Introduction

Fungi cause superficial and invasive infections. Opportunistic invasive infections,though less prevalent, are of much greater concern because of high mortality (often over50%) associated with them1. Many fungal species are responsible for theseinvasive infections, killing over one and half a million people every year, which ishigher than that due to tuberculosis or malaria1. The treatment optionsfor invasive infections are quite limited2. Amphotericin B (AmB) is acommonly used antifungal for over five decades. In spite of its toxicity, it ispreferred for its broad-spectrum and fungicidal mode of action, particularly fortreating invasive infections. Though echinocandins are also used for treating suchinfections, their use is limited in resource poor settings due to high cost. Moreover,Cryptococcus species do not respond to echinocandins and thus AmB alone (orin combination with flucytosine) is the mainstay to treat invasive infections caused bythese species2,3.

AmB is currently considered to kill fungi by forming large, extramembranous fungicidalsterol sponge that depletes ergosterol from lipid bilayers4. Leakage ofintracellular ions due to pore formation is thought to be a secondary effect of AmB5. Though AmB resistance is rare, it is seen in a significant percentage ofpathogenic Candida species and filamentous fungi6,7. The AmBresistance mechanisms reported so far mainly involve reduction in ergosterol content oralterations in cell wall7,8,9,10,11. We have recently shown thatsphingolipids also modulate AmB resistance12. A better understanding ofAmB resistance/sensitivity mechanisms would facilitate developing therapeutic strategiesto minimize evolution of AmB resistance, or to sensitize fungi to AmB such that lowerAmB dose can be used to reduce toxicity.

While investigating apparent elevated AmB resistance of yeast cells in presence offarnesol (unpublished), we identified Saccharomyces cerevisiaePMP3 gene as conferring increased AmB resistance when present in a multicopyplasmid. Deletion of this gene rendered the cells hypersensitive to AmB. During thecourse of our studies, PMP3 gene's role in AmB resistance was alsoreported by Huang et al13, but the mechanism underlying thisphenotype was not clear. PMP3 was first reported as a non-essential gene whosedeletion results in plasma membrane hyperpolarization and salt sensitivity14. It encodes a 55 amino acid hydrophophic protein of plasma membrane. Ahomologous plant protein could complement salt sensitivity of a yeast strain deleted inPMP315. Here we have explored the mechanistic basis ofPMP3 effect on AmB resistance. We show that certain prominent phenotypes ofPMP3 delete strain, namely defects in salt tolerance, actin polarity andendocytosis, are not responsible for AmB-sensitivity of this strain. Instead, wedemonstrate that modulation of AmB resistance by PMP3 is mediated throughsphingolipid biosynthetic pathway.

Results and Discussion

PMP3 modulates AmB resistance

The S. cerevisiaePMP3 gene was isolated from a multicopy overexpression library (inplasmid pFL44L) as conferring higher resistance to AmB. A PMP3 clone with165 bp ORF along with 1196 bp upstream and 275 bp downstream regions was used infurther studies. To confirm the phenotype, PMP3 deletion andoverexpression strains were compared with their parent strain for AmB resistance(Fig. 1a). While the delete strain was 8-fold moresensitive to AmB than the parent strain, the overexpression strain was about4-fold more tolerant compared to the parent strain. During the course of thisstudy, Huang et al13, while establishing a functionalvariomics tool for discovering drug-resistance genes and drug targets, alsoidentified PMP3 as conferring AmB resistance when present at more thanone copies. PMP3 (also known as SNA1) has three paralogs in S.cerevisiae, namely SNA2, SNA3 and SNA4, whichencode proteins with 40%, 34% and 41% identity, respectively, to that ofPMP316. Deletants of these genes were comparable tothe parent strain in their susceptibility to AmB (results not shown), implyingthat these genes do not have any role in this phenotype.

