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Vol. 15, Issue 8, 3811-3828, August 2004
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* Heinrich-Heine-Universität, Funktionelle Genomforschung der Mikroorganismen, 40225 Düsseldorf, Germany;
Technische Universität München, Institut für Organische Chemie und Biochemie, 85747 Garching, Germany;
|| AG Anatomie und Zellbiologie der Charite, 13353 Berlin, Germany; and
¶ MICROMUN Private Institute for Microbiological Research GmbH, Biotechnikum Greifswald, 17489 Greifswald, Germany
Submitted September 18, 2003;
Revised May 10, 2004;
Accepted May 16, 2004
Monitoring Editor: Douglas Koshland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total. Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy. FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5'-formyltetrahydrofolate). Growth of the fol1 deletion strain on folinic acid–supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth. The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Appling, 1991). In eukaryotes the cytoplasmic and mitochondrial compartments each possess a parallel array of enzymes catalyzing the interconversion of folate coenzymes, which differ by the oxidation state of their one-carbon units (Appling, 1991).
Mammalian cells are unable to carry out de novo synthesis of folate. Therefore folate is considered a vitamin in these organisms, and the cells use a carrier-mediated active transport system for the uptake of folate. Folate deficiency causes massive incorporation of uracil into the DNA and chromosome breaks during the repair by uracil-DNA glycosylase and apyrimidinic endonuclease (Blount et al., 1997). Diets with low intake of folate are associated with increased risk of developing cancer (Ames et al., 1995; Duthie, 1999). In addition, it has been shown that up to 70% of neural tube defects can be prevented by folic acid supplementation in early pregnancy (Greene and Copp, 1997). Most microbial cells and plants must synthesize folates de novo because they lack the carrier-mediated active transport system of mammalian cells. Therefore early folate biosynthetic enzymes are attractive chemotherapeutic targets for the treatment of microbial diseases (Roland et al., 1979).
The molecular and biochemical characterization of folate biosynthetic pathways and the enzymes involved has been limited to a small number of species, in particular bacteria and plants (Talarico et al., 1992; Volpe et al., 1993; Cossins and Chen, 1997; for review see Rebeille, 1999). In most bacteria the single enzymatic activities are performed by monofunctional proteins, whereas in eukaryotes enzymatic steps are carried out by bi- or trifunctional proteins. In Saccharomyces cerevisiae the pathway leading to tetrahydrofolate (THF) has so far not been elucidated completely (Figure 1). THF synthesis starts with guanosine triphosphate, which is transformed to 7,8-dihydroneopterin triphospate by the GTP-cyclohydrolase I encoded by FOL2 (Nardese et al., 1996). The phosphatase(s) that presumably removes the phosphate residues has not been identified yet. PHO8, the only alkaline phosphatase identified in S. cerevisiae so far (Kaneko et al., 1987), is probably capable of catalyzing this reaction yielding 7,8 dihydroneopterin. We show in this work that the next three enzymes needed (dihydroneopterin aldolase [DHNA], 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase [HPPK], and dihydropteroate synthase [DHPS]) to convert 7,8 dihydroneopterin to 7,8-dihydropteroate are all carried out by a single protein, the Fol1 protein. Subsequently, dihydrofolate synthase (DHFS) encoded by FOL3 adds the first glutamate to 7,8-dihydropteroate, yielding 7,8-dihydrofolate (DHF; Cherest et al., 2000). Finally, DHF is reduced by dihydrofolate reductase (DHFR) encoded by the DFR1 gene to yield tetrahydrofolate (THF; Barclay et al., 1988; Figure 1). Although all genes encoding enzymes of the THF biosynthetic pathway are nuclear, it was unclear in which subcellular compartment(s) THF is produced. In yeast the biochemical analysis had only revealed that dihydrofolate reductase (DHFR), which catalyzes the last step in THF synthesis is present in both the cytoplasm and mitochondria (Zelikson and Luzzati, 1977).
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In many fungi nutrient deprivation or changes in the environment result in a distinct morphological differentiation. This dimorphic switch to filamentous growth is thought to be adaptive for nonmotile microorganisms because it facilitates foraging through the environment for scarce nutrients. This dimorphic switch is similar to morphological transitions observed for fungal pathogens such as Candida albicans and Ustilago maydis (for reviews see Lengeler et al., 2000; Sanchez-Martinez and Perez-Martin, 2001). When starved for nitrogen diploid strains of S. cerevisiae undergo a developmental transition from a single yeast cell form to filaments of elongated pseudohyphal cells (for reviews see Lengeler et al., 2000; Palecek et al., 2002). Pseudohyphal cells exhibit an altered morphology and budding pattern and can invade the growth substrate (Gimeno et al., 1992; Kron et al., 1994). In haploid S. cerevisiae cells nutrient limitation, specifically loss of fermentable sugars such as glucose causes a related process called haploid invasive growth (Roberts and Fink, 1994; Cullen and Sprague, 2000). Presence of short-chain alcohols (fusel oils; e.g., isoamyl and isobutyl alcohols) in liquid and solid media can also induce pseudohyphal-like growth in haploid cells (Dickinson, 1996), indicating that yeast may not only sense nutrient limitation but also metabolic byproducts to regulate differentiation (Lorenz et al., 2000a). Starch-degrading haploid and diploid S. cerevisiae cells are also able to switch from a yeast cell form to pseudohyphal forms in response to carbon limitation (Lambrechts et al., 1996).
