymerization, which activates the cAMP/PKAsignalling pathway and consequently upregulates HWP1and ECE1.

The Calac1D mutant lacking glucosylceramide wasunable to form hyphae on various solid hypha-inducingmedia, but it grows as a hyphal form in liquid hypha-inducing media (Fig. S1B and Fig. 1A). This result sug-gests that, although glucosylceramide plays a role inC. albicans hyphal growth, it is not an essential one. Simi-larly, other researchers have reported that C. albicansmutants depleted of sphingolipid 8-desaturase (Sld1p),C9 methyltransferase (Mts1p) or glucosylceramide syn-thase (Hsx11p), which are required for glucosylceram-ides, exhibit a decreased hyphal growth rate comparedwith the wild-type strain (Oura and Kajiwara, 2008; 2010).Interestingly, a recent publication revealed that glucosyl-ceramide is a virulence factor of C. albicans. Mutantswith defective glucosylceramide biosynthesis grew wellin liquid culture medium and did not display defects inhyphal morphogenesis. Despite this, they could not pro-liferate in the host kidney and lost their virulence (Nobleet al., 2010). This finding is surprising because it defiesthe general understanding that the ability to switch mor-phology is associated with virulence in C. albicans. Dis-covering the in vivo function of glucosylceramide and itsassociation with host cell infection is therefore necessary.

From the sphingolipidomic analysis, we could inferseveral conclusions pertaining to sphingolipid biosynthesisin C. albicans. First, despite the distinct substrate specifici-ties of CaLag1p and CaLac1p regarding fatty acid chainlength, CaLac1p can use a broad range of substrates forceramide synthesis, especially in the absence of CaLag1p(Fig. 5), assuming that CaLag1p and CaLac1p are the onlyceramide synthases in C. albicans. However, the substratespecificities of IPC synthase, IPC mannosyltransferaseand inositolphosphotransferase limit the final molecularspecies produced. Nonetheless, the ceramides 36:0;3,36:0;4, 42:0;3 and 42:0;4 are further processed by theseenzymes in Calag1D. This observation provides an expla-nation for why CaLac1p overexpression partially restoresthe growth and hyphal morphogenesis of Calag1D (Fig. 6).

Second, our data illustrate that all ceramides with very longchain fatty acids (> Cer40:0) were saturated (Fig. 5A),indicating that these sphingolipids are not substrates ofsphingolipid D4-desaturase (CaDes1p) or sphingolipidD8-desaturase (CaSld1p). Third, the major species ofinositol-containing sphingolipids are 42:0:4 and 44:0;4,which are a-hydroxylated, while the major glucosylcer-amide species generated is 37:2;3, which must also bean a-hydroxylated form because 4-desaturation andC4-hydroxylation of the long-chain base are mutuallyexclusive. This result suggests that a-hydroxylation by thea-hydroxylase Scs7p (http://www.candidagenome.org/)may be operating for both inositol-containing sphingolipidsand glucosylceramides synthesis in C. albicans.

The Calag1D mutant is characterized by poor growthand an inability to form hyphae, which implies that thisstrain may not cause host damage following infection.However, the general assumption that slow growth in vitrocorrelates with reduced virulence and that the ability tochange morphologically associates with pathogenicitymay not be correct (Noble et al., 2010). We injected theCalag1D mutant into mice to test whether the mutant isvirulent. As shown in Fig. S8, all mice infected with theCalag1D mutant survived, suggesting that the Calag1Dmutant is avirulent. Nevertheless, further experiments arenecessary to confirm that CaLag1p is a real virulencefactor because the Calag1D mutant cells are severelyaggregated and the URA3 gene, whose expression level iscritical for virulence of C. albicans (Brand et al., 2004), wasinserted at the CaLAG1 locus that has not been demon-strated to be an appropriate locus for URA3 expression.

In conclusion, CaLag1p and CaLac1p are componentsof pathways involved in inositol-containing sphingolipidand glucosylceramide synthesis, respectively, in C. albi-cans. However, in the absence of CaLag1p, ceramidescontaining C18:0 fatty acid moieties, which are synthe-sized by CaLac1p, can be used to synthesize inositol-containing sphingolipids. Consequently, CaLag1p isimportant for the growth and hyphal morphogenesis ofC. albicans, while CaLac1p plays a minor role in hyphalgrowth of this organism (Fig. 7).

