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Originally published In Press as doi:10.1074/jbc.M510746200 on January 16, 2006

J. Biol. Chem., Vol. 281, Issue 11, 7030-7039, March 17, 2006
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Candida albicans Spt23p Controls the Expression of the Ole1p {Delta}9 Fatty Acid Desaturase and Regulates Unsaturated Fatty Acid Biosynthesis*

Chan-Seok Oh and Charles E. Martin1

From the Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey 08854-8082

Received for publication, October 3, 2005 , and in revised form, January 6, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In Saccharomyces cerevisiae the endoplasmic reticulum membrane proteins scSpt23p and scMga2p control the formation of unsaturated fatty acids by a mechanism that involves their release from the membrane by ubiquitin-mediated proteolysis. The resulting soluble polypeptides act as transcription activators that specifically control the expression of scOLE1, a gene that encodes scOle1p, a {Delta}9 fatty acid desaturase that forms cis-monounsaturated fatty acids (9Z-16:1 and 9Z-18:1) from saturated fatty acyl-CoA precursors. ScOle1p is the only long chain fatty acid desaturase in Saccharomyces and its membrane and storage lipids contain only saturated fatty acids and the monounsaturated products of that enzyme. Most other fungi, however, express multiple endoplasmic reticulum desaturases, including enzymes that form both mono- and polyunsaturated fatty acids. These typically include {Delta}12 and {Delta}15 enzymes that form the polyunsaturated species, 9Z,12Z-18:2, and 9Z,12Z,15Z-18:3, which are the most abundant fatty acids in membrane and storage lipids. An analysis of genomic DNA sequences shows that Candida albicans has a single homologue of the Saccharomyces scSPT23 and scMGA2 genes that we designate here as caSPT23. This study describes the characterization of the caSPT23 gene product and shows that it can repair the unsaturated fatty acid auxotrophy when it is expressed in a Saccharomyces scspt23{Delta};scmga2{Delta} strain. In addition we show caSPT23 is essential for the expression of one of the two {Delta}9 desaturase homologues in Candida and potentially other functions associated with fatty acid metabolism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In fungi, unsaturated fatty acids are synthesized by endoplasmic reticulum membrane-bound fatty acid desaturases that form double bonds in the acyl chains of either fatty acyl-CoAs (1) or glycerolipids (2, 3). In the yeast Saccharomyces cerevisiae, which only forms 9Z monounsaturated fatty acids, the only fatty acid desaturase is encoded by the scOLE12 gene that is under the regulatory control of a pair of homologous endoplasmic reticulum proteins, scSpt23p and scMga2p (4–6). In this system, the N-terminal fragments of scSpt23p and scMga2p control the expression of scOLE1 as transcription co-activators following their release from the endoplasmic reticulum by a mechanism that involves the ubiquitination of the membrane-bound precursor followed by a selective proteolytic cleavage by the 26 S proteosome (7). The activation and regulation of scOLE1 expression appears to be the only essential function of scSpt23p and scMga2p and disruption of both genes produces a synthetic fatty acid auxotrophy that can be repaired by including cis-unsaturated acids in the growth medium or by expression of the scOle1p desaturase gene under the control of a heterologous promoter (4).

Most fungi, however, including the opportunistic pathogen Candida albicans, express multiple fatty acid desaturases including {Delta}9, {Delta}12, and {Delta}15 enzymes that can sequentially form the monounsaturated species 9Z-16:1 and 9Z-18:1, and the polyunsaturated species 9Z,12Z-18:2 and 9Z,12Z,15Z-18:3 (8). In some fungi {Delta}5, {Delta}6, and {omega}-3 desaturases have also been identified that are used in the formation of {gamma}-linolenic acid 6Z,9Z,12Z-18:3, docosatetraenoic (arachidonic) acid 5Z,8Z,11Z,14Z-20:4, and eicosapentaenoic acid 5Z,8Z,11Z,14Z,17Z-20:5 (9–11).

An analysis of the Candida albicans genome identified only one homologue of the Saccharomyces scSPT23/scMGA2 genes. Given the highly specific and essential regulatory role of the scSpt23p/scMga2p proteins in regulating unsaturated fatty acid formation, we examined the hypothesis that caSpt23p exerts a similar control over the expression of the {Delta}9 enzyme and possibly other desaturases. This study shows that when caSpt23p is expressed in Saccharomyces it can repair the loss of scOLE1 transcription in an scspt23{Delta};scmga2{Delta} strain. We further show that repression of caSPT23 in Candida albicans blocks the expression of the caOLE1 desaturase, producing growth defects and loss of viability that can be repaired by a mixture of unsaturated fatty acids. Metabolic labeling studies also indicate that, under conditions that repress caSpt23, the synthesis of both monoenoic and dienoic acids are repressed, suggesting that it plays a fundamental role in the regulation of unsaturated fatty acid metabolism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Growth Media—C. albicans and S. cerevisiae strains and plasmids used in this study are shown in Tables 1 and 2. Saccharomyces strains were grown at 30 °C on synthetic dextrose) or SG (synthetic galactose) drop out medium as previously described (12). Candida yeast phase cells were also grown at 30 °C in synthetic dextrose or synthetic maltose drop-out medium with the addition of 80 µM uridine (13). In experiments employing fatty acid supplements, the growth medium used in liquid cultures contained 0.1% Tergitol/Nonidet P-40 (Sigma) to disperse the fatty acids. Tergitol/Nonidet P-40 is not derived from fatty acids or fatty alcohols and is apparently not metabolized by Candida. Fatty acids were obtained from NuChek Prep (Elysian, MN). Escherichia coli NovaBlue Singles® competent cells were obtained from Novagen. Growth tests were performed by monitoring A600 of the cultures or by hemocytometer counting. For viability assays, cells were subjected to brief sonication (2 x 3 s) to disperse clumped cells before plating on the appropriate agar medium.


