Isolation of a gene encoding a putative polyamine transporter from Candida albicans, GPT1.

A gene encoding a transport protein from the pathogenic yeast, Candida albicans, has been isolated during a complementation experiment utilizing an ornithine decarboxylase-negative (spe1 Delta) strain of Saccharomyces cerevisiae. This gene restores gamma-aminobutyric acid (GABA) transport to a GABA transport-negative mutant of S. cerevisiae and encodes a protein which putatively allows transport of one or more of the polyamines. We have assigned the name GPT1 (GABA/polyamine transporter) to this gene.

Polyproline binding is an essential function of human profilin in yeast.

Structural analysis of human profilin has revealed two tryptophan residues, W3 and W31, which interact with polyproline. The codons for these residues were mutated to encode phenylalanine and the mutant proteins overexpressed in Eschericia coli. The isolated proteins were diminished in their ability to bind polyproline, whereas phosphatidylinositol 4,5-bisphosphate (PIP2) binding remained unchanged. In many strains of Saccharomyces cerevisiae, disruption of the gene encoding profilin, PFY1, is lethal. It was found that expression of the gene for human profilin is capable of suppressing this lethality. The polyproline-binding mutant alleles of the human gene were cloned into various yeast expression vectors. Each of the mutant genes resulted in suppression of the lethality of pfy1Delta. It was observed that the mutant protein expression levels paralleled the growth rates of the strains. The severity of various morphological abnormalities of the strains was also attenuated with increased protein levels, suggesting that profilin polyproline-binding mutations are deleterious to cell growth unless overexpressed. Both tryptophan mutations were combined to give a third mutant allele that was found both unable to bind polyproline and to suppress the lethality of a pfy1 deletion. Immunoprecipitation experiments suggested that the mutants were unaltered in their affinity for actin and PIP2. These data strongly suggest that polyproline binding is an essential function of profilin.

The extracellular domain of the Saccharomyces cerevisiae Sln1p membrane osmolarity sensor is necessary for kinase activity.

The function of the extracellular domain (ECD) of Sln1p, a plasma membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeast Saccharomyces cerevisiae. Truncations of SLN1 that retain an intact kinase domain are capable of complementing the lethality of an sln1Delta strain. By observing levels of Hog1p phosphorylation as well as the phosphorylation state of Sln1p, the kinase activities of various SLN1 constructions were determined. In derivatives that do not contain the first TMD, Sln1p activity was no longer dependent on medium osmolarity but appeared to be constitutively active even under conditions of high osmolarity. Removal of the first TMD (DeltaTMD1 construct) gave a protein that was strongly phosphorylated whereas Hog1p was largely dephosphorylated, as expected if the active form of Sln1p is phosphorylated. When both TMDs as well as the ECD were deleted, so that the kinase domain is cytosolic, Sln1p was not phosphorylated whereas Hog1p became constitutively hyperphosphorylated. Surprisingly, this hyperactivity of the HOG mitogen-activated protein kinase signaling pathway was not sufficient to result in cell lethality. When the ECD of the DeltaTMD1 construct was replaced with a leucine zipper motif, Sln1p was hyperactive, so that Hog1p became mostly unphosphorylated. In contrast, when the Sln1p/leucine zipper construct was crippled by a mutation of one of the internal leucines, the Sln1 kinase was inactive. These experiments are consistent with the hypothesis that the ECD of Sln1p functions as a dimerization and activation domain but that osmotic regulation of activity requires the presence of the first TMD.

Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae.

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 --> Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1. 3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.

Isolation and sequence of the gene encoding ornithine decarboxylase, SPE1, from Candida albicans by complementation of a spe1 delta strain of Saccharomyces cerevisiae.

The gene encoding ornithine decarboxylase, SPE1, from the pathogenic yeast Candida albicans has been isolated by complementation of an ornithine decarboxylase-negative (spe1 delta) strain of Saccharomyces cerevisiae. Four transformants, three of which contain plasmids with the SPE1 gene, were isolated by selection on polyamine-free medium. The C. albicans ornithine decarboxylase (ODC) showed high homology with other eukaryotic ODCs at both the amino acid and nucleic acid levels.

Isolation and characterization of the Candida albicans PFY1 gene for profilin.

We have isolated the Candida albicans gene for profilin, PFY1. Degenerate oligonucleotide primers based on regions of high homology were utilized to obtain a polymerase chain reaction-amplified copy of the gene. This was then used as a probe to isolate the gene from a C. albicans genomic library. Our studies indicate that the full-length gene is unstable in Escherichia coli. Several clones were sequenced, and the predicted amino acid sequence demonstrated homology with profilin proteins from other organisms, most notably Saccharomyces cerevisiae. Northern analysis revealed that the gene is expressed in C. albicans. Attempts to express the gene in S. cerevisiae cells were unsuccessful until the C. albicans promoter was replaced with an S. cerevisiae promoter. Functional complementation of the gene was demonstrated in S. cerevisiae profilin-requiring cells. Antibodies raised to isolated C. albicans profilin protein recognized a protein of the predicted molecular weight when the gene was expressed in S. cerevisiae cells.