Although little is known about the sensors and effectors of pseudohyphal differentiation, considerably more insight has been gained about the signal transduction pathways. The two best characterized pathways are the mating Cdc42p/Ste20p/Ste12p/Tec1p MAPK cascade, including several unique filamentation specific components, and the receptor/G protein/cAMP-PKA/Flo8p signaling pathway (for reviews see Borges-Walmsley and Walmsley, 2000; Lengeler et al., 2000; D'Souza and Heitman, 2001; Palecek et al., 2002). Other transcription factors known to regulate filamentous growth include Sok2p, Phd1p, and Ash1p and there is evidence that Phd1p could act in a pathway distinct from the cAMP and MAP kinase pathways (for review see Pan et al., 2000). Few targets of these signal transduction pathways are known, but one is the FLO11 gene encoding the most important flocculin relevant for cell-cell adhesion during filamentous growth in haploids and diploids (Rupp et al., 1999; Tamaki et al., 2000). The FLO11 gene expression is highly regulated by several transcription factors; among them Ste12p and Tec1p regulate transcription from the MAPK cascade, whereas Flo8p regulates Flo11p expression downstream of PKA (Lo and Dranginis, 1998; Madhani and Fink, 1998a; Rupp et al., 1999). New transcriptional profiling data indicate that invasive growth is not controlled solely by a dedicated invasion-specific transcriptional program and thus a variety of signals may contribute to initiate the dimporphic switch (Breitkreutz et al., 2003).
In this report we show that the FOL1 gene encodes three subsequent enzymatic activities for the biosynthesis of folic acid, namely DHNA, HPPK, and DHPS. The trifunctional Fol1 protein is expressed at a very low level and is localized at mitochondrial membranes. Deletion of FOL1 leads to a nongrowth phenotype that can be suppressed by the addition of folinic acid to the media. Under these conditions haploid fol1 strains exhibited strong invasive growth on rich media containing glucose and ammonium compounds known to suppress invasive growth. Likewise haploid and diploid fol1 strains showed a switch to filamentous pseudohyphal growth on these media, which was independent of the transcription factors Ste12p, Tec1p, Phd1p, and the cell wall protein Flo11p. Thus our results indicate that loss of the C1 carrier activity mediated by folate triggers a signal for pseudohyphae development under rich nutrient supply conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). All YP media were supplemented with adenine (4 mg/l) and tryptophan (20 mg/l). G418 (geneticin, final concentration 200 µg/ml, Cal-biochem, Novabiochem GmbH, Bad Soden, Germany), phleomycin (final concentration 7.5 µg/ml, Cayla, Toulouse, France), 5'fluoro orotic acid (5-FOA, final concentration 1000 µg/ml, PCR) and folinic acid (final concentration 50–1000 µg/ml, F-7878, Sigma, Taufkirchen, Germany) were added as required.
Yeast Strains
All yeast strains used in this study are derivatives of CEN.PK2 or CGX31 (genetic background: 
1278b) as indicated in Table 1 (Gimeno et al., 1992; Entian and Kötter, 1998). Transformation of yeast cells, sporulation, and tetrad analysis was carried out as described (Sherman and Hicks, 1991).
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Databank Searches
The NCBI's protein database was searched using BLASTP (Altschul et al., 1997). Sequence alignments were performed using CLUSTAL W (Thompson et al., 1994). Pfam domains were calculated according to Bateman et al. (2002). Isolated Pfam domains were subjected to FASTA analysis (Pearson, 1994).
Gene Deletions
ORF YNL256w (FOL1) was deleted in strain CEN.PK2 resulting in strain CEN.HE4 using the PCR-based disruption strategy. Briefly the deletion cassette was created on template plasmid pFA6-kanMX4 using oligonucleotides 278 and 279 (Table 2) and transformed into CEN.PK2 (Wach et al., 1994). The N terminal 357 nucleotides (119 amino acids) of FOL1 were not deleted. The correct deletion was verified by Southern blot analysis (unpublished data).
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All gene deletions in the diploid yeast strain CGX31 were done using a PCR-generated deletion cassette carrying the loxP-kanMX-loxP marker created on template plasmid pUG6 (Güldener et al., 1996). Deletion of ORF YNL256w was verified by PCR using the genomic primers 1068 and 1069 in combination with the kanR-specific primers 363 and 335. Where appropriate the kanMX marker of the resulting diploid heterozygous deletion strain was rescued using the Cre recombinase as described (Güldener et al., 1996). Next in the FOL1/fol1::loxP strain, the STE12 gene was deleted using a kanMX deletion cassette generated using oligonucleotides 1052 and 1053. PCR verification was done using the STE12-specific genomic primers 638 and 1055 in combination with 363 and 335. Likewise in the FOL1/fol1::loxP strain the FLO11 gene was deleted using oligonucleotides 1056 and 1057. PCR verification was done using the FLO11-specific genomic primers 1058 and 1061 in combination with 363 and 335. The PHD1 and TEC1 genes were each individually deleted in the FOL1/fol1::loxP-kanMX-loxP strain using a ble deletion cassette created on template plasmid pUG66 using oligonucleotides 1425 and 1426 (PHD1) and oligonucleotides 1427 and 1428 (TEC1; Gueldener et al., 2002). PCR verification was done using the PHD1-specific genomic primers 1431 and 1432 or the TEC1-specific genomic primers 1429 and 1430 in combination with 363 and 335.