Experimental procedures

Strains, plasmids and growth conditions

The strains and plasmids used in this study are listed inTables 1 and 2 respectively. All yeast strains were grown inYPD medium (1% yeast extract, 2% peptone, 2% glucose) orsynthetic complete medium (SC; 0.67% yeast nitrogen basewithout amino acids, 2% glucose and drop-out amino acidmixture) supplemented with the required amino acids. YPDmedium supplemented with 10% serum (Newborn calf serum;Gibco BRL), Lee’s medium (Lee et al., 1975) and Spidermedium (Liu et al., 1994) were prepared for hypha induction inC. albicans. SCAA medium was used to activate the PCK



promoter in C. albicans (Stoldt et al., 1997). SLAD (nitrogenlimiting ammonia medium with 2% glucose) and YPD contain-ing 1% butanol media were used to induce filamentousgrowth of S. cerevisiae (Gimeno et al., 1992; Lorenz et al.,2000). Galactose was used to activate the S. cerevisiaeGAL1 promoter. Synthetic complete (SC) medium contain-ing 10% serum or 1% N-acetylglucosamine (SGN) were pre-pared to induce hyphal growth of Y. lipolytica (Kim et al.,2000).

To induce filamentous growth of S. cerevisiae, cells grownovernight in YPD were washed twice with steriled water andthen incubated in YPD or yeast extract peptone galactose(YPG) medium containing 1% butanol at 30°C for 8 h. Forhyphal growth of C. albicans, cells cultured in YPD at 30°Cwere harvested during the late exponential phase, washedthree times with sterilized water, and then inoculated at5 ¥ 106 cells ml-1 in hypha-induction media at 37°C.Y. lipolytica cells grown in synthetic medium at 28°C wereharvested, washed twice with sterilized water, transferred into10% serum medium at a concentration of 106 cells ml-1, andthen incubated at 28°C for 24 h.

Cloning and sequence analysis

General DNA manipulations were performed as described inSambrook and Russell (2001). DNA sequencing was per-

formed using an ABI Model 373A automated DNA sequencer(Applied Biosystems). Sequence analysis was performedusing Lasergene 5.06 (DNASTAR) and Vector NTI (Invitrogen)software, followed by homology searches in the GenBankdatabase, the Y. lipolytica genome sequencing consortiumdatabase (http://genome.jouy.inra.fr/clib/consortium/), C. albi-cans genome database CGD (http://www.candidagenome.org/) and SGD (http://www.yeastgenome.org/) using the BLAST

algorithm.

Gene disruption

Gene disruption was performed by replacing part of eachopen reading frame with a selection marker gene. Cloningwas confirmed by PCR and Southern blotting analyses. DNAprobes for Southern blotting were labelled with digoxigeninusing a DIG labelling kit (Roche Applied Science). The plas-mids and primers used in this study are listed in Table 2 andTable S1 respectively.

To construct a YlLAC1 disruption cassette, two parts (0.58kb and 0.48 kb) of the YlLAC1 gene were amplified by PCRusing two primer pairs, YlLAC1-1F and YlLAC1-2B andYlLAC1-3F and YlLAC1-4B. The amplified fragments werefused by PCR using the YlLAC1-1F and YlLAC1-4B primers.The PCR product was subcloned into the pGEM T-easyvector (Promega) and the mini-YlTRP1 blaster (Cheon et al.,

Fig. 7. A model for sphingolipid biosynthesis and the roles of CaLag1p and CaLac1p in the physiology of C. albicans. This diagram depictsonly molecular species containing the major C18:0, C24:0 and C26:0 fatty acid moieties. Enzymes and their reactions in wild-type strains areshown in solid line. Proposed reactions and their products in the Calag1D mutant are shown in dotted line. The width of the arrows indicatesthe relative contribution to each process and C. albicans physiology.



