Molecular,functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast,Schizosaccharomyces pombe

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase, hmg1+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmg1+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmg1+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMG1p, the hmg1+ protein induced membrane proliferations known as karmellae. A previously undescribed ‘feed-forward’ regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmg1+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.     

A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast

We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER-targeted proteins, pre-acid phosphatase from S. pombe and prepro-α-factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A–Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro-α-factor can be post-translationally imported into the fission yeast ER, while S. pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism.     

Malolactic fermentation by engineered Saccharomyces cerevisiae as compared with engineered Schizosaccharomyces pombe

The ability of yeast strains to perform both alcoholic and malolactic fermentation in winemaking was studied with a view to achieving a better control of malolactic fermentation in enology. The malolactic gene of Lactococcus lactis (mleS) was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The heterologous protein is expressed at a high level in cell extracts of a S. cerevisiae strain expressing the gene mleS under the control of the alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid. Malolactic enzyme specific activity is three times higher than in L. lactis extracts. Saccharomyces cerevisiae expressing the malolactic enzyme produces significant amounts of l-lactate during fermentation on glucose-rich medium in the presence of malic acid. Isotopic filiation was used to demonstrate that 75% of the l-lactate produced originates from endogenous l-malate and 25% from exogenous l-malate. Moreover, although a small amount of exogenous l-malate was degraded by S. cerevisiae transformed or not by mleS, all the exogenous degraded l-malate was converted into l-lactate via a malolactic reaction in the recombinant strain, providing evidence for very efficient competition of malolactic enzyme with the endogenous malic acid pathways. These results indicate that the sole limiting step for S. cerevisiae in achieving malolactic fermentation is in malate transport. This was confirmed using a different model, S. pombe, which efficiently degrades l-malate. Total malolactic fermentation was obtained in this strain, with most of the l-malate converted into l-lactate and CO₂. Moreover, l-malate was used preferentially by the malolactic enzyme in this strain also.     

Oxylipin Associated Co‐Flocculation in Yeasts

According to the lectin‐theory, the yeast Schizosaccharomyces pombe lacks the specific receptors (α‐mannans) necessary to facilitate co‐flocculation with Saccharomyces cerevisiae species. In this study we demonstrate oxylipin associated co‐flocculation between Sacch. cerevisiae and S. pombe strains using differential cell staining, immunofluoresence and ultrastructural studies. Using a 3‐hydroxy (OH) oxylipin specific antibody coupled to a fluorescing compound, 3‐OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3‐OH oxylipins was confirmed using gas chromatography‐mass spectrometry. Strikingly, when acetylsalicylic acid (aspirin), a 3‐OH oxylipins inhibitor, was added to Sacch. cerevisiae which was then mixed with S. pombe strains grown in complex media, co‐flocculation was significantly inhibited. We conclude that aspirin‐sensitive 3‐OH 8:0 is probably involved in co‐flocculation.     

Properties and Heterologous Expression of the Glucose Transporter GHT1 from Schizosaccharomyces pombe

Genomic DNA of the Schizosaccharomyces pombe glucose transporter, GHT1, was obtained by complementation of the glucose transport deficient Sz. pombe strain YGS-5. Here we describe the GHT1 gene that encodes a protein of 565 amino acids with a corresponding molecular mass of 62·5 kDa. This eukaryotic glucose transporter contains 12 putative transmembrane segments and is homologous to the HXT multigene family of S. cerevisiae with several amino acid motifs of this sugar transporter family. It is also homologous to other sugar carriers from human, mouse and Escherichia coli. The function of the Ght1 protein as a glucose transporter was proved both by homologous and heterologous expression in the Sz. pombe mutant YGS-5 and in the S. cerevisiae hxt mutant RE700A, respectively. Both transformed yeast strains transported d -glucose with substrate specificity similar to that in Sz. pombe wild-type cells. Moreover, the cells of the two transformed yeast strains accumulated 2-deoxy-d -glucose, a non-metabolizable d -glucose analogue, with an efficiency similar to Sz. pombe wild-type cells. The ability of the S. cerevisiae mutant RE700A to accumulate 2DG in an Δμdependent manner after transformation with GHT1 provides evidence that the Sz. pombe transporter catalyses an energy-dependent uptake of glucose. The sequence of GHT1 was deposited at EMBL, Outstation EBI, Accession Number X91218. ©1997 John Wiley & Sons, Ltd.     