Tetrad dissection of the resulting diploid heterozygous deletion strains was done on YPD + 1 mg/ml folinic acid, and the mating type was determined via PCR analysis (Huxley et al., 1990). To generate a diploid homozygous fol1/fol1 deletion strain the haploid fol1 deletion strain YUG1 was transformed with the HO plasmid Ycp50-HO-12 (gift from C. Hollenberg). Several uracil-prototrophic transformants were streaked out and diploids were identified by mating type-specific PCR. The HO plasmid was recovered by 5-FOA counterselection (Boeke et al., 1984).
Quantification of Fol1 Protein
The amount of Fol1 protein was quantified by fluorescence-activated cell analysis of a Fol1-GFP fusion protein. The genomic fusion of yEGFP to the C-terminus of FOL1 in yeast strain CEN.PK2 was done by homologous integration of a yEGFP3-loxP-kanMX-loxP integration cassette, which was created by PCR on template plasmid pUG24 using oligonucleotides 609 and 636. In the resulting strain YUG31 the yEGFP3 gene was integrated in frame at the C-terminal end of FOL1, replacing the FOL1 stop codon and the following 60 base pairs downstream. The correct integration was verified by two PCRs using 1) the yEGFP3 specific primer 604 and the FOL1 specific primer S52, and 2) the kanMX module-specific primer 335 and a primer in the FOL1 terminator region (S38). Plasmid pUG24 was obtained by cloning a PCR-generated yEGFP3 SalI fragment (template plasmid: pYGFP3; Cormack et al., 1997; oligonucleotides 556 and 557) into SalI cut pUG6 (Güldener et al., 1996).
Wild-type and yEGFP3 integration strains were grown overnight at 30°C in the appropriate liquid media as indicated. From these cultures cells were freshly inoculated in the same media (starting OD600 0,1) and grown to an OD600 of 0.3–0.5. Cells were diluted to an OD600 of 0.03 in 10 mM Tris, pH 7.5, and analyzed directly. Flow cytometry was carried out using a FACSort system (Becton Dickinson, Heidelberg, Germany; Niedenthal et al., 1996, 1999). Ten thousand particles (living cells) were analyzed per sample. The autofluorescence obtained for the wild-type (wt) strain was set electronically to channel 200 and the deletion strains were then analyzed using the same parameters. For each data point five independent cultures were measured.
Quantification of the FLO11 Promoter Activity
) were expressed in 1278b-based strains and analyzed by FACS as described before (Niedenthal et al., 1999). Wild-type and yEGFP3 integration strains were grown overnight at 30°C in liquid synthetic minimal glucose media. From these cultures cells were freshly inoculated in the same media (starting OD600 0.1) and grown to an OD600 of 0.3–0.5 and analyzed by FACS as described above. ) were pregrown in SC-ura medium containing 200 µg/ml folinic acid at 30°C O/N. Approximately 1 x 106 cells (wt) and 28 x 106 cells (fol1) were spotted on SD plates without amino acids containing 50 µg/ml folinic acid and incubated at 30°C for 25 h. On average spots carried 4 x 107 cells. The overlay assay was performed as previously described (Suckow and Hollenberg, 1998). Briefly spots were covered with 5 ml of 70°C hot agarose (0.5% agarose, 0.5 M sodium phosphate buffer, pH 7.0, 0.2% SDS, and 0.4 mg/ml 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside) and incubated for 24 h at 37°C.
Quantification of Fol1p Enzymatic Activities
) yielding pUG15 (selection marker: TRP1). pUG15 was transformed into strain CEN.HE4 (relevant genotype: FOL1/fol1::KanMX). Subsequent tetrad analysis and replica platting identified G418-resistant and Trp+ haploid segregants (YUG3). The GAL1/2 µ FOL1 overexpression vector pUG14 was constructed by subcloning the SpeI/SalI fragment of pUG15 into SpeI/SalI-cut p424GAL1 (Mumberg et al., 1994). ). To this end the FOL1 gene was first PCR-amplified from an EcoRI subclone of cosmid 14-4 using oligonucleotides 345 and 346, cleaved with EcoRV, and cloned into pUG7 (pBluescript derivative; Güldener et al., unpublished results) resulting in plasmid pSH50. pSH50 was cleaved by NcoI and SacI and the DNA fragment carrying the complete FOL1 gene was cloned into NcoI/SacI cut pHK255-I resulting in plasmid pSH51, which was transformed into E. coli strain XL1 blue for expression of the Fol1 protein.
Enzymatic Assays
The following materials were purchased from B. Schircks (Jona, Switzerland): 6-hydroxymethylpterin, 6-hydroxymethylpterin pyrophosphate, and 7,8-dihydro-D-neopterin. Pterines were hydrated with Pd-Charcoal (4 h, RT, atmospheric pressure) under the formation of 7,8-dihydropterins.