Figure 1
figure 1

S. cerevisiaePMP3 and its homologs from C. albicans and C. glabratamodulate AmB resistance.

(a) Multicopy overexpression of S. cerevisiae PMP3 (ScPMP3) andits homologs from C. glabrata (CgPMP3) and C. albicans(CaPMP3-O: PMP3 ortholog, orf19.1655.3; CaPMP3-B:PMP3 best hit, orf19.2959.1) in pmp3Δ strain ofS. cerevisiae enhance AmB resistance by about 4-fold with respectto wild-type strain (BY4741) and about 32-fold with respect topmp3Δ strain. The relative growth of the strains on 0.1μg/ml AmB (not shown) was comparable to that of respectivestrains on 0.2 μg/ml AmB. (b) AmB sensitivity of C.glabrata strain deleted in PMP3 ortholog(Cgpmp3Δ) and C. albicans strains deleted in bothalleles of PMP3 ortholog (Capmp3-OΔ/Δ) andPMP3 best hit (Capmp3-BΔ/Δ), with respectto their respective parent strains CG462 and SN95. Five μl of10-fold serial dilutions of cells were spotted starting from about105 cells/spot, as described in Methods.

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To test if PMP3 has a similar role in pathogenic yeasts, we searched forhomologs in C. albicans and C. glabrata. C. albicans hastwo homologs, which encode proteins that show 51% and 45% identity at amino acidlevel to that of S. cerevisiae. The first one is referred to asCaPMP3 ortholog (orf19.1655.3) and the second one as CaPMP3best hit (orf19.2959.1) in Candida Genome Database17. C.glabrata has a single ortholog CgPMP3 (CAGL0M08552g) encoding aprotein with 76% identity to ScPmp3p. The open reading frames of these homologswere PCR amplified and used to replace the ORF in ScPMP3 clone, therebyplacing these ORFs under the control of ScPMP3 promoter and terminator inpFL44L vector. These were tested for their ability to modulate AmB resistanceafter being transformed into pmp3Δ strain of S.cerevisiae. PMP3 ortholog from C. albicans was earlier shownto increase AmB resistance of S. cerevisiae13. Inaddition, we found CaPMP3 best hit and CgPMP3, besidescomplementing pmp3 mutation, provided resistance higher than that ofwild-type strain (Fig. 1a). While the AmB resistanceconferred by CgPMP3 and CaPMP3 best hit (CaPMP3-B) wassimilar to that of ScPMP3, i.e., 4-fold higher than that of wild-typestrain, the CaPMP3 ortholog (CaPMP3-O) provided 2-fold higherresistance (Fig. 1a).

To study the role of CaPMP3 ortholog and CaPMP3 best hit in C.albicans, we deleted both alleles of these genes in strain SN95 andconfirmed by diagnostic PCR (Fig. S1). The C.glabrata ortholog CgPMP3 (CAGL0M08552g) was also deleted andconfirmed by diagnostic PCR (Fig. S2). The AmBsusceptibility of these delete strains with respect to their parent strains wascompared (Fig. 1b). While deletion of PMP3orthologs in C. glabrata and C. albicans sensitized the cells toAmB by about 4-fold, deletion of CaPMP3 best hit did not have any effect.The AmB sensitivity of ortholog deletants in both these species provides strongevidence that PMP3 gene is important for modulation of AmB resistance inpathogenic fungi as well.

AmB resistance mediated by Pmp3p is not dependent on ergosterol or Hsp90or cell wall integrity