2003) was inserted between the amplified YlLAC1 gene frag-ments, resulting in the YlLAC1 disruption vector pTYLPT.Similarly, a disruption cassette to generate the Sclag1Dmutant was constructed using the primer pairs, ScLAG1-1Fand ScLAG1-2B and ScLAG1-3F and ScLAG1-4B, and thetc190-ScURA3-tc190 blaster to delete ScLAG1. The primersets ScLAC1-1F and ScLAC1-2B and ScLAC1-3F andScLAC1-4B, as well as the ScLEU2 gene, were used todelete ScLAC1 to generate the Sclac1D mutant. AnotherScLAG1 disruption cassette, Sclag1::ScHIS3, was used togenerate the Sclag1D Sclac1D double-deletion mutant fromthe Sclac1D mutant by amplifying the PCR product (ScHIS3flanked by short stretches of ScLAG1) obtained using theprimer pairs, ScLAG1HIS3-1F and ScLAG1HIS3-2B andScLAG1HIS3-3F and ScLAG1HIS3-4B, which were designedto extend the short stretches of ScLAG1. CaLAG1 andCaLAC1 disruption cassettes used to generate the Calag1Dor Calac1D mutant were constructed with a mini-CaURA3blaster (Wilson et al., 2000; Song et al., 2008) and the primerpairs, CaLAG1-1F and CaLAG1-2B and CaLAG1-3F andCaLAG1-4B for CaLAG1 deletion and CaLAC1-1F andCaLAC1-2B and CaLAC1-3F and CaLAC1-4B for CaLAC1deletion.

Complementation analysis

For complementation analyses in Y. lipolytica, LAG1 homolo-gous genes controlled by the constitutive Y. lipolytica TEFpromoter were cloned into pINATX1 (Cheon et al., 2003).Yllag1D mutants transformed with vectors carrying each LAG1homologue were selected on solid synthetic medium lackingleucine and tested for hyphal growth. For complementationtests in S. cerevisiae, the Sclac1D mutant (Sclac1Dt; MATaura3-52 his3::hisG leu2::hisG Sclac1::LEU2 trp1::tc190) wasco-transformed with pRS314-ScLAC1, which is a CEN-basedvector carrying the ScLAC1 gene with its own promoter andTRP1 as a reporter gene, and the Sclag1::HIS3 disruptioncassette. A selected Sclag1D Sclac1D mutant carryingpRS314-ScLAC1 (SGC97) was transformed with 2-micron(pESC-URA) or CEN-based (pRENC or pRE316) plasmids(Ura+) containing each LAG1 homologue controlled bythe GAL1 promoter. pRS314-ScLAC1 was shuffled with theplasmid expressing each LAG1 homologue by growing thetransformants on galactose medium containing 0.4 g l-1

5-fluroanthranilic acid (5-FAA). For complementation analy-ses in C. albicans, we constructed integrative vectors thatexpressed the CaLAC1 gene from the constitutive CaADH1

Table 2. Plasmids used in this study.

Plasmid Characteristics Purpose

C. albicanspDDB57H dpl200-CaURA3-dpl200 blaster (modified pDDB57) Selective markerpTCaLAG1DUm Calag1::dpl200-URA3-dpl200 Disruption of CaLAG1pTCaLAC1DUm Calac1::dpl200-URA3-dpl200 Disruption of CaLAC1pBINLAG1U2 CaLAG1-dpl200-URA3-dpl200 Reintegration of CaLAG1pBINLAC1U2 CaLAC1-dpl200-URA3-dpl200 Reintegration of CaLAC1pTUP-3HACaLAG1-T2 URA3-pPCK1-3HA-CaLAG1 Overexpression of 3HA-CaLAG1pTCUPLAC1 URA3-pPCK1-CaLAC1 Overexpression of CaLAC1pTCUALAC1 URA3-pADH1-CaLAC1 Overexpression of 3HA-CaLAC1pTUP-3HACaEFG1-T2 URA3-pPCK1-3HA-EFG1 Overexpression of 3HA-EFG1