Assay of 7,8-Dihydroneopterin Aldolase (Haussmann et al., 1998) Assay mixtures contained 100 mM Tris/HCl, pH 8.0, 5 mM -mercaptoethanol, 100 µM 7,8-dihydro-D-neopterin and protein. The mixture was incubated at 37°C for 20 min. The reaction was terminated by the addition of 30 µl 1 M HCl containing 1% I2 and 2% KI (wt/vol). The samples were incubated at room temperature (RT) in the dark for 5 min. Excess iodine was reduced by the addition of 20 µl 2% ascorbinic acid (wt/vol). Samples were analyzed by reversed-phase HPLC using a column of Nucleosil RP18 (4 x 250 mm). The eluent contained 30 mM HCOOH and 7% MeOH. The flow rate was 2 ml/min. 6-hydroxymethylpterin was detected fluorometrically (Ex: 365 nm; Em: 446 nm). The retention volume of the reaction product was 6.5 ml.
Assay of 6-Hydroxymethyldihydropterin Pyrophosphokinase Assay mixtures contained 100 mM Tris/HCl, pH 8.0, 10 mM MgCl2, 5 mM -mercaptoethanol, 50 µM 6-hydroxymethyl-7,8-dihydropterin, and protein. After incubation for 20 min the reaction was stopped as described above. The samples were then anaylzed for 6-hydroxymethylpterin pyrophosphate as described above. The retention volume of 6-hydroxymethylpterin pyrophosphate was 2.5 ml.
Assay of Dihydropteroate Synthase Assay mixtures contained 0.1 M Tris hydrochloride, pH 8.0, 10 mM MgCl2, 5 mM -mercaptoethanol, 20 µM 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, 0.04 nM [carboxyl-14C]4-aminobenzoate, and protein in a total volume of 6 µl. The mixture was incubated at 37°C for 30 min. The reaction was terminated by immersion into a boiling water bath. Aliquots (3 µl) were applied to Whatman 3MM paper that was subjected to descending paper chromatography using 0.1 M potassium phosphate as solvent. The enzyme product remained at the start of the chromatogram. Radioactivity of paper segments was determined by scintillation counting.
For the DHNA and HPPK activity measurements two independent cultures were measured in triplicate and the median and the SD was calculated.
Yeast Protein Analysis
Wild-type and fol1 strains (strain background: 1278b) were grown to an OD600 of 0.6–0.8. Then cells were incubated with 0.15 µM 
factor for 15 min. Cells from 20-ml cultures were pelleted, and crude protein extracts were produced according to a protocol provided by Miltenyi Biotec GmbH (Bergisch-Gladbach, Germany). Protein samples were immediately boiled with SDS loading buffer, aliquoted, and frozen at -20°C.
Protein samples were electrophoresed by SDS-PAGE on 12% polyacrylamide gels (acrylamide:bis-acrylamide at a ratio of 37.5:1) using the Hoefer Mighty Small gele system (Amersham Biosciences, Freiburg, Germany). After protein transfer the membranes were blocked in TBST buffer as described by the ECL Advance Western Blotting Detection Kit (Amersham Biosciences). Membranes were incubated with primary antibody (antiphospho p44/42 MAPK; Cell Signaling Technology, Inc., Beverly, MA; 9101) in TBST-2% block overnight at 4 C° followed by three washings with TBST at RT. Immunoblots were incubated with secondary antibody (rabbit Ig, HRP-linked Whole antibody; Amersham Biosciences) in TBST-2% block for 7 h at 4°C and then washed three times with TBST at RT and developed using the Amersham ECL kit.
Microscopy
The FOL1 gene was PCR amplified using oligonucleotides 327 and 391 and plasmid pUG16 as template. The SpeI/EcoRI 2.6-kb PCR product was cloned into SpeI/EcoRI cut yEGFP3 vector pUG23 (Güldener et al., unpublished results) resulting in the CEN-vector pUG38.
For Fol1p localization experiments yeast strain CEN.PK2 was transformed with plasmid pUG38. For staining of mitochondria and nuclei in living cells, exponential growth phase cells were incubated in YPD media supplemented with 4'6-diamino-2-phenylindoledihydrochloride (DAPI; stock:1 µg/µl, final concentration: 1 µg/ml) for 30 min. Cells were fixed by addition of formaldehyde (500 µl of 37% formaldehyde were added to 5 ml yeast culture) and incubation for 30 min at 30°C. Cells were washed once with PBS and then adsorbed onto a polylysine-coated coverslip. Cells were viewed using a Zeiss Axioskop (Oberkochen, Germany) microscope.
Assessment of Budding Pattern
Haploid cells with the indicated genotype grown on YPD plates containing 50 µg/ml folinic acid were taken from plates, resuspended in PBS buffer, and stained with the chitin-specific dye calcofluor white (concentration 4 µg/ml). Cells were spotted onto polylysine-coated microscope slides and viewed using a Zeiss Axioskop microscope. Cells with two or more bud scars were classified as follows: unipolar/axial (scars only at one end), bipolar (scars at both ends), or random (scars not localized to the ends; number of cells 
100).