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  		TABLE 1
Strains used in this study

 


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  		TABLE 2
Plasmids used in this study

 
Cloning, Disruption, and Modifications of caSPT23—The construction of Candida BWP17 strains in which the caSPT23 was placed under the control of the caMAL2 promoter was done in a two-step procedure as shown in Fig. 1. First, one of the caSPT23 alleles was disrupted using the linear DNA fragments containing the caHIS1 gene flanked by caSPT23 upstream and downstream gene sequences. A linear DNA cassette containing the C. albicans caARG4 gene and sequences derived from the promoter of the caMAL2 gene was then inserted before the start codon of the caSPT23 gene on the homologous chromosome. All DNA fragments used for the gene disruption and modification procedures were constructed by PCR methods from the native caSPT23 gene sequences using the oligonucleotide primers shown in Table 3. The native caSPT23 gene used in these manipulations was isolated from C. albicans strain BWP17 by PCR cloning of genomic DNA. Strain BWP17 was transformed with the disrupted or modified forms of DNA fragments encoding the caSPT23 gene using the BD Biosciences Yeastmaker® Transformation System 2 method.


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  		TABLE 3
PCR primers used in this study

 
Construction of caspt23{Delta}HIS1 Cassette—The caSPT23 gene was isolated using the PCR primer pair SPT-BamHI-ATG and SPT-R2, which amplified a fragment containing the open reading frame and 538 bp of 3'-untranslated region (UTR) of the chromosomal gene. The amplified fragments were ligated into vector pSTBlue-1 (Novagen) yielding pSTBlue-SPT23. The caHIS1 gene was amplified from plasmid pGEM-HIS1 (14) with primer pair SPT-5DR and SPT-3DR, which contained 79 bp of 5' and 3' flanking sequence of the SPT23 open reading frame. The amplified fragments were cloned into vector pBluescript II SK(+) (Stratagene) to create pBlue-spt23{Delta}HIS1. To make the final pSTBlue-caspt23{Delta}HIS1 mod plasmid (psH), an EcoNI-NotI fragment of pSTBlue-SPT23 was subcloned into EcoNI/NotI cut pBlue-spt23{Delta}HIS1. The caspt23{Delta}HIS1 disruption cassette was liberated from the plasmid, psH, by digestion with HindIII and ScaI and transformed into BWP17 to make BWP17-spt23{Delta}::HIS1 (s{Delta}H).

Construction of the caARG4/caMAL2p/SPT23 Cassette—The 5'-UTR of caSPT23 was amplified with PCR primer pair S1-F and BamHI-SPT-R, which amplifies a 1040-bp fragment of the 5'-UTR of the chromosomal gene. Amplified fragments were cloned into vector pSTBlue-1 to create pSTBlue-SPT23(5'UTR). A BamHI fragment of this plasmid was inserted into BamHI cut and dephosphorylated plasmid pFA-ARG4-MAL2p (15) to create pFA-SPT23(5'UTR)-ARG4-MAL2p (pSAM). The SPT23/ARG4/MAL2p/caSPT23 cassette was amplified with PCR primer pair S1-F and S2-Prom MAL2. The PCR product was transformed into s{Delta}H to make BWP17-caspt23{Delta}::HIS1;ARG4/MAL2p/SPT23 (s{Delta}H-M2pS).

Construction of the ARG4/MAL2p/3xHA/SPT23 Cassette—A double-stranded oligonucleotide containing the triple HA epitope tag primers, SpeI-HA-F and SpeI-HA-R, was ligated into the dephosphorylated SpeI site of plasmid pSAM to make pFA-SPT23(5'UTR)-ARG4-MAL2p-3xHA (pSAMHA). The SPT23/ARG4/MAL2p/3xHA/SPT23 cassette was then amplified with the PCR primer pair S1-F and HA-SPT-R and the resulting PCR product was transformed into Candida strain s{Delta}H to make strain BWP17-spt23{Delta}::HIS1;ARG4/MAL2p/3xHA/SPT23 (s{Delta}H-M2pHAS).

Heterologous Expression of the C. albicans caSPT23 Gene in Saccharomyces—The protein coding sequences of the caSPT23 gene were subcloned into a BamHI-XhoI site of vector pESC-TRP (Stratagene) to make vector pEGS (pESC-GAL1-caSPT23-TRP1). This plasmid, which contains the caSPT23 gene under control of the scGAL1 promoter, was transformed into S. cerevisiae strain m{Delta}s{Delta} (mga2::kanMAX4;spt23::kanMAX4), which contains disrupted alleles of the scSPT23 and scMGA2 genes. Expression assays of this strain were performed on tryptophan dropout synthetic glucose agar medium with 1% Tergitol/Nonidet P-40 with no fatty acids or 0.5 mM 16:1 + 0.5 mM 18:1.

Preparation of Radiolabeled Probes—The entire open reading frames of the caOLE1, caOLE2, and caACT1 genes were isolated using PCR primer pairs (Table 3) and cloned into plasmid pSTBlue-1 to make plasmids pSTBlue-OLE1, pSTBlue-OLE2, and pSTBlue-ACT1 (Table 2). The resulting plasmids were opened with restriction enzymes, NcoI (for pSTBlue-OLE1), ScaI (for pSTBlue-OLE2), and NdeI (for pSTBlue-ACT1), and the linearized plasmid DNAs were separated by agarose gel electrophoresis in 1x Tris acetate-EDTA buffer and purified by the GENECLEAN® Kit (Q-Biogene). Radiolabeled RNA probes from the linearized plasmids were synthesized in vitro with [{alpha}-32P]dUTP (PerkinElmer Life Sciences) by SP6 polymerase using the Strip-EZ® RNA kit from Ambion. Unincorporated nucleotides were removed from the samples using a MicroSpin® G-25 column (GE Healthcare). The specific activities of the labeled probes were determined by liquid scintillation counting.