Characterization of the Saccharomyces cerevisiae cdc42-1ts Allele and New Temperature-Conditional-Lethal cdc42 Alleles

Cdc42p is a highly conserved GTPase involved in controlling cell polarity and polarizing the actin cytoskeleton. The CDC42 gene was first identified by the temperature-sensitive cell-division-cycle mutant cdc42-1^ts in Saccharomyces cerevisiae. We have determined the DNA and predicted amino-acid sequence of the cdc42-1^ts allele and identified multiple mutations in the coding region and 5′ promoter region, thereby limiting its usefulness in genetic screens. Therefore, we generated additional temperature-conditional-lethal alleles in highly conserved amino-acid residues of both S. cerevisiae and Schizosaccharomyces pombe Cdc42p. The cdc42^W97R temperature-sensitive allele in S. cerevisiae displayed the same cell-division-cycle arrest phenotype (large, round unbudded cells) as the cdc42-1^ts mutant. However, it exhibited a bud-site selection defect and abnormal bud morphologies at the permissive temperature of 23°C. These phenotypes suggest that Cdc42p functions in bud-site selection early in the morphogenetic process and also in polarizing growth patterns leading to proper bud morphogenesis later in the process. In S. pombe, the cdc42^W97R mutant displayed a cold-sensitive, loss-of-function phenotype when expressed from the thiamine-repressible nmt1 promoter under repressing conditions. In addition, cdc42^T58A and cdc42^S71P mutants showed a temperature-sensitive loss-of-function phenotype when expressed in S. pombe; these mutants did not display a conditional phenotype when expressed in S. cerevisiae. These new conditional-lethal cdc42 alleles will be important reagents for the further dissection of the cell polarity pathway in both yeasts. © 1997 John Wiley & Sons, Ltd.     

Mdm31 protein mediates sensitivity to potassium ionophores but does not regulate mitochondrial morphology or phospholipid trafficking in Schizosaccharomyces pombe

Mdm31p is an inner mitochondrial membrane (IMM) protein with unknown function in Saccharomyces cerevisiae. Mutants lacking Mdm31p contain only a few giant spherical mitochondria with disorganized internal structure, altered phospholipid composition and disturbed ion homeostasis, accompanied by increased resistance to the electroneutral K⁺/H⁺ ionophore nigericin. These phenotypes are interpreted as resulting from diverse roles of Mdm31p, presumably in linking mitochondrial DNA (mtDNA) to the machinery involved in segregation of mitochondria, in mediating cation transport across IMM and in phospholipid shuttling between mitochondrial membranes. To investigate which of the roles of Mdm31p are conserved in ascomycetous yeasts, we analysed the Mdm31p orthologue in Schizosaccharomyces pombe. Our results demonstrate that, similarly to its S. cerevisiae counterpart, SpMdm31 is a mitochondrial protein and its absence results in increased resistance to nigericin. However, in contrast to S. cerevisiae, Sz. pombe cells lacking SpMdm31 are also less sensitive to the electrogenic K⁺ ionophore valinomycin. Moreover, mitochondria of the fission yeast mdm31Δ mutant display no changes in morphology or phospholipid composition. Therefore, in terms of function, the two orthologous proteins appear to have considerably diverged between these two evolutionarily distant yeast species, possibly sharing only their participation in ion homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

A new method to efficiently induce a site-specific double-strand break in the fission yeast Schizosaccharomyces pombe