As far as the mechanistic basis of PMP3 effect on AmB resistance isconcerned, Huang et al13 showed that it is not related toits role in ion homeostasis. Absence or severe reduction in the amount ofergosterol in the fungal membranes and its replacement with certain othersterols results in AmB resistance in fungi7,10,11. To addressthis possibility total cellular content of ergosterol was estimated, asdescribed18. The ergosterol content, as % wet weight ofcells, of parent, delete and overexpression strains, was 0.021 ±0.001, 0.023 ± 0.002 and 0.023 ± 0.001, respectively.Though these values are comparable, it is possible that the intracellulardistribution of ergosterol might be affected. To check this, cells were stainedwith filipin, which is specific for sterols19 and observed (Fig. S3a). While wild-type and PMP3 overexpressionstrains showed intense fluorescent spots within cells, pmp3Δstrain lacked such spots. Thus, it is possible that more ergosterol isdistributed in the plasma membrane of the delete strain, rendering it moreaccessible for AmB binding and killing. If this is true, then the delete strainshould be more sensitive to other polyenes which also act by binding toergosterol. However, the sensitivity pmp3Δ strain to the polyenesnystatin, natamycin and filipin was found to be comparable to that of wild-typeand PMP3 overexpression strains (Fig. S3b), rulingout ergosterol distribution or content having any role in modulation of AmBresistance by PMP3. Huang et al13 have also ruled out theinvolvement of ergosterol in modulation of AmB resistance by PMP3, sincethis gene did not affect the resistance against other polyenes.

A recent report suggested that AmB resistance of ergosterol biosynthetic pathwaymutants is highly dependent on Hsp90 chaperone and these mutants arehypersensitive to Hsp90 inhibitors radicicol and geldanamycin as well asoxidative stress20. To check the Hsp90 dependence of AmBresistance conferred by PMP3, the sensitivity of this strain to radicicoland oxidative stress was checked along with erg6Δ strain aspositive control (Table 1). The AmB resistance oferg6Δ strain and PMP3 overexpression strain wascomparable. However, while erg6Δ strain was 8-fold and 4-fold,respectively, more sensitive to radicicol and oxidative stress, the sensitivityof PMP3 overexpression strain was comparable to wild-type, implying thatPmp3p is not dependant on Hsp90 for conferring AmB resistance. Cell wallalterations also can affect AmB resistance7. Compared to parentstrain, PMP3 delete strain showed normal chitin deposition (Fig. S4a), as well as similar resistance to cell wall disruptingagents calcofluor white, sodium dodecyl sulphate and congo red (Fig. S4b), implying that AmB sensitivity of delete strain is notrelated to cell wall integrity.

Table 1 AmB resistance mediated by PMP3 is not dependant onHSP90
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Actin polarity and endocytosis, though impaired in pmp3Δstrain, are not responsible for its AmB sensitivity

To gain further insight into PMP3 mechanism of action, we tried to predictits possible functions on the basis of biological roles of genes that interactwith PMP3. The list of interacting genes was analyzed using DAVIDBioinformatics Resources21 for enrichment of gene ontology termsfor biological processes. The top-two annotation clusters corresponded toendocytosis and actin cytoskeleton (Table 2). To checkif impaired endocytosis would result in AmB sensitivity, we screened mutants ofseveral genes having role in endocytosis for their AmB sensitivity. Deletants ofRVS161 and RVS167 were about 4-fold more sensitive to AmBcompared to the parent strain (Fig. S5). These strains,besides defects in endocytosis have several other phenotypes including saltsensitivity and altered actin cytoskeleton22,23,24,25.SUR7, encoding an eisosome protein involved in endocytosis, partiallysuppresses several of these phenotypes upon multicopy overexpression26,27,28. Thus, we exploited overexpression of SUR7 tounderstand if AmB sensitivity of pmp3Δ strain is a consequence ofdefects in actin cytoskeleton or endocytosis, or it is an independentphenotype.