S. cerevisiaepTScLAG1DU Sclag1::tc190-URA3-tc190 Disruption of ScLAG1pTScLAC1DL Sclac1::tc190-URA3-tc190 Disruption of ScLAC1pTScTRP1DU Sctrp1::tc190-URA3-tc190 Disruption of ScTRP1pRS314-ScLAC1 ScLAC1, CEN-TRP1 Complementation of ScLAC1pRENC-ScLAG1 pGAL1-cMyc-ScLAG1, CEN-URA3 Overexpression of ScLAG1pRENC-ScLAC1 pGAL1-cMyc-ScLAC1, CEN-URA3 Overexpression of ScLAC1pRENC-CaLAG1 pGAL1-cMyc-CaLAG1, CEN-URA3 Overexpression of CaLAG1pRENC-CaLAC1 pGAL1-cMyc-CaLAC1, CEN-URA3 Overexpression of CaLAC1pRENC-YlLAG1 pGAL1-cMyc-YlLAG1, CEN-URA3 Overexpression of YlLAG1pRENC-YlLAC1 pGAL1-cMyc-YlLAC1, CEN-URA Overexpression of YlLAC1pRE316-AnlagA pGAL1-AnlagA-cMyc, CEN-URA3 Overexpression of AnlagApRE316-AnBarA pGAL1-AnbarA-cMyc, CEN-URA3 Overexpression of AnbarA

Y. lipolyticapTYLPT Yllac1::mini-TRP1 Disruption of YlLAC1pTYLPU Yllac1::tc-URA3-tc Disruption of YlLAC1pINA52-18 YlLAC1, ARS68-LEU2 Complementation of YlLAC1pINATX1-YlLAG1 pYlTEF-YlLAG1, ARS68-LEU2 Overexpression of YlLAG1pINATX2-YlLAC1 pYlTEF-YlLAC1, ARS68-LEU2 Overexpression of YlLAC1pINATX2-ScLAG1 pYlTEF-ScLAG1, ARS68-LEU2 Overexpression of ScLAG1pINATX2-ScLAC1 pYlTEF-ScLAC1, ARS68-LEU2 Overexpression of ScLAC1pINATX2-CaLAG1 pYlTEF-CaLAG1, ARS68-LEU2 Overexpression of CaLAG1pINATX2-CaLAC1 pYlTEF-CaLAC1, ARS68-LEU2 Overexpression of CaLAC1pINATX2-AnLagA pYlTEF-AnLagA, ARS68-LEU2 Overexpression of AnlagApINATX2-AnBarA pYlTEF-AnBarA, ARS68-LEU2 Overexpression of AnbarA



promoter (1208 bp) or the inducible PCK1 promoter (1398 bp)instead of the endogenous CaLAC1 promoter. A portion ofCaLAC1 (425 bp from theATG) was cloned into the NheI–SphIsites downstream of the CaADH1 and CaPCK1 promoters inpTCaADH1 and pTCaPCK1 respectively. The CaURA3 genewas inserted into the SpeI site upstream of each promoter,resulting in pTCUALAC1 for the ADH promoter andpTCUPLAC1 for the PCK1 promoter. pTCUALAC1 andpTCUPLAC1 were linearized with XbaI and NcoI, respectively,and used to transform the Calag1D mutant.

Staining and microscopy

Cells grown in YPD at 30°C to mid-logarithmic phase werestained with Calcofluor white (1 mg ml-1; Sigma). For visualiza-tion of sterol-rich domains, cells grown in YPD or YPD+serummedium for 3 h at 30°C or 37°C, respectively, were stainedwith filipin [10 mg ml-1 prepared in dimethyl sulphoxide(DMSO); Sigma] for 10 min at room temperature. Nuclei werestained with DAPI (4′, 6′-diamidino-2-phenylindole; Sigma).Vacuole morphology was visualized with the lipophilic fluores-cent dye FM4-64 (Molecular Probes). Cells were suspended in50 ml of YPD plus 1 ml of 1.6 mM FM4-64 (in DMSO) andincubated at 30°C for 20 min. Cells were then chased in freshYPD for 0, 30 and 120 min. For phalloidin staining, cells weretreated with a 1:500 dilution of 0.2 mg ml-1 tetramethyl-rhodamine B isothiocyanate (TRITC)-conjugated phalloidin(Sigma) overnight at 4°C. Images were acquired using anOlympus BX61 microscope equipped with differential interfer-ence contrast optics, appropriate filters and a camera(Olympus DP71). Image contrast and brightness wereadjusted using ImageJ (National Institutes of Health).

