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.     

A DNA region of Torulaspora delbrueckii containing the HIS3 gene: sequence, gene order and evolution

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae his3 mutant strain. DNA sequence analysis revealed that the fragment contained two complete ORFs, which share a high similarity with S. cerevisiae His3p and Mrp51p, respectively. The cloned TdHIS3 gene fully complemented the his3 mutation of S. cerevisiae, confirming that it encodes for the imidazoleglycerol-phosphate dehydrate of T. delbrueckii. Two additional ORFs, with a high homology to S. cerevisiae PET56 and DED1 genes, were mapped upstream and downstream from TdHIS3 and TdMRP51, respectively. This genetic organization is analogous to that previously found in Saccharomyces kluyveri and Zygosaccharomyces rouxii. The evolutionary significance of gene order in this chromosomal region is analysed and discussed.     

Isolation and characterization of the TIM10 homologue from the yeast Pichia sorbitophila: a putative component of the mitochondrial protein import system

The Saccharomyces cerevisiae TIM10 gene encodes one of the few essential mitochondrial proteins that are required for the import of nuclear-encoded precursor proteins from the cytosol and their subsequent sorting into the different mitochondrial compartments. We have isolated and characterized a putative homologue of TIM10 from the halotolerant yeast Pichia sorbitophila. The Pichia TIM10 gene encodes a protein of 90 amino acids with 66% identity to S. cerevisiae Tim10p. It was capable of suppressing the temperature sensitivity of tim10-1 mutant in S. cerevisiae, suggesting that Pichia TIM10 is both a functional and structural homologue of S. cerevisiae TIM10. The putative Pichia TIM10 gene product contains all the four conserved cysteine residues and the two CX(3)C motifs typical of the Tim family proteins in the mitochondrial intermembrane space. Using anti-Tim10p serum, Western blots detected a protein of about 10 kDa, suggesting that the Pichia Tim10p is a mitochondrial protein. The results suggest that mitochondrial import and sorting systems might be also strongly conserved in other fungi. The coding sequence of the P. sorbitophila TIM10 has been deposited in the EMBL Nucleotide Sequence Database under Accession No. AJ243940.     

Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.     

Functional characterization of the Candida albicans homologue of secretion-associated and Ras-related (Sar1) protein

Secretion-associated and Ras-related protein (Sar1p) plays an essential role during the protein transport from the endoplasmic reticulum to the Golgi apparatus. The cDNA sequence of the Sar1 gene has been identified and characterized from the human yeast pathogen, Candida albicans. This cDNA encodes a protein of 190 amino acids, which shares a 78% sequence identity with Saccharomyces cerevisiae Sar1p and contains the conserved GTP-binding motifs of the small GTPase superfamily. Complementation studies confirmed that this cDNA encodes the functional homologue of ScSar1p. The recombinant C. albicans Sar1p exhibits GTP-binding activity in vitro that was abolished by deletion of one of the three GTP-binding motifs.     

Isolation and sequencing of the Candida albicans MSI3, a putative novel member of the HSP70 family

We have reported previously that the expression of CGR1 increased at an early stage of the yeast-mycelial transition (morphogenesis) in Candida albicans. We now show that Cgr1p interacts in a yeast two-hybrid system with the C. albicans Msi3p (CaMsi3p), a putative novel member of the heat shock protein 70 (HSP70) family. The DNA sequence of CaMSI3 encodes a predicted protein of 702 amino acids with a molecular mass of 78.6 kDa. The amino acid sequence of CaMsi3p is 63% identical to Msi3p/Sse1p of the HSP70 family of Saccharomyces cerevisiae. Further, CaMSI3 complemented the temperature-sensitive phenotype of the msi3(-) mutant of S. cerevisiae. Other heat shock proteins of C. albicans are required for morphogenesis and are highly antigenic. These observations suggest that CaMSI3 may well provide functions for this organism unrelated to a heat shock function. The DDBJ Accession No. for the sequence reported in this paper is AB061274.     

Mutation of the homologue of GDP-mannose pyrophosphorylase alters cell wall structure, protein glycosylation and secretion in Hansenula polymorpha

A Hansenula polymorpha mutant with enhanced ability to secrete a heterologous protein has been isolated. The mutation defines a gene, designated OPU24, which encodes a protein highly homologous to GDP-mannose pyrophosphorylase Psa1p/Srb1p/Vig9p of Saccharomyces cerevisiae and CaSrb1p of Candida albicans. The opu24 mutant manifests phenotypes similar to those of S. cerevisiae mutants depleted for GDP-mannose, such as cell wall fragility and defects in N- and O-glycosylation of secreted proteins. The influence of the opu24 mutation on endoplasmic reticulum-associated protein degradation is discussed. The GenBank Accession No. for the OPU24 sequence is AF234177.     

