Candida albicans SOU1 encodes a sorbose reductase required for L-sorbose utilization

Previous work in our laboratory showed that L-sorbose utilization in Candida albicans is subject to a novel form of regulation which involves a reversible increase or decrease in the copy number of chromosome 5. Furthermore, the structural gene SOU1 is required for L-sorbose utilization and encodes a member of the short chain dehydrogenase family. However, the precise function of SOU1 was not known and neither was the pathway for L-sorbose utilization. We have now expressed SOU1 at a high level from a replicative plasmid having a constitutive ADH1 promoter and purified a version of Sou1p tagged with the FLAG epitope at the N-terminus. Sou1FLAGNp has a sorbose reductase activity which utilizes NADPH as a co-factor and converts L-sorbose to D-sorbitol. It can also less efficiently utilize fructose as a substrate with NADPH as a co-factor, converting fructose to mannitol. In agreement with prediction, the purified enzyme has a subunit molecular weight of 31 kDa and a pI of about 4.8. It probably consists of four identical subunits and has a pH optimum of 6.2. The L-sorbose utilization pathway in C. albicans probably converts L-sorbose to fructose-6-phosphate via D-sorbitol as an intermediate. The first step is catalysed by Sou1p. We also found that C. albicans extracts have a D-sorbitol-6-phosphate dehydrogenase activity, not encoded by SOU1, which utilizes NADP as a co-factor. This activity has not been described previously in yeasts and may be involved in the conversion of phosphorylated D-sorbitol to fructose-6-phosphate or glucose-6-phosphate.     

The Candida albicans 14-3-3 gene, BMH1, is essential for growth.

The 14-3-3 proteins are a family of conserved small acidic proteins that have been implicated in playing major roles in a wide variety of signalling cascades. In Saccharomyces cerevisiae, the 14-3-3 genes (BMH1 and BMH2) are essential for normal pseudohyphal induction and normal bud cell development. The Bmh proteins function in the cAMP-dependent RAS/MAPK and rapamycin-sensitive signalling cascades. Deletion of only one BMH gene demonstrates no phenotypic differences under normal growth conditions. Strains deleted of both BMH1 and BMH2 are either non-viable or demonstrate sensitivity to environmental stresses. In Schizosaccharomyces pombe, the BMH homologues (RAD24 and RAD25) are essential for cell cycle control after DNA damage and deletion of both genes renders the cell inviable. The 14-3-3 gene in Candida albicans (BMH1) was identified using a novel adherence assay and differential display RT-PCR. Unlike other yeasts, C. albicans has only one 14-3-3 gene (BMH1). It was not possible to construct double knockouts by routine methods. These results suggested that the C. albicans BMH1 gene is essential. The essentiality of C. albicans BMH1 was confirmed by a PCR disruption technique. The C. albicans bmh1 Delta/BMH1 heterozygotes exhibit growth and morphogenetic defects. Therefore, the BMH1 gene in C. albicans (Accession No. AF038154) is an excellent candidate to improve our understanding of the coordinate regulation of cell cycle and morphogenesis.     

Yeast 14-3-3 proteins

14-3-3 proteins form a family of highly conserved proteins which are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. They interact with more than 200 different, mostly phosphorylated proteins. The molecular consequences of 14-3-3 binding are diverse: this binding may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization, to the interaction with other proteins or to shielding of binding sites. The binding partners, and hence the 14-3-3 proteins, are involved in almost every cellular process and 14-3-3 proteins have been linked to several diseases, such as cancer, Alzheimer's disease, the neurological Miller-Dieker and spinocerebellar ataxia type 1 diseases and bovine spongiform encephalopathy (BSE). The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both have two genes encoding 14-3-3 proteins, BMH1 and BMH2 and rad24 and rad25, respectively. In these yeasts, 14-3-3 proteins are essential in most laboratory strains. As in higher eukaryotes, yeast 14-3-3 proteins bind to numerous proteins involved in a variety of cellular processes. Recent genome-wide studies on yeast strains with impaired 14-3-3 function support the participation of 14-3-3 proteins in numerous yeast cellular processes. Given the high evolutionary conservation of the 14-3-3 proteins, the experimental accessibility and relative simplicity of yeasts make them excellent model organisms for elucidating the function of the 14-3-3 protein family.     