Immunocytochemistry and Electron Microscopy
For electron microscopy a gfp-FOL1 fusion was constructed by cloning a SpeI/SalI FOL1 fragment (out of pUG15) into plasmid pGFP-N-FUS (Niedenthal et al., 1996) resulting in pUG16. Yeast strain CEN.PK2 was transformed with plasmid pUG16. Cells were grown in selective minimal media, harvested, and fixed with a mixture of 0.5% glutaraldehyde and 4% freshly depolymerized paraformaldehyde in 0.1 M sodium citrate buffer, pH 4.7, for 1.5 h at 30°C (Zimmer et al., 1995). Subsequent treatment with 1% sodium metaperiodate (Sigma) for 1 h at 4°C was performed to support penetration of the cell wall by the cryoprotectant sucrose. Cryoprotection of the cells was achieved by infusion with a mixture containing 25% polyvinylpyrrolidone (PVP, MW 10,000, Sigma) and 1.6 M sucrose for 3 h at 30°C (Tokuyasu, 1989). Cells were mounted on specimen holders and immediately frozen in liquid nitrogen. Ultrathin cryosections were prepared at -100°C and deposited on formvar/carbon-coated copper grids after thawing. Immunolabeling with polyclonal anti-GFP antibody (Clontech, Palo Alto, CA) or polyclonal anti-Fol1p antibody (D. Becher, unpublished results) and secondary colloidal gold (10 nm) goat anti-rabbit IgG conjugate (British BioCell, Cardiff, Wales) was performed according to Griffiths et al., (1984) with minor modifications. As controls, the sections were incubated directly with gold IgG conjugate, omitting the primary antibody step, or cells without GFP-fusion protein were used. Finally, the frozen-thawed sections were stabilized and stained with a mixture of 0.3% uranyl acetate and 2% methylcellulose (25 cp; Sigma). Specimens were examined with a Zeiss 906 electron microscope at 60 kV acceleration voltage.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Therefore we named ORF YNL256w FOL1 according to the standard yeast genetic nomenclature. Furthermore a folate auxotroph mutant named fol1 had been mapped previously to this region (Little and Haynes, 1979).
Detailed analysis of the Fol1p protein sequence indicated the presence of three putative enzymatic activities: DHNA, HPPK, and DHPS (Figure 2). These three enzymatic activities perform three subsequent steps in the biosynthesis of folate (Figure 1). In bacteria these three enzymatic activities are usually encoded by three separate ORFs (e.g., Bacillus subtilis; Slock et al., 1990). In contrast in filamentous fungi and probably in other higher eukaryotes only the HPPK and DHPS domains are fused to one protein, e.g., Toxoplasma gondii and in pea (Triglia and Cowman, 1994; Rebeille et al., 1997; Figure 2). However, in the fungi Schizosaccharomyces pombe and C. albicans and in Pneumocystis carinii (zygomycete), all three enzyme activities have been found to be located on a single polypeptide (Volpe et al., 1993; Figure 2). Detailed FASTA alignments of Fol1p indicated that its N-terminus carries two domains with 24 and 32% identity, respectively, to ORF1 from B. subtilis encoding DHNA. This organization has also been found in the P. carinii fas protein, where the two domains FasA and FasB are both required for DHNA function (Volpe et al., 1995; Figure 2). The S. cerevisiae DHNA domains show the lowest identity (21%) to the corresponding domain from Neurospora crassa and the highest identity (43%) to the domain from C. albicans. The central Fol1p domain exhibits identities between 31 and 48%, respectively, to the HPPK carrying domains from T. gondii and P. carinii. The C-terminal domain of Fol1p shares between 25% identity with the corresponding domain from T. gondii and 44% identity with the C-terminal domain from C. albicans (Figure 2). Taken together these data indicate that Fol1p shares its trifunctional nature with the corresponding proteins from a limited number of fungi, whereas in most other eukaryotes this protein is bifunctional.
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FOL1 Codes for the Enzyme Activities DHNA, HPPK, and DHPS
To confirm that the three enzymatic activities inferred from homology were indeed encoded by FOL1, we performed enzymatic tests for all three enzyme activities. First the FOL1 gene was expressed in E. coli via the inducible tac promoter. As expected, control E. coli cells exhibited all three endogenous enzyme activities (Table 3; XL1 Blue). However in E. coli cells expressing the S. cerevisiae FOL1 gene all three enzyme activities increased significantly. The DHNA and HPPK enzyme levels were increased fivefold compared with the enzyme levels in protein extracts from control cells, whereas the DHPS enzyme level increased about ninefold (Table 3; ptac:FOL1). These data demonstrate that FOL1 encodes all three enzyme activities.
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We next characterized the FOL1 enzyme activities in yeast. Protein extracts prepared from a haploid wt strain exhibited low but significant levels for all three enzymatic reactions (Table 3; FOL1). Analysis of an isogenic FOL1 deletion strain (kept alive by the presence of folinic acid in the media; see later) revealed severely reduced activities for all three enzyme reactions (Table 3; fol1). In contrast moderate overexpression of FOL1 from the GALS promoter in a wt strain resulted in a strong increase of all three enzymatic activities (Table 3; FOL1 + pGALS:FOL1).
These data demonstrate that Fol1p carries three enzymatic activities for the biosynthesis of folic acid, namely DHNA, HPPK, and DHPS (Figure 1).