Figure 1
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  		FIGURE 1.
Disruption of caSPT23 allele 1 and integration of caMAL2 promoter elements into allele 2 of Candida strain BWP17. The diagram shows the relevant PCR primers and integration cassettes used to construct strains that express the MAL2 promoter-regulated native or an HA epitope-tagged form of caSPT23. A, relevant chromosomal loci for the MAL2-regulated native allele of caSPT23. B, relevant loci for the MAL2-regulated 3x HA epitope-tagged form of caSPT23.

 
beta-Galactosidase Assays—beta-Galactosidase reporter gene assays were performed as previously described (16) with the modification that cells were disrupted prior to the assay by three cycles of freezing and thawing at –80 and 30 °C.

RNA Isolation and Northern Blot Analysis—The Candida strain, s{Delta}H-M2pS, was cultured in His/Arg dropout synthetic dextrose medium at 30 °C overnight and the cells were washed with and transferred to His/Arg dropout synthetic maltose medium for induction of the caSPT23 gene. The cultures were incubated at 30 °C with shaking and harvested by centrifugation. The cell pellet was washed with cold water twice and 2 volumes of RNAlater® (Ambion) was added to the pellet prior to freezing at –80 °C. Total RNA was isolated using the RiboPure® YeastKit from Ambion. The amount of RNA was measured in a 96-well microplate with the Quanti-iT® RNA Assay Kit (Molecular Probes) and the fluorescence signal was quantified by fluorescence imaging with a STORM 860 (GE Healthcare) using the ImageQuant program (Amersham Biosciences). For Northern blot analysis equal amounts of heat-denatured total RNA were fractionated on 1% formaldehyde gels (17) and the separated RNA was transferred to Hybond®-N+ membrane (GE Healthcare) in 10x SSC for 90 min at 7 inches of mercury using a Vacuum Blotter (model 785), according to instructions from the manufacturer. The membrane was pre-hybridized at 65 °C for 2 h in 1 mM EDTA, 0.25 M Na2HPO4, pH 7.2, and 7% SDS and hybridized at 65 °C overnight with 1 x 106 cpm/ml of radiolabeled RNA probe. After incubation the membrane was washed twice at 65 °C for 30 min with 1 mM EDTA, 40 mM Na2HPO4, pH 7.2, and 5% SDS. This was followed by two 30-min wash cycles at 65 °C with 1 mM EDTA, 40 mM Na2HPO4, pH 7.2, and 1% SDS. Northern blot radioactivity was quantified by phosphorimaging.

Protein Isolation and Western Blot Analysis—Candida strain s{Delta}H-M2pS was grown at 30 °C in supplemented minimal media to a 1 x 107 cells/ml and lysis of cells for immunoblotting was performed as described (18). Cells were resuspended in 300 µl of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride) + protease inhibitor mixture (Sigma)) and disrupted with 300 µl of acid-washed glass beads (Sigma) in a BioSpec bead beater at full speed 3 times for 1 min. Cells were allowed to cool on ice for 1 min between cycles to avoid overheating the sample. Glass beads were removed from the total lysate by centrifugation for 10 s and the supernatant was mixed with 2x urea sample buffer (8 M urea, 4% SDS, 10% beta-mercaptoethanol, 0.125 M Tris, pH 6.8) with incubation at 65 °C for 10 min. The liquid lysate was then clarified by centrifugation for 5 min. The amount of protein in the final lysate was quantified with the Quanti-iT Protein Assay Kit (Molecular Probes).

Proteins were fractionated by SDS-PAGE using a Mini-PROTEAN® 3 electrophoresis cell (Bio-Rad) as described (19). Equal amounts of heat-denatured total lysate in urea sample buffer were applied onto 8% SDS-PAGE gels and separated by electrophoresis at a constant 100 V for 1.2 h or until the bromphenol blue dye was just passed through the gel. Proteins were then electroblotted to a Hybond-P polyvinylidene difluoride membrane. The blotted membrane was placed in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) with 5% ECL® Blocking Agent (GE Healthcare) for 1 h at room temperature. Blots were incubated with either a 1/10,000 dilution of monoclonal anti-HA antibody (Sigma) or a 1/500 dilution of monoclonal anti-{alpha}-tubulin antibody (Sigma) in TBST (with 5% ECL Blocking Agent) at 4 °C overnight or for 2 h at room temperature. The membrane was then washed with TBST and reacted with a 1/10,000 dilution of anti-mouse IgG horseradish peroxidase conjugate (Promega) in TBST (with 5% ECL Blocking Agent) for 1 h at room temperature. The secondary antibody-treated membrane was washed with TBST and developed with ECL Plus Western blotting detection reagents (GE Healthcare). The chemifluorescence signal was detected by a STORM 860 imager (GE Healthcare) and quantified with the ImageQuant program (Amersham Biosciences).