Table 2 Functional Annotation Clustering of PMP3 interacting genes
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A large scale survey using GFP-Snc1-Suc2 reporter has indicated that endocytosisis decreased in pmp3Δ strain29. We monitored rateof endocytosis with a different reporter, namely methionine permease (Mup1)tagged with ecliptic pHluorin, which is a pH-sensitive green fluorescent proteinvariant that does not fluoresce after internalization to an acidic compartmentlike vacuole30,31. Mup1-pHluorin is internalized rapidly uponexposure to methionine. Wild-type cells showed substantial decrease inMup1-pHluorin intensity within 20 min after adding methionine (Fig. 2a). However, in pmp3Δ strain 40 min was neededfor a similar decrease, confirming that the rate of endocytosis is slowed downin this strain. SUR7 expressed from a multicopy plasmid restored the rateof endocytosis of pmp3Δ strain to normal level (Fig. 2a). Mup1-pHluorin fluorescence was also monitored by flowcytometry (Fig. 2b). Though background fluorescence washigh for all the strains, the rate of decrease in fluorescence is indicative ofrate of endocytosis. While it was slow in the pmp3Δ strain, itwas restored to wild-type level upon SUR7 overexpression.

Figure 2
figure 2

Slow rate of endocytosis of pmp3Δ strain is restored to normallevel by overexpression of ScSUR7.

(a) Wild type strain 3818 (SEY6210-Mup1pHluorin) and pmp3Δstrain (3818 pmp3Δ::HIS3) transformed with eithervector or ScSUR7, were grown without methionine to promoteaccumulation of Mup1-pHluorin in the plasma membrane. After addition of 20μg/ml methionine, random fields of cells were imaged atdifferent time intervals. All images were obtained at identical exposureconditions. (b) After addition of methionine, Mup1-pHluorin fluorescence wasmeasured at indicated time intervals in a flow cytometer, as described inMethods. The values shown are average of two replicates from onerepresentative experiment. Experiments were repeated thrice with comparableresults.

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Actin cytoskeleton plays a central role in endocytosis25 andrvs161Δ and rvs167Δ strains impaired inendocytosis also have actin polarization defects23. Moreover, asPMP3 interacts with genes having role in actin cytoskeleton (Table 2), we visualized actin in PMP3 strains. Thepmp3Δ strain showed pronounced defect in actin polarity,which is suppressed by overexpression of SUR7 (Fig.3 and Fig. S6). SUR7 also suppressed thesensitivity of pmp3Δ, rvs161Δ andrvs167Δ strains to NaCl (Fig. 4a). However,it could not reverse the sensitivity of these strains to AmB (Fig. 4b), demonstrating that AmB sensitivity of these mutants is notmediated by defects in actin polarity, endocytosis or NaCl tolerance.

Figure 3
figure 3

Actin polarization defect of pmp3Δ strain is suppressed bymulticopy SUR7 overexpression.

Cells were grown to log phase and actin was visualized by rhodaminephalloidin staining. About 200 cells with small buds were scored accordingto their polarization state. Cells with actin patches concentrated in thesmall bud, with fewer than four patches in the mother cell, were classifiedas polarized cells. Other cells with more actin patches in the mother cellthan in the small bud were classified as depolarized cells. Representativeimages are shown in Figure S6. Mean values of two independent experimentsare given. The error bars indicate the range.

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Figure 4
figure 4

SUR7 overexpression can suppress salt sensitivity (a), but not AmBsensitivity (b) of strains deleted in PMP3, RVS161 or RVS167.Wild-type (BY4741) and PMP3 overexpression strains are included ascontrols.

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Sphingolipid biosynthetic pathway is essential for PMP3 mediatedincrease in AmB resistance