Negative regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by a Candida albicans orthologue of OPI1

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are coordinately regulated by a UAS element, designated ICRE (inositol/choline-responsive element). Opi1 is a negative regulator responsible for repression of ICRE-dependent genes in the presence of an excess of inositol and choline. Gene regulation by phospholipid precursors has been also reported for the pathogenic yeast Candida albicans. Screening of a data base containing raw sequences of the C. albicans genome project allowed us to identify an open reading frame exhibiting weak similarity to Opi1. Expression of the putative CaOPI1 in an opi1 mutant of S. cerevisiae could restore repression of an ICRE-dependent reporter gene. Similar to OPI1, overexpression of CaOPI1 strongly inhibited derepression of ICRE-driven genes leading to inositol-requiring transformants. Previous work has shown that Opi1 mediates gene repression by interaction with the pleiotropic repressor Sin3. The genome of C. albicans also encodes a protein similar to Sin3 (CaSin3). By two-hybrid analyses and in vitro studies for protein-protein interaction we were able to show that CaOpi1 binds to ScSin3. ScOpi1 could also interact with CaSin3, while CaOpi1 failed to bind to CaSin3. Despite of some conservation of regulatory mechanisms between both yeasts, these results suggest that repression of phospholipid biosynthetic genes in C. albicans is mediated by a mechanism which does not involve recruitment of CaSin3 by CaOpi1.     

Studies on the ATP3 gene of Saccharomyces cerevisiae: presence of two closely linked copies,ATP3a and ATP3b,on the right arm of chromosome II

In this paper, we present evidence that there are two closely linked copies of the ATP3 gene coding for the gamma subunit of the F(1)F(0)-ATPase complex (EC3.6.1.34) in four laboratory strains of Saccharomyces cerevisiae, even though the yeast genome project has reported that ATP3 is a single-copy gene on chromosome II. We previously reported that the gene dosage (three copies) of ATP1 and ATP2 is coincident with the subunit number of F(1)-alpha and F(1)-beta, but that the gene dosage of ATP3 was not consistent with the subunit stoichiometry of F(1)F(0)-ATPase. By applying long PCR and gene walking analyses, we estimated that the two copies of ATP3 were approximately 20 kb apart, and we designated that which is proximal to the centromere ATP3a, while we named that which is distal ATP3b. The nucleotide sequences of the two copies of ATP3 were identical to the reported sequence in the W303-1A, W303-1B and LL20 strains, while only the DC5 strain had a single base substitution in its ATP3a. With the exception of this substitution, the other nucleotide sequences were identical to the upstream 860 bp and the downstream 150 bp. The differences between ATP3 with the single base substitution (Ser(308) to Phe) and ATP3 without the substitution on the complementation of the ATP3 disruptant and on the maintenance of the mitochondrial DNA were observed, suggesting that Atp3ap and Atp3bp in the DC5 strain might have different functions. However, it should not always be necessary for yeast cells to carry different types of ATP3 because the other three strains carry the same type of ATP3. It was also demonstrated that the disruption of the ATP3 genes basically leads to a loss of wild-type mtDNA, but the stability of the mtDNA is not dependent on the ATP3 alone.     