Fatty Acid Analysis and Labeling Studies—Fatty acid composition of C. albicans cultures were determined by HCl methanolysis of cells harvested by centrifugation and washed two times with ice-cold distilled water as previously described for Saccharomyces cultures (20). The resulting fatty acid methyl esters were analyzed by gas chromatography as previously described (21). For fatty acid radiolabeling studies, overnight cultures of strain s{Delta}H-M2pS were harvested by centrifugation, washed with an equal volume of synthetic medium with no carbon source, and inoculated at a density of 2.5 x 106 in 15 ml of synthetic maltose medium or 5 x 106/ml in 15 ml of SG medium. After incubation at 30 °C for 4 h, 10 µCi of 1-14C-labeled 16:0 or 18:1 were added to each culture flask. After a 4-h incubation, cells were harvested by centrifugation and washed two times in cold 0.7% NaCl. Washed cell pellets were then frozen at –80 °C overnight. Cell pellets were transferred to Tefloncapped glass tubes in 2 x 1.0-ml aliquots of methanol and the lipids were extracted by the Bligh-Dyer procedure as previously described (22). The lipid fraction was washed two times according to the Folch procedure (23), evaporated under nitrogen, and transesterified by HCl methanolysis as previously described (20). Fatty acid methyl ester fractions were concentrated under nitrogen prior to thin layer chromatography analysis. Argentation TLC analysis was performed using a 70:30 (v/v) hexane:ethyl ether solvent system on Silica Gel G plates presoaked in a solution of methanolic 4% AgNO3 in methanol:H2O (9:1, v/v) and activated for 30 min at 110 °C in the dark.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A comparison of the Candida caSPT23 gene with its Saccharomyces scSPT23 and scMGA2 homologues reveals significant conservation within their protein domains despite the divergence of these two species over 700 million years (Fig. 2A). Although the length of the Candida protein is about 75% of the Saccharomyces homologues, all three proteins show regions of high sequence identity in the N-terminal transcription activation domains, a highly homologous protein-protein interaction domain and near-perfect identities in regions that contain the predicted nuclear export signal and the transmembrane domains. These regions of high homology are located at identical positions on all three proteins (Fig. 2B). Unlike the Saccharomyces homologues, however, the Candida protein lacks the ankyrin repeat sequences located at a C-terminal position to the protein-protein interaction domain.

To test whether the Candida protein can activate scOLE1 transcription in Saccharomyces, a plasmid containing the caSpt23p protein coding sequence under the control of the yeast scGAL1 promoter was introduced into the scspt23{Delta};scmga2{Delta} strain. Fig. 3A shows that the expression of the Candida gene in medium containing galactose relieves the fatty acid auxotrophy of the strain, indicating that it can function in activating the expression of the scOLE1 gene. Fig. 3B shows that a beta-galactosidase reporter gene fused to the full-length Saccharomyces OLE1 promoter is activated in the Saccharomyces spt23{Delta};mga2{Delta} cells by caSpt23 and can be repressed ~4.5-fold by exogenous unsaturated fatty acids. This attenuation of reporter gene activity requires a longer time of exposure to unsaturated fatty acids than that observed in wild type cells containing functional copies of the native scSPT23 and scMGA2 genes.


Figure 2
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  		FIGURE 2.
Comparison of domain structure and homologous regions between the Saccharomyces Spt23p/Mga2p and Candida caSpt23p. A, scale diagram showing conserved domains between the Saccharomyces and Candida proteins. TXN, transcription activation region; I, protein interaction (IPT) domain (gray ovals); NE, putative nuclear export signal (rounded gray boxes); A, ankyryn repeat regions (striped rounded boxes); NL, nuclear localization signal (pentagons); TM, transmembrane domain (open boxes). Black filled boxes within the transcription activation region show the positions of regions with >50% identity between the Saccharomyces proteins. Boxes within the same region of the Candida protein represent regions with greater than 25% identities between the Candida and Saccharomyces proteins. B, homologous sequences within the protein interaction regions, predicted nuclear export regions, and transmembrane domains of the Candida and Saccharomyces proteins. Numbers preceding each sequence refer to the position of the first residue in the polypeptide chain.

 
Genetic and Biochemical Characterization of Strains Containing Glucose Repressible Forms of caSPT23—Because C. albicans is diploid, tests of the function of the gene requires the inactivation of both alleles. To examine the functions of the caSpt23 gene, we constructed integrating plasmids that placed the native and an N-terminal HA epitope-tagged form of its protein coding sequence under control of the maltose-inducible and glucose-repressible caMAL2 promoter. These were integrated into the remaining caSPT23 locus of a strain that contains a deletion of one of the caSPT23 alleles. The caMAL2 gene encodes a maltase that is the homologue of the Saccharomyces MAL62 gene. Previous studies have shown that the caMAL2 mRNA is undetectable by Northern blotting in C. albicans cells grown on glucose or glycerol, but is induced when cells are grown on maltose (24). Other studies have shown that a green fluorescent protein (25) and the caURA3 gene (26) are expressed when cells are grown on maltose but not on glucose when they are placed under the control of the caMAL2 promoter. Although it cannot be ruled out that a very low level of caMAL2-driven expression occurs in glucose-grown cells, studies of the caURA3 gene under control of caMAL2 promoter in cells grown under glucose is not sufficient to confer 5-fluoroorotic acid sensitivity on the strain, which is a sensitive indicator of weak basal gene activity (24).