Ascosteroside, a new antifungal agent from Ascotricha amphitricha. I. Taxonomy, fermentation and biological activities.

Ascosteroside, a novel antifungal compound, was isolated from the culture broth of Ascotricha amphitricha. This compound is an alpha-linked glycoside of a lanostane type triterpenoid. It is active against yeasts such as Candida albicans and Saccharomyces cerevisiae and against filamentous fungi but shows no activity against bacteria. It is not toxic to mammalian cells at concentrations up to 150 microM. In a mouse model, the compound afforded protection comparable to that of ketoconazole.

The sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differential gene expression in Candida albicans.

The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reinformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1 alpha 2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters.

Regulation of profilin localization in Saccharomyces cerevisiae by phosphoinositide metabolism.

Profilin is an actin- and phosphatidylinositol 4,5-bisphosphate-binding protein that plays a role in the organization of the cytoskeleton and may be involved in growth factor signaling pathways. The subcellular localization of profilin was examined in the yeast Saccharomyces cerevisiae. Immunoblot analysis showed that profilin was localized in both the plasma membrane and cytosolic fractions of the cell. Actin was bound to the profilin localized in the cytosol. The association of profilin with the membrane was peripheral and mediated through interaction with phospholipid. The phospholipid dependence of profilin for membrane binding was examined in vitro using pure profilin and defined unilamellar phospholipid vesicles. The presence of phosphatidylinositol 4,5-bisphosphate in phospholipid vesicles was required for maximum profilin binding. Moreover, the binding of profilin to phospholipid vesicles was dependent on the surface concentration of phosphatidylinositol 4,5-bisphosphate. The subcellular localization of profilin was examined in vivo under growth conditions (i.e. inositol starvation of ino1 cells and glucose starvation of respiratory deficient cells) where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were depleted. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate levels resulted in a translocation of profilin from the plasma membrane to the cytosolic fraction. Profilin translocated back to the membrane fraction from the cytosol under growth conditions where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were replenished. These results suggested that phosphoinositide metabolism played a role in the localization of profilin.

Characterization of the Candida albicans TRP1 gene and construction of a homozygous trp1 mutant by sequential co-transformation.

The Candida albicans TRP1 gene has been isolated by complementation of an Escherichia coli trpC mutant. Sequence analysis has revealed a single ORF (open reading frame) of 678 nucleotides (nt). The amino acid (aa) sequence deduced from this coding region demonstrates a high degree of homology with PRAI (phosphoribosylanthranilate isomerase) enzymes of other fungi, as well as bacterial species. The gene is also analogous to other yeast TRP1 genes in that it encodes a unifunctional enzyme, whereas TRP1 in filamentous fungi encodes a tri-functional enzyme. Both chromosomal copies of the gene were disrupted by sequential integrative transformation employing co-transformation of an ade1 mutant in order to create a homozygous auxotrophic trp1,ade1 C. albicans strain. This double auxotroph was used to test the ability of the Saccharomyces cerevisiae TRP1 gene to complement the C. albicans trp1 mutation; no expression of the S. cerevisiae gene was detectable.

Isolation and sequence of the t-RNA ligase-encoding gene of Candida albicans.

The gene encoding tRNA ligase from Candida albicans was isolated from a genomic library by complementation of a Saccharomyces cerevisiae strain containing a disrupted structural gene, RLG1, encoding tRNA ligase. The cloned gene also complements a temperature-sensitive allele of RLG1. Sequence analysis revealed a single 2499-nt coding region. The gene encodes a protein of 833 amino acids that is 42% identical to S. cerevisiae tRNA ligase. Hybridization to chromosomes of C. albicans separated by pulsed-field gel electrophoresis located the gene to chromosome 1, the smallest C. albicans chromosome.

Identification, characterization and sequence of Candida albicans repetitive DNAs Rel-1 and Rel-2.

Two moderately repetitive DNA elements, Rel-1 and Rel-2, were identified in a screen for clones that hybridized to a Candida albicans minichromosome. Rel-1, a 223-bp sequence, is C. albicans-specific. The 2789-bp Rel-2 sequence hybridizes weakly to C. stellatoidia DNA but not to DNA from several other yeast species. Genomic Southern-blot analysis indicated that Rel-1 and Rel-2 are often closely associated in the genome, suggesting that they may be subsequences of a larger repetitive element. Small subrepeats are located in the nucleotide sequence of both clones. Hybridization demonstrated that Rel-2 contains both repetitive and unique DNA sequences. The repetitive DNA is present on most, and perhaps all, C. albicans chromosomes. The unique sequence maps to chromosome 7; however, in some strains, it is also present on additional chromosomes.

Molecular cloning and characterization of the Candida albicans enolase gene.