Characterization of Candida albicans orthologue of the Saccharomyces cerevisiae signal-peptidase-subunit encoding gene SPC3

The Candida albicans orthologue of the SPC3 gene, which encodes one of the subunits essential for the activity of the signal peptidase complex in Saccharomyces cerevisiae, was isolated by complementation of a thermosensitive mutation in the S. cerevisiae SEC61 gene. The cloned gene (CaSPC3) encodes a putative protein of 192 amino acids that contains one potential membrane-spanning region and shares significant homology with the corresponding products from mammalian (Spc22/23p) and yeast (Spc3p) cells. CaSPC3 is essential for cell viability, since a hemizygous strain containing a single copy of CaSPC3 under control of the methionine-repressible MET3 promoter did not grow in the presence of methionine and cysteine. The cloned gene could rescue the phenotype associated with a spc3 mutation in S. cerevisiae, indicating that it is the true C. albicans orthologue of SPC3. However, in contrast with results previously described for its S. cerevisiae orthologue, CaSPC3 was not able to complement the thermosensitive growth associated with a mutation in the SEC11 gene. The heterologous complementation of the sec61 mutant suggests that Spc3p could play a role in the interaction that it is known to occur between the translocon (Sec61 complex) and the signal peptidase complex, at the endoplasmic reticulum membrane.     

The centromere-binding factor Cbf1p from Candida albicans complements the methionine auxotrophic phenotype of Saccharomyces cerevisiae

A gene encoding the centromere binding factor 1 (Cbf1p) of the human pathogenic yeast Candida albicans was cloned and characterized. An open reading-frame was detected which encoded a 223 amino acid protein with a calculated molecular weight of 25.8 kDa and a relative isoelectric point of 5.55. It shares 39% overall amino acid sequence identity with Saccharomyces cerevisiae Cbf1p. We localized the CaCBF1 gene on chromosome 4. Southern analysis indicated that CaCBF1 is probably present as a single copy gene per haploid genome. The CaCBF1 gene under the control of its own promoter was able to complement the methionine auxotrophic growth, the increased mitotic instability of CEN plasmids and the slow growth of a Saccharomyces cerevisiae cbf1Delta mutant strain.     

Common industrial sake yeast strains have three copies of the AQY1-ARR3 region of chromosome XVI in their genomes

Genomic analysis of industrial yeast strains is important for exploitation of their potential. We analysed the genomic structure of the most widely used sake yeast strain, Kyokai no. 7 (K7), by DNA microarray. Since the analysis suggested that the copy number of the AQY1-ARR3 region in the right arm of chromosome XVI was amplified, we performed Southern blot analysis using the AQY1 gene as a probe. The probe hybridized to three bands in the widely used sake strains derived from K7, but only to one band of 1.4 kb in the laboratory strains. Since the extra two bands were not observed in old sake strains, or in other industrial strains, the amplification of this region appeared to be specific for the widely used sake strains. The copy number of the AQY1-ARR3 region appeared to have increased by chromosomal translocation, since chromosomal Southern blot analysis revealed that the AQY1 probe hybridized to chromosomes IV and XIII, in addition to chromosome XVI, in which AQY1 of the laboratory strain is encoded. The chromosomal translocation was also confirmed by PCR analysis using primers that amplify the region containing the breakpoint. Cloning and sequencing of cosmids that encode the AQY1-ARR3 region revealed that this region is flanked by TG(1-3) repeats on the centromere-proximal side in chromosomes IV and XIII, suggesting that amplification of this region occurred by homologous recombination through TG(1-3) repeats. These results demonstrate the genomic characteristics of the modern widely used sake strains that discriminate them from other strains.     

Localization of the FAR3 gene: Genetic mapping and molecular cloning using a chromosome walk-‘n’-roll strategy

FAR3 is a newly-discovered yeast gene required specifically for pheromone-mediated cell cycle arrest. I have used strains harboring the far3-1 mutation to map the gene to the right arm of chromosome XIII, establishing the gene order CEN13-LYS7-MCM1-FAR3. I cloned the FAR3 gene based on its genetic map position using a strategy that combined chromosome walking and a related technique termed ‘chromosome rolling’. In addition to the genetic and physical localization of FAR3, I present data that suggest corrections to the tentative map positions of VAN1 and ARG80.     