Repression of caSPT23 Blocks Growth and Viability of Strains That Express Native and Epitope-tagged Forms of the Protein—Tests of strains that only express the caMAL2-controlled forms of either the wild type or the HA epitope-tagged caSPT23 genes show that after transfer from maltose to glucose medium there is a brief growth period and then cell division is strongly repressed. Fig. 4A shows the results of a representative set of experiments in which cells grown on maltose medium were washed and transferred to glucose containing medium at an inoculum of 2 x 106 cells/ml. The cells that were exposed to glucose grew at the same rate as those transferred to fresh maltose carbon source for ~4–6 h (corresponding to about 3.5 generations). At that time, growth of the glucose-repressed cells slows dramatically and progresses into a stationary phase. By 6 h, the glucose-treated cell population begins to exhibit significant clumping and the formation of elongated cells. By 10 h, very few cells with buds are observed and by 24 h a significant fraction of the clumped cells have formed pseudohyphal structures (Fig. 5, A–C). The timing of the initial growth phase, growth arrest, and pseudohyphal formation is consistent over a wide range of inoculum densities ranging from 2 x 104 to 5 x 106 cells/ml. Viability is also dramatically reduced in the glucose-repressed cultures, resulting in ~12% viable cells at 6 h, 5% at 10 h, and 3% after 24 h of growth (Fig. 4C). By contrast, cells containing either the native or HA-tagged genes that were maintained on maltose retained 60–90% viability during the same period and remained as yeast phase cells as they progressed into stationary phase during the 24-h test period.


Figure 3
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  		FIGURE 3.
A, repair of the unsaturated fatty acid requirement of a Saccharomyces spt23{Delta}; mga2{Delta} strain by caSpt23p. Growth of a Saccharomyces spt23{Delta};mga2{Delta} strain containing an empty vector (top), or vectors containing the Candida caSPT23 gene under the control of the Saccharomyces GAL1 promoter (bottom, left and right). Cells were grown for 6 days at 30 °C in synthetic glucose complete medium containing a mixture of unsaturated fatty acids (UFA, left panel) or no fatty acids (NFA, right panel). B, unsaturated fatty acid repression of the Saccharomyces OLE1 gene in cells that only express the Candida caSPT23 gene. Wild type (WT) (11A) and spt23{Delta};mga2{Delta};GAL1-caSPT23 cells containing beta-galactosidase reporter gene plasmid p62::–938 (16) were grown with and without a mixture of unsaturated fatty acids in galactose-containing medium. Cells were assayed for beta-galactosidase activity at the indicated times following transfer to medium containing no fatty acids or a 1 mM mixture of unsaturated fatty acids. Black bars, wild type, no fatty acids; open bars, wild type with 1 mM fatty acid mixture; gray bars, cells expressing the Candida gene with no fatty acids; striped bars, Candida gene with unsaturated fatty acids. Data represents average beta-galactosidase activities from three experiments using three independent transformants for each strain.

 
Glucose-repressed Growth, Loss of Viability, and Morphogenic Changes in the caMAL2::caSPT23 Strain Can Be Repaired by Combinations of Unsaturated Fatty Acids—To test the specificity of the caSPT23 on the expression of fatty acid biosynthesis and desaturase genes, we monitored the growth of cells expressing either the MAL2-regulated native or HA-tagged forms of caSpt23p subjected to glucose repression in the presence fatty acids. The effects of fatty acids on the growth of the strain that expresses the native form of caSpt23p are shown in Fig. 4B and the effects of fatty acids on the viability of that strain are shown in Fig. 4C. A mixture of saturated fatty acids did not have any effect on the inhibition of growth resulting from the glucose repression of caSPT23 (data not shown). Growth was not repressed in cells incubated in glucose media that contained either a mixture of saturated and unsaturated fatty acids or a mixture of unsaturated fatty acids. Furthermore, the addition of the unsaturated fatty acid mixture to the glucose medium prevented the loss of viability as well as the formation of pseudohyphae (Fig. 5C) that is characteristic of caMAL2::caSPT23 cells grown under glucose repressed conditions. An identical pattern of unsaturated fatty acid repair of growth, viability, and maintenance of the yeast phase was observed with cells that expressed the caMAL2-regulated HA-tagged form of caSPT23p (data not shown).

Glucose Inhibition of caMAL2::caSPT23 Represses the Synthesis of caSpt23p—Quantitative PCR measurements showed that glucose repression of the caMAL2-regulated transgene resulted in a 2.6-fold (standard deviation (S.D.) = 0.05, n = 3) reduction of the transcript by 6 h. The effects of this repression are also shown by a Western blot analysis of the HA-tagged protein under control of the caMAL2 promoter (Fig. 6). Like the Saccharomyces homologues, a high molecular mass form of the protein (corresponding to the 83-kDa membrane-bound precursor) and a lower molecular mass species (the proteolytically processed, soluble form) can be found in normally growing cells. Both forms are abundant in cells grown under derepressing conditions (in maltose medium) and semi-quantitative comparisons of protein levels using the tubulin gene as a control showed that the highest total levels of the caSpt23p protein were present immediately following the transfer of cells to fresh maltose medium. Under those conditions ~20% of caSpt23p was present as the 85-kDa membrane-bound precursor and 80% as the soluble, 55-kDa proteolytically cleaved form. During the 6 h of logarithmic phase growth, however, total caSpt23p levels declined to about 25% of their initial value and the levels of the membrane-bound and soluble forms of the protein were approximately equal.

By contrast, we were unable to detect the 85-kDa membrane-bound form of the protein in cells that were transferred to glucose containing medium during the course of the experiment. The absence of the membrane-bound caSpt23p in the glucose-repressed cells suggests that synthesis of the protein is strongly repressed and that the remaining membrane-bound precursor is rapidly converted to the soluble, 55-kDa form whose levels are also markedly reduced. We used the ratio of the 83- and 55-kDa forms of caSpt23p to {alpha}-tubulin to monitor the differences between the total caSpt23 protein levels between glucose- and maltose-derepressed cells at each time point. Two hours after the transfer to fresh medium the glucose-repressed cells had ~10–20% of the total caSpt23p found in the corresponding 2-h maltose-grown control cells and by 6 h the levels of the remaining caSpt23p had declined to 5–10% of the corresponding maltose grown control values. These data show that repression of caMAL2-caSPT23 causes a rapid reduction in the active form of the protein, which results in the cessation in growth and reduced viability.