The FOL1 Gene Is Required for Growth
Deletion of one allele of FOL1 in a diploid yeast strain (Figure 3A) and subsequent tetrad analysis revealed that FOL1 is an essential gene, as spores carrying the FOL1 deletion were unable to form visible colonies on YPD (Figure 3B, left panel). The terminal phenotype of the fol1 deletion on rich media (YPD) was a microcolony of uneven shape containing 50–100 slightly elongated cells. The nongrowth phenotype of the fol1 strain could be rescued by addition of 5-formyl tetrahydrofolate (folinic acid, FA) as an alternative one-carbon source into the media (Figure 3B, middle and right panels). Folinic acid can only be metabolized by 5,10-methenyl-THF synthetase yielding 5,10-methenyl-THF, a folate coenzyme that can be converted into the other folate coenzymes (Holmes and Appling, 2002). However although the fol1 strain could be rescued by addition of folinic acid, the colonies never reached wt size (Figure 3B; 1 mg/ml FA), probably because folinic acid is known to be an inhibitor of several folate-dependent enzymes (Girgis et al., 1997).
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Additionally, although the fol1 deletion strains could grow on media supplemented with folinic acid, they were unable to grow on the nonfermentable carbon sources ethanol, glycerol, or acetate (supplemented with folinic acid), indicating that Fol1p and thus 1C carrier are also required for mitochondrial function in yeast and that addition of exogenous folinic acid cannot fill up the mitochondrial one-carbon carrier pools adequately (unpublished data).
Moderate But Not High FOL1 Expression Rescues the Nongrowth Phenotype of a fol1 Strain
Interestingly, strong overexpression of FOL1 by expression from the GAL1 promoter on a 2-µ–based plasmid did not complement the nongrowth phenotype of the fol1 strain. Likewise strong overexpression of FOL1 in a diploid wt strain was toxic and resulted in a significant increase of cells with a 2C DNA content (Figure 4A; unpublished data). However FOL1 expression from the moderate GALS promoter on a CEN-based plasmid exhibited almost wt-like growth (Figure 4A). These data suggest that high levels of THF-synthesizing enzymes and/or of C1 carrier units are detrimental to the yeast cell. Thus it can be assumed that the Fol1p level in the cell is low, and indeed our own data indicate that the Fol1 promoter activity is low (unpublished data).
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Next we wanted to know whether the amount of Fol1 protein is regulated under conditions where the intracellular C1 carrier concentration changes (here: addition of folinic acid). A genomic FOL1-yEGFP fusion construct replaced one of the two chromosomal wt FOL1 alleles in a diploid strain (Figure 4B). Quantification of the background fluorescence of the parental wt strain (FOL1) by flow cytometry revealed a relative green fluorescence (RGF) of 10.23 U, while the Fol1-yEGFP fusion strain exhibited 11.2 U, which represents a small increase of 0.97 RGF units (see MATERIALS AND METHODS for details; Figure 4C). Addition of folinic acid to the media led to a reduction in background fluorescence for the wt control strain (RGF 9.30) of 
0.93 RGF units; however, the FOL1-yEGFP fusion strain showed a stronger reduction (RGF 9.85) of 1.35 RGF units, suggesting that the FOL1 promoter might be autoregulated (Figure 4C).
Together these data indicate that excess amounts of Fol1p in yeast hampers growth and that cells therefore may have evolved sensing and regulating mechanisms to adjust the Fol1p levels adequately.
Fol1p Is Localized at the Mitochondrial Membrane
To study the subcellular localization of the Fol1 protein, we expressed a C-terminal FOL1-yEGFP3 fusion protein from a CEN plasmid under control of the regulatable MET25 promoter. The fusion protein was functional as it was able to rescue the nongrowth phenotype of the fol1 deletion strain (unpublished data). The Fol1p-yEGFPp fusion protein showed a punctate staining and colocalized with DAPI-stained mitochondria (Figure 5).
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This localization was confirmed by electron microscopy of yeast cells expressing a N-terminal GFP-Fol1 fusion protein using anti-GFP antibodies (Figure 6, a–c). Expression of this fusion protein again complemented the nongrowth phenotype of the fol1 deletion strain. However sometimes clumped mitochondria were observed in this strain. In cells expressing the GFP-Fol1 fusion protein the gold-particles labeled mainly the mitochondria (Figure 6a). A detailed inspection at higher resolution revealed that the gold particles were found associated mainly with both mitochondrial membranes and rarely with the mitochondrial matrix (Figure 6, b and c). To exclude that the GFP part of the fusion protein influenced the localization we also localized the Fol1 protein in a diploid wt strain using peptide antibodies against an antigenic epitope of Fol1p itself. Although the Fol1 protein level is very low in yeast and the antibody exhibited only limited specificity, most of the gold particles were again located at the mitochondrial membrane (Figure 6d). Taken together the data indicate that the Fol1 protein is located at the mitochondrial membrane system.
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Absence of FOL1 Induces Filamentous and Adhesive Growth
Rescue of the nongrowth phenotype of fol1 cells was seen on media containing folinic acid. The degree of rescue observed was dependent on the concentration of folinic acid (Figure 3B). Microscopic inspection of haploid fol1 cells grown for only 8 h on rich YPD media in the presence of folinic acid revealed elongated connected cells forming branching filamentous asters (Figure 7A). The extent of filamentation was dependent on the amount of folinic acid present: on media with 50 µg/ml all cells formed filaments consisting of elongated and bigger cells (Figure 7A, top panel), on media with 200 µg/ml 50–80% of the cells formed filaments (Figure 7A, middle panel, both types shown), whereas on media with 800 µg/ml, most cells showed an almost regular yeast-like morphology (Figure 7A, bottom panel). After growth for 24 h filamentous growth of fol1 cells grown on media with 50 and 200 µg/ml folinic acid had continued, whereas cells on media with 800 µg/ml folinic acid were yeast-like but had formed uneven microcolonies (Figure 7B). These data indicated that deletion of the FOL1 gene induces a filamentous growth phenotype and prompted us to test if fol1 cells showed adhesive growth. Haploid fol1 cells grown for 48 h on YPD media supplemented with varying amounts of folinic acid had invaded the agar, whereas isogenic wt cells did not show agar invasion (Figure 7C).
