Functional analysis of Candida albicans genes whose Saccharomyces cerevisiae homologues are involved in endocytosis

PCR-based techniques for directed gene alterations have become standard tools in Candida albicans. To help to increase the speed of functional analysis of Candida albicans genes, we previously constructed and updated a modular set of pFA-plasmid vectors for PCR-based gene targeting in C. albicans. Here we report the functional analyses of C. albicans ORFs whose homologues in S. cerevisiae are involved in endocytosis, to explore their potential involvement in polarized cell growth. Three C. albicans genes, ABP1, BZZ1 and EDE1, were found to be non-essential. Yeast and hyphal morphogenesis were not affected by the individual deletions and the mutant strains appeared wild-type-like under the different growth conditions tested. On the other hand, deletion of both alleles of the C. albicans PAN1 homologue was not feasible. Promoter shut-down experiments using a MET3p-PAN1/pan1 strain indicated severe growth defects and abolished endocytosis, indicating that PAN1 is an essential gene. Subcellular distribution of CaAbp1 and CaPan1 was analysed via GFP-tagged proteins. Both proteins were found to localize at the cortex and at hyphal tips in a patch-like manner, supporting their role in endocytosis. Localization patterns of Abp1 and Pan1, however, were distinct from that of the FM4-64 stained Spitzenk?rper.     

Isolation and characterization of Candida albicans homologue of RAP1, a repressor and activator protein gene in Saccharomyces cerevisiae

To study the function of RAP1, a Candida albicans gene (CaRAP1) that shows sequence similarity to RAP1 of Saccharomyces cerevisiae was isolated by colony hybridization. DNA sequencing predicted an open reading frame of 429 amino acids with an overall identity of 24% to the ScRap1p. The DNA binding domain (DBD) was highly conserved, and EMSA using a GST-CaRap1p fusion protein confirmed its binding ability to the RPG-box of S. cerevisiae ENO1. In contrast, the N-terminus was less conserved and a moderate homology was observed in the BRCT domain. Interestingly, CaRap1p did not contain the C-terminal activation/repression region of ScRap1p.     

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.     

Cloning and sequence of a 3·835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5-like gene from Candida albicans

We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3′-terminal half of a gene encoding a PEX5-like protein. The EMBL/DDJB/GenBank Accession Number of this sequence is AJ003115. © 1998 John Wiley & Sons, Ltd.     

Sequencing of a 4.3 kbp region of chromosome 2 of Candida albicans reveals the presence of homologues of SHE9 from Saccharomyces cerevisiae and of bacterial phosphatidylinositol-phospholipase C

The nucleotide sequence of a 4.3 kb fragment downstream of the LIG4 gene of Candida albicans has been determined. This fragment contains two entire ORFs (ORF1 and ORF2) and a truncated one (ORF3). ORF1 (1029 bp; EMBL databank, Accession No. AJ277539) encodes a putative protein of 343 amino acids with a high degree of similarity to phosphatidylinositol-specific phospholipases C (PI-PLC) of bacterial origin and, to a lesser degree, to similar proteins from trypanosome, fly and human. Isolated ORF1 confers PI-PLC activity to Escherichia coli transformants. ORF2 (1572 bp; EMBL databank, Accession No. AJ277538) predicts a protein of 524 amino acids with high similarity along most of the entire length to Ydr393w from Saccharomyces cerevisiae. This protein carries a domain with significant similarity to several cytoskeleton proteins of different origins. YDR393w (SHE9) is an orphan gene whose overexpression compromises cell growth. ORF3 appears to encode the homologue of the well-conserved proteasomal 26S regulatory subunit.     

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.     

Isolation and sequence of the gene encoding ornithine decarboxylase,SPE1, from Candida albicans by complementation of a spe1Δ ctrain 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 (spe1Δ) 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. The GenBank accession number for this gene is U85005. © 1997 John Wiley & Sons, Ltd.     

A 12·8 kb segment,on the right arm of chromosome II from Saccharomyces cerevisiae including part of the DUR1, 2 gene,contains five putative new genes

A 12 820 bp fragment from the right arm of chromosome II of Saccharomyces cerevisiae was sequenced and analysed. This fragment contains six non-overlapping long open reading frames (ORFs) designated from the centromere- to the telomere-proximal ends as: YBR1441, 1443, 1444, 1445, 1446 and 1448. YBR1441 encodes a polypeptide of 845 amino acids which shares a long consensus domain with products of S. cerevisiae MCM2, MCM3, CDC46 and Schizosaccharomyces pombe cdc21⁺ genes. These genes are involved in DNA replication. YBR1445 encodes a polypeptide of 404 amino acids which has strong similarity with the S. cerevisiae KRE2/MNT1, YUR1, KTR1 gene products. The KRE2/MNT1 protein is an α-1,2-mannosyltransferase. The product of YBR1444, which encodes a protein of 375 amino acids, presents a lipase signature sequence and a peroxisomal targeting signal. YBR1448, whose sequence extends further on the telomere-proximal end of the fragment, is identical to the 3′ end of the DUR1,2 gene encoding urea amidolyase. The two ORFs, YBR1443 and YBR1446, exhibit no significant similarity with any known gene.     