Fatty Acid Composition of Cells following Growth under caSPT23 Derepressed and Repressed Conditions—To determine the effects of derepressed and repressed caSpt23p expression on fatty acid synthesis and desaturation, total cellular fatty acid levels were monitored during the 6-h period following the shift to maltose- or glucose-containing growth medium. Following overnight growth in maltose, the monounsaturated acid products of {Delta}9 desaturase activity (14:1, 16:1, and 18:1) in BWP17 (wild type) cells and those containing the caMAL2-regulated caSpt23p comprise ~28 and 35% of the total fatty acid mass, respectively (Table 4). Under the same conditions, monounsaturated acid levels in cells that expressed the caMAL2-regulated HA-tagged form of caSpt23 were consistently higher and comprised ~40% of the total fatty acid mass (Table 4). In all three strains, 18:1 was the most abundant of the monenoic acids (25–30%) followed by 16:1 (8%) and 14:1 (1–2%). The polyunsaturated species ({Delta}9,12–18:2 and {Delta}9,12,15–18:3) vary between 39 and 45%, and the saturated acids (14:0, 16:0, and 18:0) ranged between 19 and 20% of the total mass.


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  		TABLE 4
Comparison of fatty acid composition of strains that express caMAL2-regulated native and N-terminal HA epitope-tagged forms of caSPT23 following transfer to fresh maltose- or glucose-containing medium

Data expressed as wild type % total C14-C18 fatty acid methyl ester mass derived from total cellular lipids. Values are mean ± S.D. for three independent experiments for each strain (values in bold face type show significant deviations).

 
After a shift to either fresh maltose or glucose medium there is a rapid increase in saturated fatty acid levels. This apparently reflects a general increase in fatty acid synthesis relative to desaturation as the cells progress into logarithmic growth phase with either carbon source. Following glucose repression of caSPT23, however, there were highly significant changes in fatty acid levels involving the monounsaturated and polyunsaturated acids and the saturated species 18:0. Cells containing the MAL2-regulated native and HA-tagged forms of caSpt23p showed dramatic decreases in their monoenoic and dienoic fatty acid levels in the first 6 h of growth (Table 4). The largest decline involved the monoenoic acids, changing from 35 to 24.7% (native caSpt23p) and 40 to 27.9% (HA-tagged caSpt23p), starting 4 h after the shift. These were accompanied by large increases in the saturated fatty acids (19–34%) in cells containing the native caSpt23p, whereas, there were increases in both saturated and polyunsaturated species in the cells that contained the HA-tagged form of the protein. By contrast, in the BWP17 wild type cells that were exposed to glucose for 6 h there was a significant increase in monounsaturated fatty acid levels from 32 to 40% and the polyenoic acids decreased from 49 to 36% over cells maintained on maltose (data not shown).


Figure 4
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  		FIGURE 4.
Fatty acid-mediated repair of growth and viability of C. albicans strains following glucose repression of MAL2-regulated native and HA epitope-tagged caSPT23. A, comparison of growth on maltose and glucose between strains expressing the native and HA-tagged forms of caSpt23p at 30 °C. Figure 4, strain s{Delta}H-M2pS (native protein); {blacktriangleup}, strain s{Delta}H-M2pHAS (HA epitope-tagged protein). B, comparison of growth of strain s{Delta}H-M2pS (expressing the native protein) at 30 °C in fatty acid deficient (NFA) and 1 mM unsaturated fatty acid-supplemented medium. Filled symbols indicate growth on maltose, open symbols indicate growth on glucose. C, viability of cells expressing the native caSpt23p after transfer at 6, 10, and 24 h to either maltose medium or glucose medium with and without a 1 mM mixture of unsaturated fatty acids. To monitor the effects of cell density on the timing of the growth arrest, experiments represented in panels A and B were performed using a range of initial inocula from 2 x 104 to 5 x 106 cells/ml. Data and error bars in panels A and B show the range of duplicate experiments performed with a starting inocula of 2 x 106 cells/ml. Error bars in panel C represent S.D. for three independent experiments using an inoculum density of 2 x 106/ml.

 
Glucose Repression of MAL2::caSPT23 Reduces mRNA Levels of caOLE1; caOLE2 Expression Is Not Affected—A Northern blot analysis was performed to determine the effects of caSPT23 repression on {Delta}9 fatty acid desaturase mRNA levels (Fig. 7). C. albicans has two genes with homology to {Delta}9 desaturases. The caOLE1 gene is highly homologous to a number of fungal {Delta}9 enzymes and disruption of both alleles in Candida produces an unsaturated fatty acid requirement. Furthermore, it has been shown by heterologous expression in a S. cerevisiae ole1{Delta} strain to encode an authentic {Delta}9 fatty acid desaturase (27). The caOLE2 gene has less homology to fungal desaturases and does not produce an unsaturated fatty acid auxotrophy when disrupted in Candida or function as a desaturase when it is expressed in yeast (27). Quantitative Northern blot analysis using the Candida actin gene as a loading control showed that caOLE1 mRNA levels at 2, 4, and 6 h were <20% of the levels found in control cells grown in maltose at the same times. By contrast, the caOLE2 levels in the glucose-grown cells did not change significantly from the controls over the same period. These results were further confirmed by QPCR analysis of the two genes at the 6-h point using the Candida tubulin gene (TUB2) as an internal control. Those results showed that the caOLE1 mRNA levels had undergone a 5.6-fold decrease (S.D. = 0.03, n = 3), whereas the caOLE2 transcript levels did not show a statistically significant difference from the maltose-grown cells.