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These growth phenotypes are reminiscent of the filamentous growth phenotype observed for diploid cells under nitrogen limitation, or for haploid cells exhibiting invasive growth when grown on rich media for an extended period of time or when starved for glucose (Cullen and Sprague, 2000; Lengeler et al., 2000; Palecek et al., 2002).
The wt and fol1 strains used were derived from CEN.PK2 and have a W303 background. We next assayed the consequences of a FOL1 deletion in a 1278b/CGX31 strain background which is commonly used for filamentation and invasion studies (Gimeno et al., 1992; Palecek et al., 2002). Haploid strains of this strain background show invasive growth on rich YPD media but only after prolonged incubation for 4–6 days (Roberts and Fink, 1994; Rupp et al., 1999; Braus et al., 2003). As expected, microcolonies of the 1278b-based haploid wt strain showed yeast-like growth forming round colonies on rich YPD media supplemented with folinic acid (Figure 8, a and b) and showed the typical substrate invasiveness after growth for 5 days (Figure 9, c and d, wt), whereas after growth for 24 h no agar invasion could be observed (Figure 9, a and b, wt). In contrast, deletion of the FOL1 gene in this strain background gave rise to slightly larger and often elongated cells with filamentous structures and uneven microcolony morphologies (Figure 8, g and h). Moreover, in contrast to wt cells the fol1 strain showed strong adhesive growth after 24 h of growth on media supplemented with folinic acid (Figure 9, a and b, fol1). After prolonged growth for 5 days the fol1 strain despite its significantly reduced overall growth behavior showed adhesive growth similar to that observed for the wt strain (Figure 9, c and d, fol1).
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In diploid strains, starvation for nitrogen also induces pseudohyphal growth. Therefore we tested the consequences of the deletion of both alleles of the FOL1 gene in a diploid 1278b-based strain. Growth of this strain for 24 h on rich YPD media supplemented with folinic acid resulted in filamentous growth and after growth for 72 h an uneven colony was formed (Figure 8m and n, diploid). However, after growth for 24 h the adhesive growth properties of the fol1 fol1 strain were similar to those of the diploid wt strain (Figure 9, a and b, diploid).
The haploid fol1 cells exhibited an altered budding pattern in comparison to wt cells. Haploid wt cells typically exhibit an axial budding pattern in which they choose the location of the new bud adjacent to the sites of the previous ones (for review see Madden and Snyder, 1998; Chant, 1999). The haploid fol1 strain showed mainly bipolar distal bud site selection (91%), where most of the buds emerged at the distal pole opposite to the birth end, whereas only 47% of the wt cells exhibited bipolar budding (Table 4).
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The actin cytoskeleton plays a central role in fungal dimorphism (Kron et al., 1994). Therefore we also analyzed the actin cytoskeleton in a fol1 strain grown on YPD media supplemented with 50 µg/ml folinic acid. Cells of the fol1 strain showed an enhanced polarity of actin patches toward the distal tip of daughter cells throughout bud emergence relative to the distribution of actin patches in wt cells. Furthermore the actin cables were more pronounced in fol1 cells when compared with wt cells (unpublished data). These findings indicate that in a fol1 strain the actin cytoskeletal structures have adopted characteristics of pseudohyphal growth (Kron et al., 1994; Cali et al., 1998).
Filamentous Growth of fol1 Strains Is Independent of Ste12p, Tec1p, Pdh1p, and Flo11p
The Ste12p and Tec1p transcription factors are critical for the induction of filamentous and invasive growth stimulated by the MAPK cascade and strains lacking these genes are deficient in both filamentous and invasive growth (Liu et al., 1993; Gavrias et al., 1996; Madhani et al., 1997; Palecek et al., 2002). To test whether the folate starvation signal is mediated by the Ste12 and/or Tec1p proteins fol1 ste12 and fol1 tec1 double mutant strains were analyzed for their ability to show pseudohyphal and adhesive growth. Surprisingly the haploid fol1 ste12 as well as the fol1 tec1 double mutant strain still showed increased bipolar budding and filamentous growth as well as uneven colony morphologies (Figure 8, c and d, k and l; Table 4). However, adhesive growth could no longer be observed for the fol1 ste12 double mutant strain after 24 h of growth (Figure 9, a and b). Likewise, no invasive growth could be observed for the fol1 ste12 strain after growth for 5 days, whereas the single ste12 mutant strain showed residual substrate invasiveness (Figure 9, c and d). In contrast the fol1 tec1 double mutant strain showed adhesive growth like the single fol1 mutant strain, whereas the single tec1 mutant strain showed some residual invasiveness (Figure 9, e and f). Thus our data suggest that the filamentation process induced by loss of the Fol1p activity and presence of folinic acid in the media does not require the Ste12p and Tec1p transcription factors, whereas invasiveness is dependent on the presence of the Ste12 protein.