We had recently shown that sphingolipid biosynthetic pathway genes FEN1(ELO2) and SUR4 (ELO3) modulate AmB resistance12. While inhibition of sphingolipid biosynthesis with myriocinsensitized cells to AmB, addition of phytosphingosine, a sphingolipid pathwayintermediate, reversed this phenotype12. To check the importanceof this pathway for PMP3 mediated increase in AmB resistance, multicopyScPMP3 was transformed into a few sphingolipid pathway mutants andthe resistance was checked (Fig. 5a). In the wild-typeparent strain (BY4741) ScPMP3 could increase AmB resistance at least by4-fold. However, it increased AmB resistance by 2-fold or less in mutants ofsphingolipid biosynthetic genes FEN1 and SUR4 and regulatorygenes YPK132,33 and SAC134. IfPMP3 overexpression effect is independent of sphingolipid pathway,then fold-increase in AmB resistance by PMP3 in these mutants should havebeen comparable to that of the parent strain. Only 2-fold or less increase inresistance shows that PMP3 is dependent on this pathway for enhancing AmBresistance. Even this increase appears to be due to genetic redundancy.FEN1 and SUR4 are involved in fatty acid elongation and canpartly compensate for each other's loss, since double deletion islethal35. YPK1 and YPK2 are syntheticlethal36 and arose from the whole genome duplication37. Sac1p is a phosphatidylinositol phosphate phosphatase and itscatalytic domain (Sac1-like domain) is seen among several phosphatases withpartially overlapping function38. Sac1p is known to modulatesphingolipid metabolism34,39. Physical interaction of Pmp3p andSac1p has also been reported in a large-scale study40. Thus itappears likely that Pmp3p modulates sphingolipid biosynthesis and AmB resistanceby interacting with Sac1p. Dependence of Pmp3p on Sac1p provides possible linkbetween Pmp3p and sphingolipid pathway.

Figure 5
figure 5

PMP3 modulates AmB resistance through sphingolipid biosyntheticpathway.

(a) Sphingolipid biosynthetic pathway genes FEN1and SUR4 and regulatory genes YPK1 and SAC1 areimportant for PMP3 mediated increase in AmB resistance. Wild-type(BY4741) and pmp3Δ strains overexpressing ScPMP3 serveas positive controls. (b) PMP3 modulates tolerance to myriocin, asphingolipid biosynthetic pathway inhibitor. While strains overexpressingPMP3 are about 4-fold more tolerant, the strain deleted inPMP3 is about 2-fold more sensitive to myriocin, compared to thewild-type strain BY4741.

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Myriocin inhibits the first committed step of sphingolipid biosynthesis catalyzedby serine palmitoyltransferase33. Sphingolipid pathway regulatorygenes YPK132,33 and SAC134 modulatemyriocin resistance. To test if PMP3 also regulates sphingolipid pathway,we checked myriocin resistance of deletion and overexpression strains. Whiledeletion of PMP3 decreased myriocin resistance by 2-fold, itsoverexpression increased myriocin resistance by 4-fold, both with respect toparent strain (Fig. 5b), indicating that PMP3 ispossibly involved in regulation of this pathway in S. cerevisiae. We alsochecked the myriocin sensitivity of C. glabrata strain deleted inPMP3 ortholog and C. albicans strains deleted in PMP3ortholog or best hit. However, the sensitivity of these strains was found to becomparable to that of their respective parent strains (Fig.S7). Another approach used to establish the role or dependence ofgenes on sphingolipid pathway is by supplementing with phytosphingosine (PHS), asphingolipid pathway intermediate33,41. Addition of PHSincreased the AmB resistance of pmp3Δ strain of S.cerevisiae to wild type level. It also decreased the AmB resistance ofPMP3 overexpression strain to nearly wild type level (Fig. 6a), perhaps by its known antifungal activity at highconcentration42. PHS also suppressed AmB sensitivity of C.glabrata and C. albicans strains deleted in PMP3 orthologs(Figs. 6b and 6c). These resultsfurther establish that PMP3 modulates AmB resistance through sphingolipidpathway in S. cerevisiae as well as in pathogenic Candidaspecies.

Figure 6
figure 6

Phytosphingosine (PHS), a sphingolipid pathway intermediate, modulates AmBresistance.