Isolation and sequence analysis of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer

The URA3 gene of Candida glycerinogenes WL2002-5, an industrial glycerol producer encoding orotidine-5'-phosphate decarboxylase enzyme, was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 786 bp, encoding a 262 amino acid protein, which shares 71.65% amino acid sequence similarity to the S. cerevisiae URA3 protein. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the C. glycerinogenes Ura3 (CgUra3) protein.     

Isolation and characterization of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Torulaspora delbrueckii

A DNA fragment containing the URA3 gene from Torulaspora delbrueckii was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an ORF of 795 bp, encoding a 264 amino acid protein, which shares a high similarity to the Saccharomycetaceae Ura3 proteins. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the TdUra3 protein. The GeneBank Accession No. for TdURA3 is AF518402.     

Cloning and analysis of the Kluyveromyces lactis TRP1 gene: a chromosomal locus flanked by genes encoding inorganic pyrophosphatase and histone H3

The TRP1 gene of the yeast Kluyveromyces lactis has been cloned from a genomic library by complementation of the Saccharomyces cerevisiae trp1-289 mutation. The gene was located within the clone by transposon mutagenesis and the coding region identified by DNA sequencing. This has indicated that K. lactis TRP1 encodes a 210-amino acid polypeptide which shows 53% identity to the homologous S. cerevisiae protein. The K. lactis TRP1 gene has been disrupted by substituting the S. cerevisiae URA3 gene for a large part of the TRP1 coding sequence. Replacement of the chromosomal TRP1 locus with this construction has enabled the production of non-reverting trp1- strains of K. lactis, while a genetic analysis of the disrupted allele confirmed that the TRP1 gene had been cloned. DNA sequencing has also shown that the K. lactis TRP1 sequence is flanked by genes encoding inorganic pyrophosphatase and histone H3, which we have designated IPP and HHT1 respectively. Hybridization studies have shown that in common with S. cerevisiae, K. lactis has two copies of the histone H3 gene. Each H3 gene is closely linked to a gene encoding histone H4 and in both yeast species the IPP gene is tightly linked to one of the histone gene pairs.     

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae PMR1 gene

A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca²⁺-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.     

Ynl038wp (Gpi15p) is the Saccharomyces cerevisiae homologue of human Pig-Hp and participates in the first step in glycosylphosphatidylinositol assembly.

Glycosylphosphatidylinositols (GPIs) are found in all eukaryotes and are synthesized in a pathway that starts with the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol (PI). This reaction is carried out by a protein complex, three of whose subunits in humans, hGpi1p, Pig-Cp and Pig-Ap, have sequence and functional homologues in the Saccharomyces cerevisiae Gpi1, Gpi2 and Gpi3 proteins, respectively. Human GlcNAc-PI synthase contains two further subunits, Pig-Hp and PigPp. We report that the essential YNL038w gene encodes the S. cerevisiae homologue of Pig-Hp. Haploid YNL038w-deletion strains were created, in which Ynl038wp could be depleted by repressing YNL038w expression using the GAL10 promoter. Depletion of Ynl038wp from membranes virtually abolished in vitro GlcNAc-PI synthetic activity, indicating that Ynl038wp is necessary for GlcNAc-PI synthesis in vitro. Further, depletion of Ynl038wp in an smp3 mutant background prevented the formation of the trimannosylated GPI intermediates that normally accumulate in this late-stage GPI assembly mutant. Ynl038wp is therefore required for GPI synthesis in vivo. Because YNL038w encodes a protein involved in GPI biosynthesis, we designate the gene GPI15. Potential Pig-Hp/Gpi15p counterparts are also encoded in the genomes of Schizosacchomyces pombe and Candida albicans.     

Molecular cloning of the CRM1 gene from Candida albicans

In a screen for Candida albicans genes capable of supressing a ste20Delta mutation in Saccharomyces cerevisiae, a homologue of the exportin-encoding gene CRM1 was isolated. The CaCRM1 gene codes for a protein of 1079 amino acids with a predicted molecular weight of 124 029 and isoelectric point of 5.04. Crm1p from C. albicans displays significant amino acid sequence homology with Crm1p from Saccharomyces cerevisiae (65% identity, 74% similarity), Schizosaccharomyces pombe (55% identity, 66% similarity), Caenorhabditis elegans (45% identity, 57% similarity), and Homo sapiens (48% identity, 59% similarity). Interestingly, CaCRM1 encodes a threonine rather than a cysteine at position 533 in the conserved central region, suggesting that CaCrm1p is leptomycin B-insensitive, like S. cerevisiae Crm1p. CaCRM1 on a high copy vector can complement a thermosensitive allele of CRM1 (xpo1-1) in S. cerevisiae, showing that CaCrm1p and S. cerevisiae Crm1p are functionally conserved. Southern blot analysis suggests that CaCRM1 is present at a single locus within the C. albicans genome. The nucleotide sequence of the CaCRM1 gene has been deposited at GenBank under Accession No. AF178855.     