The ALD6 gene of Saccharomyces cerevisiae encodes a cytosolic,Mg2+-activated acetaldehyde dehydrogenase

The deduced translation product of an open reading frame on the left arm of chromosome XVI of Saccharomyces cerevisiae, with the systematic name of YPL061w, is 500 amino acids in length and shares significant homology with aldehyde dehydrogenases. Amino acids 2 to 16 of the protein encoded by YPL061w were found to be identical to the N-terminal 15 amino acids of the purified cytosolic, Mg²⁺-activated acetaldehyde dehydrogenase (ACDH) of S. cerevisiae. This enzyme is thought to be involved in the production of acetate from which cytosolic acetyl-CoA is then synthesized. Deletion of YPL061w was detrimental to the growth of haploid strains of yeast; an analysis of one deletion mutant revealed a maximum specific growth rate (in complex medium containing glucose) of one-third of that displayed by the wild-type strain. Mutants deleted in YPL061w were also unable to use ethanol as a carbon source. As expected, the cytosolic, Mg²⁺-activated ACDH activity had been lost from the mutants, although the mitochondrial, K⁺-activated ACDH was readily detected. YPL061w has been registered with the name of ALD6 in the Saccharomyces Genome Database and the nucleotide sequence submitted to GenBank as part of accession number U39205. © 1997 John Wiley & Sons, Ltd.     

X. Yeast sequencing reports. Sequence and function analysis of a 9·74 kb fragment of Saccharomyces cerevisiae chromosome X including the BCK1 gene

In the framework of the European BIOTECH project for sequencing the Saccharomyces cerevisiae genome, we have determined the nucleotide sequence of the cosmid clone 233 provided by F. Galibert (Rennes Cedex, France). We present here 9743 base pairs of sequence derived from the left arm of chromosome X. This sequence reveals three new open reading frames and includes the published sequence (5′ end and open reading frame) of the gene BCK1/SLK1/SSP31 also identified as ORFAA. Deletion mutants of two earlier unknown open reading frames J0840 and J0904 are viable and the open reading frame J0902 is essential for yeast growth. The sequence has been entered in the EMBL data library under accession number X77923.     

The nucleotide sequence of a 2·1 kb fragment from chromosome VI of Saccharomyces cerevisiae identifies a tRNAGly gene,part of a delta element and a palindromic sequence

The nucleotide sequence was determined of a 2·1 kb DNA fragment located at approximately 35 kb to the right of the centromere of chromosome VI from Saccharomyces cerevisiae. Analysis revealed the presence of a tRNA^GLy gene, part of a delta element and a remarkable palindromic sequence. The longest open reading frame found encodes a putative protein of 195 amino acids. Although the fragment was isolated by hybridization to a human diacylglycerol kinase cDNA, no evidence was obtained for the presence of a gene encoding diacylglycerol kinase.     

The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene

We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology. The complete nucleotide sequence was submitted to the EMBL Data Library under Accession Number X83690.     

Functional analysis of six genes from chromosomes XIV and XV of Saccharomyces cerevisiae reveals YOR145c as an essential gene and YNL059c/ARP5 as a strain-dependent essential gene encoding nuclear proteins

We report here basic functional analysis of strains deleted for six open reading frames (ORFs), YNL059c and YNL148c from chromosome XIV and YOR145c, YOR152c, YOR161c and YOR162c from chromosome XV of Saccharomyces cerevisiae. ORFs were replaced with the KanMX4 resistance marker using a long flanking homology PCR strategy in FY1679 and W303 diploid strains. Replacement cassettes were constructed in plasmid pUG7 and the cognate wild-type genes were recovered by gap repair. Sporulation and tetrad analysis revealed that deletion of YNL059c/ARP5 was lethal for vegetative growth in strain W303 and caused severe growth defects in strain FY1679 while YOR145c was essential for growth in both strains. Fusion of the green fluorescent protein (GFP) gene to the 3' ends of the YNL059c/ARP5 and YOR145c coding sequences created functional chimeric genes at the respective chromosomal loci. Both Arp5-GFP and Yor145-GFP localized to the nucleus, Yor145-GFP concentrating in the nucleolus. The vectors containing the deletion cassettes and the cognate wild-type genes, the oligonucleotides, and the deletant strains are available from the EUROFAN resource centre EUROSCARF (Frankfurt).