Northern blot analysis and qRT-PCR

Candida albicans strains were cultured in liquid YPD mediumovernight at 30°C. Cultures were resuspended in 50 ml (in250 ml flasks) of fresh YPD and YPDS at an initial OD600 of0.3–0.6, followed by incubation at 30°C and 37°C for 3 hrespectively. Total RNA was extracted using RNeasy Mini Kit(QIAGEN). For Northern blot analysis, 10 mg of total RNA wasresolved on a 1% agarose-formaldehyde gel, capillary blottedonto a nylon membrane (Schleicher & Schuell), and hybridizedaccording to standard procedures (Sambrook and Russell,2001). The hybridization probes (500 bp fragment of CaLAG1,458 bp fragment of CaLAC1, 475 bp fragment of HWP1 and807 bp fragment of ACT1 ORF) were amplified and radiola-belled using the Rediprime II DNA Labelling System (Amer-sham Bioscience). For qRT-PCR, RNA was isolated using theRNeasy Mini kit (QIAGEN), purified, and then subjected tocDNA synthesis using SuperScriptase III (Invitrogen) accord-ing to the manufacturer’s recommendations. qRT-PCR wasperformed using 1¥ SYBR Green Mix (Bio-Rad) and theCFX96 C1000 thermal cycler system (Bio-Rad). Primers forqRT-PCR are listed in Table S1.

Sphingolipid analysis using mass spectrometry

Lipids were extracted as previously described (Ejsing et al.,2009; Zech et al., 2009). Inositol-containing sphingolipids

were monitored by direct infusion using an LTQ Orbitrap XLmass spectrometer equipped with the automated nanoflow ionsource Triversa NanoMate (Advion Biosciences) (Ejsing et al.,2009). Ceramide and hexosylceramide species were analy-sed by normal-phase liquid chromatography using a PVA-SILcolumn (YMC Europe GmbH) interfaced with the TriversaNanoMate and the LTQ Orbitrap XL mass spectrometer. Lipidspecies were monitored in negative ion mode by recordingFourier transform mass spectra with a target mass resolutionof 100 000. Lipid species were quantified by extracting theirpeak intensities as previously described (Ejsing et al., 2009)and normalizing to the total intensity of all monitored lipidspecies within the monitored lipid class.

Glucosylceramide analysis using TLC

Glucosylceramide analysis was performed as previouslydescribed (Takakuwa et al., 2005) with slight modification.Lyophilized cells (0.3 g) were homogenized using an equalvolume of glass beads and mixed with 2 ml of chloroform-:methanol (1:1, v/v) and 2 ml of 0.8 M KOH-methanol for5 min by vortexing. After further incubation at 42°C for30 min, 5 ml of chloroform and 2.25 ml of distilled water wereadded to the mixture. The organic phase was dried on arotary evaporator and dissolved in 200 ml of chloroform-:methanol (2:1, v/v). The extract (50 ml) was spotted ontoSilica gel 60 TLC glass plates (Sigma) and developed usingchloroform:methanol:acetic acid:water (20:3.5:2.3:0.7, v/v)as a mobile phase. Spots corresponding to glucosylceramidewere detected using orcinol-sulphuric acid reagent [0.1 g oforcinol in 45 ml of a solution of sulphuric acid:water:ethanol(5:13:27, v/v)]. Soy-glucosylceramide (Avanti Polar Lipids)was used as a standard.

Acknowledgements

We would like to thank P.J. Maeng for providing A. nidulanscDNA. This work was supported by National Research Foun-dation of Korea Grant funded by the Korean Government(NRF-2009-0076215 and NRF-2009-0066195). Work inH.A.K.’s laboratory was supported by the Microbial Genomicsand Applications R&D Program grant (No. 11-2008-09-003-00) from the Korean Ministry of Education, Science, andTechnology. Work in C.S.E.’s laboratory was supported bythe Danish Council for Independent Research (09-072484,C.S.E.) and Lundbeckfonden (R45-A4342, C.S.E.).

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Distinct roles of two ceramide synthases, CaLag1p and CaLac1p, in the morphogenesis of Candida...

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