Identification of a Candida albicans homologue of the PHO85 gene, a negative regulator of the PHO system in Saccharomyces cerevisiae

In a screen for the protein kinase genes of the human pathogenic yeast Candida albicans, a putative homologue (CaPHO85) of PHO85, a negative regulator of the PHO system of Saccharomyces cerevisiae, which is one of the cyclin-dependent protein kinases (CDKs), was isolated. An open reading frame (ORF) of this gene was identified encoding a predicted protein of 326 amino acids with a calculated molecular weight of 37.6 kDa. The amino acid sequence is highly homologous to S. cerevisiae Pho85 (62% identity) and its Aspergillus nidulans homologue (70% identity), but less homologous to Cdc28 (50% identity) of S. cerevisiae and to its C. albicans homologue CaCdc28 (49% identity), both of which are also CDK. The coding region for the C. albicans gene was interrupted by an intron of 81 nucleotides near the sequence encoding the N-terminal region, similarly to the case of the S. cerevisiae PHO85 gene. Alignment of CaPho85 with various yeast CDKs revealed that most of the domains for ATP-binding and protein kinase activity are conserved among fungal species. Southern blot analysis indicated that CaPHO85 is most likely present as a single copy gene. This gene complemented the pho85 mutation of S. cerevisiae by transformation.     

Candida albicans TDH3 gene promotes secretion of internal invertase when expressed in Saccharomyces cerevisiae as a glyceraldehyde-3-phosphate dehydrogenase-invertase fusion protein

We have checked the ability of the Candida albicans GAPDH polypeptide, which lacks a conventional N-terminal signal peptide, to reach the cell wall in Saccharomyces cerevisiae by using an intracellular form of the yeast invertase as a reporter protein. A hybrid TDH3-SUC2 gene containing the C. albicans TDH3 promoter sequences and a coding region encoding a fusion protein formed by the C. albicans GAPDH polypeptide, fused at its C-terminus with the yeast internal invertase, was constructed in a centromer derivative plasmid and transformed into a Suc(-) S. cerevisiae strain. Transformants displayed invertase activity measured in intact whole cells, and were able to grow on sucrose as the sole fermentable carbon source. Northern blot analysis with both TDH3 and SUC2 probes detected a single mRNA species of the expected size (about 2.7 kb), and Western immunoblot analysis of cell-free extracts, using a monoclonal antibody (mAb49) against a C. albicans GAPDH epitope, showed the presence of a 90 kDa polypeptide corresponding to the GAPDH-invertase fusion protein. This indicates that the TDH3 gene is able to direct part of the encoded gene product to the cell wall, and that any putative motifs for this targeting should be within the GAPDH amino acid sequence. Further analysis, using the same approach, of a panel of seven N- and C-terminal GAPDH truncates revealed that the region required for the cell wall targeting is located within the N-terminal half of the protein.     

A 37·5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes,a TCP-1-related gene,an open reading frame similar to the DAL80 gene,and a tRNAArg

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromsome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding γ-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Ccη subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNA^Arg3 (AGC) gene. The nucleotide sequence data reported in this paper have been deposited in the EMBL and GenBank databases under the accession number X85021.     

TOR kinase pathway and 14-3-3 proteins regulate glucose-induced expression of HXT1, a yeast low-affinity glucose transporter

Expression of HXT1, a gene encoding a Saccharomyces cerevisiae low-affinity glucose transporter, is regulated by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are scarce. In this study we show that 14-3-3 proteins are involved in the regulation of the expression of HXT1 by glucose. We also demonstrate that 14-3-3 proteins, in complex with Reg1, a regulatory subunit of Glc7 protein phosphatase, interact physically with Grr1 (a component of the SCF-Grr1 ubiquitination complex), a key player in the process of HXT1 induction by glucose. In addition, we show that the TOR kinase pathway participates actively in the induction of HXT1 expression by glucose. Inhibition of the TOR kinase pathway by rapamycin treatment abolishes HXT1 glucose induction. A possible involvement of PP2A protein phosphatase complex, through the Cdc55 B-subunit, in the glucose induction of HXT1 is also discussed.     