(a) Growth of wild-type (BY4741), PMP3 deletion and overexpressionstrains of S. cerevisiae on indicated concentrations of AmB alone orin combination with 5 μM phytosphingosine (PHS). Relative growthof strains at 0.8 μg/ml AmB (not shown) was comparable to theirgrowth at 1.6 μg/ml. (b) Growth of wild-type (CG462) andPMP3 delete (Cgpmp3Δ) strains of C.glabrata on indicated concentrations of AmB alone or in combinationwith 5 μM PHS. (c) Growth of C. albicans strains deletedin both alleles of PMP3 ortholog (Capmp3-OΔ/Δ)or PMP3 best hit (Capmp3-BΔ/Δ), with respectto their parent SN95 on indicated concentrations of AmB alone or incombination with 10 μM PHS.

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Sphingolipid bases and complex sphingolipids have multiple roles in cells, bothas structural components and as signalling molecules43,44.Mutants of sphingolipid pathway show pleiotropic phenotypes44, ofwhich those affected in actin cytoskeleton45, endocytosis46 and AmB resistance12 are pertinent here. Sinceactin is critical for endocytosis25, defective endocytosis couldbe a consequence of impaired actin polarity. Thus, impaired actin cytoskeletonand slow rate of endocytosis of pmp3Δ strain are consistent withthe regulatory role played by PMP3 in sphingolipid pathway.

In conclusion, we have shown that a few striking phenotypes of PMP3mutant, such as impaired actin polarity, endocytosis and salt tolerance are notrelated to its AmB-sensitivity. Rather, we show that modulation of AmBresistance by PMP3 is dependent on sphingolipid biosynthetic pathway,since AmB sensitivity of PMP3 deletants is suppressed byphytosphingosine, a sphingolipid pathway intermediate. Moreover, enhanced AmBresistance conferred by overexpression of PMP3 is dependent on functionalsphingolipid biosynthetic and regulatory genes. Efforts are underway toelucidate the precise mechanism underlying PMP3 effect or dependence onsphingolipid pathway for modulating AmB resistance.

Methods

Fine chemicals and yeast synthetic drop-out medium supplements without uracil wereprocured from Sigma. All other media components were obtained from BD (Difco).Oligonucleotides were custom synthesised from Sigma-Genosys, India. Restrictionenzymes, DNA polymerases and other DNA modifying enzymes were obtained from NewEngland Biolabs and DNA purification kits were obtained from Qiagen.

Strains, media and growth conditions

S. cerevisiae and Candida strains and plasmids used in this studyare listed in Table S1 and S2. The Escherichia coli strainDH5α was used as a cloning host. YPD and Synthetic complete (SC)media were prepared and used as described12. Uracil supplement isomitted in SC medium to provide SC-ura medium. Yeast transformations werecarried out using the modified lithium acetate method47. Stocksolutions of AmB (2 mg/ml), myriocin (5 mM), phytosphingosine (15 mM) andradicicol (5 mM) were prepared in DMSO. Stock solutions of nourseothricin (200mg/ml) and tert-butyl hydroperoxide (500 mM) were made in water.

Growth assays by dilution spotting

For dilution spotting assays, the strains/transformants were grown overnight inSC or SC-ura medium, reinoculated in fresh medium to an A600of 0.1 and grown for 6 h. The exponential phase cells were harvested, washed andresuspended in sterile water to an A600 of 1.0 (~2× 107 cells/ml). Ten-fold serial dilutions were madein water and 5 μl of each dilution was spotted on SC or SC-uraplates with desired concentration of compounds, as mentioned in Figures. DMSOalone was included in control plates, corresponding to its concentration inexperimental plates, where appropriate. Plates were incubated for 2 days at30°C before taking photographs. These experiments were repeated atleast three times with comparable results.