Figure 5
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  		FIGURE 5.
Morphology of caspt23{Delta};MAL2:: caSPT23 cells 24 h after transfer to medium containing: A, maltose; B, glucose + 1 mM unsaturated fatty acids; and C, glucose, no fatty acids.

 


Figure 6
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  		FIGURE 6.
Western blot of MAL2-regulated 3x HA epitope-tagged forms of caSpt23p in a caspt23{Delta};MAL2::3XHA-caSPT23 heterozygote at the indicated times following transfer to fresh maltose or glucose medium. Total cellular proteins were isolated by glass bead breakage and solubilized with sodium dodecyl sulfate prior to gel electrophoresis as described under "Experimental Procedures."

 
Glucose Repression of the caMAL2::caSPT23 Gene Results in the Repression of {Delta}9 and {Delta}12 Fatty Acid Desaturase Activity—To test the effects of repressed caSPT23 expression on fatty acid desaturase activities, cells grown in either maltose- or glucose-containing medium for 4 h were exposed to trace amounts (2.75 µmol/1.5 x 108 cells) of radiolabeled 16:0 and 18:1 and allowed to grow for an additional 4 h. Quantification of the radiolabel in the Bligh-Dyer lipid extracts showed that there were marked differences in the amounts of label that were incorporated from the two fatty acids under the different growth conditions. In the maltose-grown cultures, ~14 and 3% of the total radioactivity derived from 16:0 and 18:1, respectively, were incorporated into chloroform-soluble lipids, whereas in the glucose-grown cultures ~0.8% of both the 16:0 and 18:1 dpm were found in the lipid fraction.

To determine the relative amounts of conversion of the incorporated fatty acids by endogenous desaturase activities, the radiolabeled lipids were subjected to acid methanolysis and the resulting fatty acid methyl esters were analyzed by chromatography on AgNO3-impregnated thin layer plates (Fig. 8). In cells that were grown in maltose medium, ~63% of the incorporated radiolabeled 16:0 was desaturated by the {Delta}9 enzyme and ~2/3 of its products remained as monoenes (16:1 + 18:1, or 40.7% total incorporated dpm). The remaining labeled monenes were converted by a {Delta}12 activity to 18:2 (20% of the total incorporated dpm) and a fraction of that species was then desaturated by a {Delta}15 activity to 18:3 (2% total incorporated dpm). In comparison, sharply reduced levels of the {Delta}9 desaturase activity were observed with the glucose-repressed cells. Only 28% of the total incorporated 16:0 was desaturated. Twenty percent of the incorporated radiolabel was found in the monene fraction, 4.5% was found as 18:2 and 3.5% as 18:3. These data indicate that repression of caSpt23p results in a reduction in both {Delta}9 and {Delta}12 fatty acid desaturase activities.

The effects of Spt23p repression on the {Delta}12 desaturase activity were also supported by the labeling patterns produced from the 14C-labeled 18:1 species. In the maltose-grown cells ~70% of the incorporated 18:1 was converted by {Delta}12 desaturase activity to 18:2. Fifty-one percent of the total incorporated label remained as 18:2 and the rest of that species was converted by a {Delta}15 enzyme to 18:3, representing 19% of the total incorporated dpm. By contrast, in the glucose-repressed cells only 14% of the incorporated 18:1 was converted to 18:2 and <1% of the incorporated radiolabel was found in the 18:3 fraction.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
An analysis of the current genome data bases suggests that homologues of the Spt23p/Mga2p family are found in all fungi, but not in other organisms. This indicates that these genes emerged early in the history of the fungal kingdom, well before the divergence of Candida and Saccharomyces some 730 million years ago (28). When viewed with respect to this length of evolutionary time, the high regional sequence identities found in the Spt23p/Mga2p homologues of these two widely diverged species and the highly conserved order of domains within those proteins suggest that the SPT23/MGA2 family controls basic functions that are necessary for fungal growth and survival.

The data presented here shows that the Candida caSpt23p protein has retained a similar function to that of its Saccharomyces homologues. The loss of viability when caSPT23 is repressed indicates that it is an essential gene and the ability of unsaturated fatty acids to repair the resulting growth and viability phenotypes seems to indicate that its primary function in yeast phase cells is to control the formation of unsaturated fatty acids. Furthermore, the ability of the Candida protein to substitute for its Saccharomyces homologues in yeast indicates that there is a remarkable conservation in its mechanism of function with respect to its ability to activate scOLE1 transcription. We should note that although we have recently demonstrated that the Saccharomyces scMga2p homologue has a secondary function associated with the regulation of OLE1 mRNA stability (6), we currently do not have the reagents to determine whether the Candida protein has similar dual functions in its native host.

S. cerevisiae does not form polyunsaturated fatty acids and the scOle1p desaturase is the only enzyme responsible for the formation of long chain unsaturated fatty acids: its 9Z-(14–18):1 products comprise ~80% of the fatty acid chains found in membrane lipids. In Candida the {Delta}9 enzyme also plays a central role in unsaturated fatty acid formation because it catalyzes the initial step in the formation of unsaturated fatty acids. Unlike Saccharomyces, however, the majority of its monounsaturated products are converted to dienoic and trienoic acids by endogenous {Delta}12 and {Delta}15 desaturase activities to 18:2 and 18:3.


Figure 7
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  		FIGURE 7.
Northern blot of mRNA of OLE1, OLE2, and ACT1 in caspt23{Delta};MAL2::caSPT23 cells grown on maltose- or glucose-containing medium.