Cloning, sequencing and characterization of the alpha-aminoadipate reductase gene (LYS2) from Saccharomycopsis fibuligera

The gene putatively encoding alpha-aminoadipate reductase (AAR) was isolated successfully by degenerate PCR and chromosome walking, based on cassette PCR methods, from the dimorphous yeast Saccharomycopsis fibuligera PD70 and was named SfLYS2. Sequence analysis revealed that it contained a putative open reading frame (ORF) of 4161 bp and encoded a polypeptide of 1386 amino acids. The deduced translation product shared an identity of 53% and 51% to the Lys2p homologues of Candida albicans and Saccharomyces cerevisiae, respectively. An atypical TATA box and a GCN4-box element were found in the 5'-upstream region. Genomic Southern hybridization suggested the presence of a single locus of SfLYS2 in the S. fibuligera genome. Expression of the ORF of SfLYS2 in a lys2(-) strain of S. cerevisiae could functionally complement the lysine mutant of the S. cerevisiae strain. S. fibuligera could use lysine as the sole nitrogen source but its growth was inhibited on the alpha-aminoadipate (AA) medium. Approximately 90% of the mutants of S. cerevisiae resistant to AA are lysine auxotrophs; in contrast all the mutants of S. fibuligera resistant to AA recovered in this work were not lysine auxotrophs.     

Analysis of the expression and secretion of the Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae

The alpha-glucosidase gene of Candida tsukubaensis is contained within a 3.47 kb BamH1-Mlul fragment which, when introduced into Saccharomyces cerevisiae AH22 on a yeast-Escherichia coli shuttle vector, allows the transformants to utilize maltose as sole carbon source. Thus, the cloned gene confers a dominant selectable phenotype on transformed strains of S. cerevisiae which are otherwise unable to grow in nutrient media containing maltose, dextrin or other alpha-1.4-linked alpha-D-glucopyranosides, specifically hydrolysed by the alpha-glucosidase. The cloned enzyme expressed in yeast is secreted into the extracellular medium in a glycosylated form which accounts for up to 60% of the secreted protein and has a molecular size of 70-80 kilodalton (kDa). Deglycosylation of the alpha-glucosidase showed that the enzyme is composed of two distinct polypeptides with subunit molecular weights of 63-65 kDa (peptide 1) and 50-52 kDa (peptide 2). An increase in the level of expression of the alpha-glucosidase by yeast transformants in selective minimal medium was obtained by using a vector with increased copy number containing the leu2-d gene as selectable marker. The alpha-glucosidase gene promoter functions more effectively than the Gall-10 promoter in directing alpha-glucosidase expression in S. cerevisiae. It also directs the expression of high levels of beta-galactosidase activity in yeast when fused to a promoterless E. coli lacZ gene. Expression of the alpha-glucosidase gene under the control of its own promoter is constitutive, orientation dependent and not subject to catabolite repression.     

V. Yeast mapping reports. The MAG1 3-methladenine DNA glycosylase gene is closely linked to the SPT15 TATA-binding TFIID gene on chromosome V-R in Saccharomyces cerevisiae

The MAG1 gene encodes a 3-methyladenine DNA glycosylase, which is involved in DNA alkylation repair in Saccharomyces cerevisiae. The mag1 mutant is deficient in 3-methyladenine DNA glycosylase activity and shows enhanced sensitivity to several monofunctional alkylating agents. MAG1 is allelic to MMS5. This gene has been previously located on chromosome V by chromosomal hybridization. We present physical and genetic mapping data here showing that the MAG1 gene is located on chromosome V-R, proximal to and about 10 kilobase pairs away from the SPT15 gene coding for the yeast TATA-binding protein TFIID.