Next we tested the role of the transcription factor Phd1p, which also is thought to be involved in pseudohyphal growth by acting in a pathway distinct from the cAMP and MAP kinase pathways (Gimeno and Fink, 1994; Palecek et al., 2002). The haploid fol1 phd1 double mutant strain exhibited filamentous growth and increased bipolar budding pattern and an uneven colony shape to the same extent as the fol1 single mutant strain (Figure 8, e and f; Table 4). Furthermore in adhesion assays the fol1 phd1 double mutant strain after 24 h of growth exhibited invasiveness similar to that observed for the phd1 single mutant strain despite its significantly reduced overall growth behavior (Figure 9, e and f). These results suggest that Phd1p is not required for pseudohyphal growth and for agar invasive growth of a fol1 mutant strain.
One of the few known Ste12p target genes leading to and required for the induction of pseudohyphal growth and agar invasion is the FLO11 gene encoding a cell surface flocculin protein (Lo and Dranginis, 1998). We therefore tested whether the Flo11 protein is required for the filamentous and invasive growth behavior of the fol1 strain. The haploid fol1 flo11 double mutant strain exhibited filamentous growth and increased bipolar budding and an uneven colony shape to the same extent as the fol1 single mutant strain (Figure 8, i and j; Table 4). However, it showed no adhesive growth after 24 h and 5 days of growth (Figure 9, a–d). These data suggest that Flo11p similar to Ste12p is not required for the fol1 induced filamentous growth but required for agar invasion.
The Phosphorylation Level of the Fus3p and Kss1p MAP Kinases Is Not Increased in a fol1 Strain
Filamentous growth depends in part on the mitogen-activated protein kinase (MAPK) cascade, which results in activation of Ste12p (for review see Madhani and Fink, 1998b). It has been shown that activation of the final MAP kinase Kss1p by phosphorylation increases filamentation, whereas the kinase activity of Fus3p appears to suppress filamentous growth (Madhani and Fink, 1997; Sabbagh et al., 2001). Stimulation of the mating pheromone response pathway also leads to phophorylation and thus activation of both Kss1p and Fus3p (Sabbagh et al., 2001). We tested whether deletion of the FOL1 gene results in an increased phosphorylation level of Kss1p and/or Fus3p. The fol1 single mutant strain showed a Kss1p and Fus3p phosphorylation level very similar to that of the corresponding wt strain (Figure 10, wt and ). The level of Kss1p and Fus3p phosphorylation found in the fol1 strain did not change when cells were grown in media containing different amounts of folinic acid. However in control experiments Kss1 and Fus3p phosphorylation could be induced in both strains by addition of factor mating pheromone, indicating that the MAPK pathway can be activated (Figure 10). This experiment indicates that deletion of FOL1 does not result in an increased level of Kss1p and Fus3p phosphorylation.
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Deletion of FOL1 Results in Increased Flo11p Expression Levels
Our data suggested that the fol1 induced invasive growth was dependent on Ste12p and Flo11p. This would imply that Flo11p transcription is activated in a fol1 strain. The FLO11 promoter, the largest known promoter in S. cerevisiae, is regulated by several transcription factors including Ste12p and Tec1p (Lo and Dranginis, 1998; Rupp et al., 1999; Palecek et al., 2002). We thus assayed the FLO11 promoter activity in a haploid fol1 background. We placed a 3-kb-long DNA fragment containing the FLO11 promoter in front of the yEGFP encoding gene on a CEN plasmid and quantified the FLO11 promoter-dependent green fluorescence by flow cytometry in haploid strains grown in liquid synthetic glucose media supplemented with 250 µg/ml folinic acid. Cells were analyzed in early log phase when they were not depleted for glucose or ammonium. In the wt strain the FLO11 promoter showed 22 relative green fluorescence (RGF) units, whereas in a fol1 deletion strain the promoter was found to be strongly activated, exhibiting 104 (RGF) units (Table 5). A ste12 strain showed a FLO11 promoter activity similar to that of a wt strain, indicating that the basal FLO11 promoter activity is not dependent on Ste12p. The fol1 ste12 double deletion strain exhibited a promoter activity of 24 ± 6 RGF, indicating that the elevated FLO11 promoter activity observed in the flo11 strain is indeed dependent on Ste12p (Table 5). Thus these data indicate that depletion of the cellular folate pool triggers the activation of FLO11 gene expression in a Ste12p-dependent manner.
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To assay which FLO11 promoter regions were affected in a fol1 strain, we used a set of seven plasmid-born reporter constructs containing individual nonoverlapping 400-base pair FLO11 promoter regions covering the first 2800 base pairs of the FLO11 promoter cloned in front of a CYC1-lacZ reporter unit (Rupp et al., 1999). Wild-type or fol1 cells carrying these plasmids were plated onto synthetic glucose media containing 50 µg/ml folinic acid, incubated for 24 h, and analyzed by a -gal overlay assay. The full-length FLO11 promoter showed a small but significant increase in -galactosidase activity in the fol1 strain when compared with wt confirming our GFP measurements (Figure 11, full-length prom.) Furthermore the FLO11 promoter segments 6/5 and 12/11 exhibited a strong increase in -galactosidase activity in the fol1 deletion strain compared with wild type, indicating that these regions become activated in the absence of Fol1p (Figure 11).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTS |
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