 
We had previously observed that the deletion of the Saccharomyces scOLE1 gene causes a fatty acid auxotrophy. When those cells are deprived of unsaturated fatty acids growth is arrested after 3.5 generations (6 h) and is accompanied by rapid cell death (29). During that period, the unsaturated fatty acyl content of membrane lipids is reduced from >80% to about 10% (29). In this study, we observed a similar pattern after the Candida caSPT23 gene is repressed. But, whereas growth arrest and loss of viability occurs in Candida over a similar time scale of 2.0–2.2 generations (6 h), there is a much smaller reduction (from 75 to about 73%) in the levels of total unsaturated fatty acids. During that period, however, there are significant rearrangements within the total cellular fatty acyl composition. Most notably, repression of the native form of caSpt23p for 6 h results in a 25% reduction of monoenoic (16:1 and 18:1) acids, a 20% reduction in dienoic (18:2) species, and a 50% increase in the trienoic (18:3) species.

Whereas statistically significant differences in total saturated (c14–18) fatty acid levels occur only at the 4-h point, within those species there are also highly significant (>50%) increases in 18:0 at 4 and 6 h after repression of caSpt23p. Taken together with the observed changes in unsaturated fatty acids, these results are consistent with the expected effects if caSpt23p exerts direct control over caOLE1 transcription. Repression of caSPT23 causes a rapid reduction in the caOle1p {Delta}9 desaturase activity, blocking further synthesis of 16:1 and 18:1 and resulting in an accumulation of 18:0 through continued activity of fatty acid synthase and the ELO1-specific (21) 16:0 -> 18:0 elongation pathway. In addition, the continuing conversion of a significant fraction of the pre-existing 18:1 to 18:2 and 18:3 by residual {Delta}12 and {Delta}15 enzyme activities results in the depletion of 18:1 and 18:2 and an accumulation of 18:3. Whereas these data offer strong support to the hypothesis that caSPT23 exerts direct control over the expression of the {Delta}9 desaturase, the radiolabeling studies presented here also suggest that it may also be involved in the regulation of the {Delta}12 enzyme.


Figure 8
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  		FIGURE 8.
Conversion of [14C]palmitate (16:0) or [14C]oleate (18:1) to unsaturated fatty acids in caspt23{Delta};MAL2::caSPT23 cells 4 h after transfer to either fresh maltose or glucose-containing growth medium. A, AgNO3 Silica Gel G thin-layer chromatography of total fatty acid methyl esters extracted from cells after 4 h incubation with the labeled fatty acids; B, relative conversion of radiolabeled 16:0 to mono-, di-, and trienoic fatty acids, and radiolabeled 18:1 to di- and trienoic species in maltose-grown (solid bars) and glucose-grown (open bars) cells. In the left panel, activities are compared with the {Delta}9 desaturase activity (% counts/min of incorporated label in 16:1, 18:1, 18:2, and 18:3) in maltose-grown cells. In the right panel activities are compared with the {Delta}12 desaturase activity (% count/min of incorporated label in 18:2 + 18:3).

 
It is not clear whether the growth and viability defects produced by the repression of caSpt23p is caused by the inhibition of functions that are associated with the reduction of monoenoic or dienenoic acids, the increased levels of 18:3 and/or 18:0 or changes in critical molecular species of membrane lipids that result from altered ratios of all or some of these fatty acyl species. The relatively small reduction in total unsaturated fatty acid content associated with the inhibition of growth in Candida compared with that required to block Saccharomyces growth is striking, however, and illustrates how these two organisms have diverged in their ability to adapt to changes in the fatty acyl content of their membrane lipids. These differences in the adaptive abilities of the two organisms undoubtedly reflect the different physiological and nutritional conditions they encounter.

Although C. albicans can grow over a wide range of temperature conditions that are known to affect unsaturated fatty acid biosynthesis and mobilization it is a commensal organism whose primary habitat consists of mammalian tissues that are maintained over a narrow range of temperatures. (An extensive study of the literature has failed to identify a C. albicans isolate from an environment other than a warm-blooded host.) Within the domain of host tissues, ranging from the intestine, to various mucosal surfaces and deep organ tissues, however, there are significant differences in the availability of lipid nutrients that might be exploited as a source of fatty acids. Candida also exists in a number of different morphogenic states (30, 31), including the budding yeast and pseudohyphal forms described here as well as the filamentous hyphal form associated with the promotion of pathogenesis. All of these conditions may require differential regulation of fatty acid synthetic and desaturation activities. Although this study suggests that the primary function of caSPT23 in the budding yeast form under strictly defined growth conditions is to regulate {Delta}9 desaturase gene expression, we cannot rule out that caSPT23 may exert control over other genes, including the {Delta}12 and {Delta}15 enzymes when it grows under different environmental or developmental conditions.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM45768 (to C. E. M.) and a grant from the Charles Johanna Busch Memorial Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Nelson Laboratories, 604 Allison Rd., Piscataway, NJ 08854-8082. Tel.: 732-445-1633; E-mail: martin{at}biology.rutgers.edu.

2 The abbreviations used are: scOLE1, S. cerevisiae gene encoding the {Delta}9 desaturase; sc, prefix designating a Saccharomyces cerevisiae gene or gene product; ca, prefix designating a Candida albicans gene or gene product; caOLE1, C. albicans gene encoding a {Delta}9 fatty acid desaturase; caOle1p, protein encoded by caOLE1; caOLE1, gene encoding a protein of unknown function with high homology to caOle1p; caSPT23, C. albicans gene encoding caSpt23p with homology to the S. cerevisiae proteins scSpt23p and scMga2p; scOle1p, protein encoded by scOLE1; HA, hemagglutinin; UTR, untranslated region. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Aaron Mitchell for C. albicans strain BWP17 and Dr. J. Wendland for the PCR module MAL2 plasmids used in this study.



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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
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