Cloning methods

The ORFs of putative homologs of ScPMP3 in C. albicans[CaPMP3-ortholog (orf19.1655.3), CaPMP3-Best hit(orf19.2959.1)] and C. glabrata (CAGL0M08552g) werePCR amplified from the genomic DNA of C. albicans and C. glabratawith specific primers sets (Table S3). The PCR products were then used toreplace the ScPMP3 ORF in a ScPMP3 clone in multicopy vectorpFL44L, using Circular Polymerase Extension Cloning (CPEC) method48,49, thereby retaining the ScPMP3 promoter and terminatorregions for all PMP3 orthologs as well. For cloning ScSUR7 gene,the SUR7 ORF of S. cerevisiae along with its promoter andterminator (+568 to −326 bp) was amplified from strain BY4741 withforward primer ScSUR7-OCS1 and reverse primer ScSUR7-OCA1 (Table S3) and clonedin pFL44L by CPEC method48,49.

Construction of C. albicans strains deleted inCaPMP3-ortholog and CaPMP3-best hit

Both alleles of CaPMP3-ortholog (orf19.1655.3) and CaPMP3-Best hit(orf19.2959.1) were deleted in C. albicans, using HAH2 cassetteand gene-specific primers, as described12 and confirmed bydiagnostic PCR with appropriate primers (Table S3).

Construction of C. glabrata strain deleted inCgPMP3

PMP3 ortholog in C. glabrata (CAGL0M08552g) was deletedusing a selection cassette conferring nourseothricin resistance containingCaNAT1 gene with codon usage adapted for Candida species50. A 508 bp region upstream of and 472 bp region downstream ofCgPMP3 ORF were PCR amplified from wild type genomic DNA usingprimers for upstream (CgPMP3-US1 and CgPMP3-UA1) and downstream regions(CgPMP3-DS1 and CgPMP3-DA1). The upstream flanking region was fused with the5′ region of CaNAT1 cassette using amplified upstream regionand plasmid (pCR2.1-NAT51) with CaNAT1 as templates andprimers CgPMP3-US1 and CaNAT1-US-R1 to generate upstream split marker.Similarly, the downstream flanking region was fused to 3′ region ofCaNAT1 cassette with amplified downstream region and pCR2.1-NAT astemplates and primers CaNAT1-DS-F1 and CgPMP3-DA1 to generate downstream splitmarker. These fusion products, which share 401 bp homology between them in thecassette, were mixed together, transformed52 into C.glabrata wild type strain CG462 and plated on YPD plate. Afterincubation at 30°C for 24 h, cells were replica-plated onto YPDplate with 200 μg/ml nourseothricin and further incubated for 24 h.Nourseothricin resistant colonies were purified and checked for gene deletion bydiagnostic PCR using cassette specific primers and primers outside the flankingregion of homology (Table S3).

Fluorescence microscopy

Mup1-pHluorin internalization assay was performed as reported31,53. Mup1- pHluorin localization was visualized using a Nikon A1R confocalmicroscope using FITC optics and 100X oil immersion objective. Images wereanalysed using NIS Elements software. Visualization of actin by rhodaminephalloidin staining was carried out as described54. Calcofluorstaining of cell wall was done as described55. The subcellularlocalization of sterols was monitored by staining with filipin as described19 with slight modification. Exponentially growing cells (0.5 ODcells/ml) were fixed with 3.7% paraformaldehyde for 10 min at 30°C,washed with phosphate-buffered saline (PBS) and incubated with 5μg/ml of filipin (Sigma F9765) in the dark at 30°C for 5min. The stained cells were directly observed under a confocal laser scanningmicroscope (Nikon A1R) using 405 nm laser and images were analysed using NISelement software.

Flow cytometry

Log-phase cells were grown in SC medium without uracil and methionine for 6hours and then methionine was added to 20 μg/ml finalconcentration. At different time intervals cells were collected bycentrifugation, washed and resuspended in PBS. Mup1-pHluorin fluorescence wasmeasured with BD Accuri™ C6 flow cytometer in FL1 channel.Excitation and emission wavelengths were 488 nm and 530 nm, respectively. Foreach sample 104 cells were analysed. Three independentexperiments were done with two replicates each time.