Schizosaccharomyces pombe gmd3(+)/alg11(+) is a functional homologue of Saccharomyces cerevisiae ALG11 which is involved in N-linked oligosaccharide synthesis

The oligosaccharide of glycoproteins in the fission yeast Schizosaccharomyces pombe is unique in containing galactose. We isolated four mutants that had reduced amounts of galactose residues on their cell surface glycoproteins by fluorescence-activated cell sorter. The isolated four recessive mutants, gmd1 to gmd4, showed a defect in glycosylation of acid phosphatase, a cell surface glycoprotein. In gmd3 mutant cells, the amounts of both mannose and galactose residues were decreased on the cell surface galactomannoproteins, suggesting an underglycosylation of galactomannoproteins. The gmd3(+) gene encodes a protein that has significant similarity with Saccharomyces cerevisiae Alg11p and is likely to be involved in N-linked core oligosaccharide synthesis. ALG11 suppressed the gmd3 mutation, indicating that gmd3(+) gene is a functional homologue of the ALG11 gene. We therefore designated gmd3(+) as alg11(+).     

Identification of YPD1, a gene of Candida albicans which encodes a two-component phosphohistidine intermediate protein

We have identified the YPD1 phosphohistidine intermediate two-component gene of Candida albicans. YPD1 has an open reading frame of 552 bp. It is located on chromosome 1 and an mRNA specific for YPD1 is detected under both yeast and hyphal growth. YPD1 encodes a protein of 184 amino acids with an estimated molecular mass of 20.5 kDa. A search for similarities with other proteins in databases showed that CaYpd1p exhibits the greatest overall similarity with Ypd1p from Saccharomyces cerevisiae (34.2% identity; 49.4% similarity) as well as with the C-terminus half of a protein from Schizosaccharomyces pombe (Accession No. CAA22174). However, CaYpd1p also shows similarity with other eukaryotic and prokaryotic proteins which function as phosphohistidine intermediates in two-component phospho-relay systems. In these cases, similarity was restricted to the amino acid sequences which surround the conserved histidine residue that is phosphorylated. In addition, CaYPD1 (but not CaYPD1(H69Q)) complements the lack of YPD1 in S. cerevisiae. This observation supports the premise that CaYpd1p also may function as a phosphohistidine intermediate protein in C. albicans.     

Molecular cloning of CaYRB1, the Candida albicans RanBP1/YRB1 homologue.

The yeast Ran binding protein 1 (Yrb1p) is a small protein of 23 kDa that is highly conserved among eukaryotes. It stimulates the GTPase activity of Gsp1p in the presence of the GTPase activating protein Rna1p. In addition to its role in nucleocytoplasmic transport of macromolecules, YRB1/RanBP1 could be involved in the regulation of microtubules structure and dynamics. Since microtubules are tightly associated with morphological changes, we have been interested to study the role and function of YRB1 in the pathogenic fungus Candida albicans, where there is regulated change in cellular morphology. The gene product of CaYRB1 encodes a 212 amino acid protein displaying 73% homology to the S. cerevisiae homologue. The bacterially expressed gene product has an apparent molecular weight of 35.7 kDa. We show that it can complement a S. cerevisiae yrb1 null mutant and that its mRNA does not appear to be regulated in response to conditions inducing morphological changes in C. albicans.     

Glucose signalling pathway controls the programmed ribosomal frameshift efficiency in retroviral-like element Ty3 in Saccharomyces cerevisiae

Ty3 elements of S. cerevisiae contain two overlapping coding regions, GAG3 and POL3, which are functional homologues of retroviral gag and pol genes, respectively. Pol3 is translated as a Gag3-Pol3 fusion protein dependent on a +1 programmed frameshift at a site with the overlap between the two genes. We show that the Ty3 frameshift frequency varies up to 10-fold in S. cerevisiae cells depending on carbon source. Frameshift efficiency is significantly lower in cells growing on glucose as carbon source than in cells growing on poor alternative carbon sources (glycerol/lactate or galactose). Our results indicate that Ty3 programmed ribosomal frameshift efficiency in response to glucose signalling requires two protein kinases: Snf1p and cAMP-dependent protein kinase A (PKA). Increased frameshifting on alternative carbon sources also appears to require cytoplasmic localization of Snf1p, mediated by the Sip2p protein. In addition to the two required protein kinases, our results implicate that Stm1p, a ribosome-associated protein involved in nutrient sensing, is essential for the carbon source-dependent regulation of Ty3 frameshifting. These data indicate that Ty3 programmed ribosomal frameshift is not a constitutive process but that it is regulated in response to the glucose-signalling pathway.