A DNA clone containing the putative Candida albicans enolase gene (ENO1) was isolated from a genomic DNA library. The sequenced insert contained a continuous open reading frame of 1,320 bp. The predicted 440-amino-acid protein is 78 and 76% identical, respectively, to Saccharomyces cerevisiae enolase proteins 1 and 2. Only one enolase gene could be detected in C. albicans genomic DNA by Southern analysis with a homologous probe. Northern (RNA) analysis detected a single, abundant C. albicans ENO1 transcript of approximately 1,600 nucleotides. When cells were grown on glucose, levels of ENO1 mRNA were markedly increased by comparison with ENO1 mRNA levels in cells grown on ethanol, a gluconeogenic carbon source. In contrast to this glucose-mediated transcriptional induction, the carbon source had no dramatic effect on the levels of enolase protein or enzyme activity in the C. albicans strains tested. These results suggest that posttranscriptional mechanisms are responsible for modulating expression of the C. albicans enolase gene.

DNA translocations contribute to chromosome length polymorphisms in Candida albicans.

Rotating-gel electrophoresis and DNA hybridization were used to compare the electrophoretic karyotype of six Candida albicans isolates. The hybridization pattern for 22 cloned sequences, including eight previously unmapped genes, indicates that there are eight pair of homologous chromosomes in each strain. However, since homologous chromosomes can differ in length, it is possible to resolve more than eight bands in some strains. The mapping data demonstrate that linkage groups are generally conserved suggesting that, in spite of gross karyotype differences, there is an underlying similarity in the genome organization of different isolates. The hybridization data also provide direct evidence that DNA translocations and reciprocal translocations contribute to chromosome length polymorphisms in C. albicans.

Direct selection of galactokinase-negative mutants of Candida albicans using 2-deoxy-galactose.

The galactose analogue 2-deoxy-galactose (2DG) has been widely used to select for mutations in the gene encoding the galactose pathway enzyme galactokinase (GalK). We have tested the effect of 2DG on Candida albicans to see if it could be used to obtain GalK- mutants in this diploid asexual yeast. 2DG was shown to be toxic to wild-type cells. Enzyme assays demonstrated that 2DG can induce GalK as efficiently as galactose. Examination of the initial rate of galactose uptake indicated that the galactose transport system is constitutive. 2DG-resistant mutants were isolated from mutagenized cultures and shown to have very low levels of GalK activity. The potential genetic applications of this system of direct mutant selection are discussed.

Repeated use of GAL1 for gene disruption in Candida albicans.

A technique which has the potential to allow repeated use of the same selectable marker to create gene disruptions in Candida albicans has been developed. In this approach, originally described for Saccharomyces cerevisiae, the selectable marker is flanked by direct repeats. Mitotic recombination between these repeats leads to elimination of the selectable marker. A module in which the GALq1 gene is flanked by direct repeats of the bacterial CAT gene was constructed and used to disrupt one copy of the URA3 gene in a gal1 mutant. Gal- revertants were selected by plating on 2-deoxy-D-galactose (2DOG). The frequency of 2DOG-resistant colonies recovered was 20 times higher than that obtained with a similar construct not flanked by direct repeats. Of these, 20% had lost the GAL1 gene by recombination between the direct repeats. The GAL1 gene was used again to disrupt the remaining wild-type copy of the URA3 gene of one of these gal1 isolates, resulting in a stable ura3 mutant. This technique should be generally applicable to derive homozygous gene disruptions in this diploid organism.

Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity.

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.

Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate.

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including beta-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.

Functional expression of the Candida albicans beta-tubulin gene in Saccharomyces cerevisiae.

Expression of the beta-tubulin-encoding gene (TUB2) of Candida albicans has been examined in Saccharomyces cerevisiae. Overexpression of the TUB2 gene of C. albicans, as well as that of S. cerevisiae, was found to be lethal. Chromosomal integration of the C. albicans TUB2 gene into a strain in which the native TUB2 gene had been deleted led to functional complementation. The results demonstrate that correct splicing of the two introns present in the C. albicans TUB2 gene occurs in the heterologous host strain containing this gene. Such strains are supersensitive to the tubulin-binding agent benomyl, indicating that the natural resistance of C. albicans to benomyl is not related to the structure of its beta-tubulin.

Increasing gene expression in yeast by fusion to ubiquitin.

Heterologous gene expression in yeast can be increased up to several hundred-fold by expressing a foreign gene as a fusion to the ubiquitin gene. An endogenous yeast endoprotease (Ub-Xase) removes the ubiquitin from the fusion product to produce the authentic protein. The utility of this technique has been demonstrated by expression of three different proteins in yeast as both unfused and ubiquitin-fused forms: 1) the alpha subunit of the mammalian stimulating G-protein of the adenylate cyclase complex (Gs alpha); 2) a soluble fragment of the T cell receptor protein (sCD4); and 3) the protease domain of human urokinase (UKP). The sequence specificity of the Ub-Xase was demonstrated by mutagenesis of the carboxyl-terminal glycine of ubiquitin to an alanine, which inhibited ubiquitin removal in vivo. Processing of the ubiquitin-Gs alpha fusion protein (ub-Gs alpha) in vivo resulted in Gs alpha which could be reconstituted in mammalian membrane preparations and had the same specific activity as the authentic Gs alpha expressed in yeast. The yeast Ub-Xase has also been shown to work in vitro by the processing of a ub-sCD4 fusion protein synthesized in Escherichia coli. This technology should greatly enhance the utility of yeast for heterologous protein production.