Double‐strand DNA breaks are a serious threat to cellular viability and yeast systems have proved invaluable in helping to understand how these potentially toxic lesions are sensed and repaired. An important method to study the processing of DNA breaks in the budding yeast Saccharomyces cerevisiae is to introduce a unique double‐strand break into the genome by regulating the expression of the site‐specific HO endonuclease with a galactose inducible promoter. Variations of the HO site‐specific DSB assay have been adapted to many organisms, but the methodology has seen only limited use in the fission yeast Schizosaccharomyces pombe because of the lack of a promoter capable of inducing endonuclease expression on a relatively short time scale (~1 h). We have overcome this limitation by developing a new assay in which expression of the homing endonuclease I‐PpoI is tightly regulated with a tetracycline‐inducible promoter. We show that induction of the I‐PpoI endonuclease produces rapid cutting of a defined cleavage site (> 80% after 1 h), efficient cell cycle arrest and significant accumulation of the checkpoint protein Crb2 at break‐adjacent regions in a manner that is analogous to published findings with DSBs produced by an acute exposure to ionizing irradiation. This assay provides an important new tool for the fission yeast community and, because many aspects of mammalian chromatin organization have been well‐conserved in Sz. pombe but not in S. cerevisiae, also offers an attractive system to decipher the role of chromatin structure in modulating the repair of double‐stranded DNA breaks. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification and characterization of the KlCMD1 gene encoding Kluyveromyces lactis calmodulin

The KlCMD1 gene was isolated from a Kluyveromyces lactis genomic library as a suppressor of the Saccharomyces cerevisiae temperature-sensitive mutant spc110-124, an allele previously shown to be suppressed by elevated copy number of the S. cerevisiae calmodulin gene CMD1. The KlCMD1 gene encodes a polypeptide which is 95% identical to S. cerevisiae calmodulin and 55% identical to calmodulin from Schizosaccharomyces pombe. Complementation of a S. cerevisiae cmd1 deletion mutant by KlCMD1 demonstrates that this gene encodes a functional calmodulin homologue. Multiple sequence alignment of calmodulins from yeast and multicellular eukaryotes shows that the K. lactis and S. cerevisiae calmodulins are considerably more closely related to each other than to other calmodulins, most of which have four functional Ca²⁺-binding EF hand domains. Thus like its S. cerevisiae counterpart Cmd1p, the KlCMD1 product is predicted to form only three Ca²⁺-binding motifs. The KlCMD1 sequence has been assigned Accession Number AJ002021 in the EMBL/GenBank database. © 1998 John Wiley & Sons, Ltd.     

X. Yeast sequencing reports. Sequence analysis of a 40·2 kb DNA fragment located near the left telomere of yeast chromosome X

We have sequenced on both strands a 40,257 bp fragment located near the left telomere of chromosome X of Saccharomyces cerevisiae. The sequenced segment contains 21 open reading frames (ORFs) at least 100 amino acids long. Five of the ORFs correspond to known amino acid sequences: two hypothetical proteins in the subtelomeric Y′ repeat region of 65·4 and 12·8 KDa, the cytochrome B pre-mRNA processing CBP1 protein, the mitochondrial nuclease NUC1 and the CRT1 protein. Of the 16 remaining ORFs, eight show highest homologies with the S. cerevisiae hexose transporters family (two ORFs), the yeast α-glucosidase (two ORFs), the yeast PEP1 precursor, the Escherichia coli galactoside O-acetyltransferase, the S. cerevisiae 137·7 KDa protein located in the Y′ region and a protein of unknown function of Schizosaccharomyces pombe. Finally, eight of the ORFs exhibit no significant similarity with any amino acid sequences described in data banks. DNA sequence comparison has revealed the presence of different repeated elements characteristic of yeast chromosome ends. Disruption studies have been performed on two ORFs encoding putative proteins of unknown function. The sequence has been entered in the EMBL Data Library under Accession Number Z34098.     

Review: The Cct eukaryotic chaperonin subunits of Saccharomyces cerevisiae and other yeasts

All eight of the CCT1-CCT8 genes encoding the subunits of the Cct chaperonin complex in Saccharomyces cerevisiae have been identified, including three that were uncovered by the systematic sequencing of the yeast genome. Although most of the properties of the eukaryotic Cct chaperonin have been elucidated with mammalian systems in vitro, studies with S. cerevisiae conditional mutants revealed that Cct is required for assembly of microtubules and actin in vivo. Cct subunits from the other yeasts, Candida albicans and Schizosaccharomyces pombe, also have been identified from partial and complete DNA sequencing of genes. Cct8p from C. albicans, the only other completely sequenced Cct protein from a fungal species other than S. cerevisiae, is 72% and 61% similar to the S. cerevisiae and mouse Cct8 proteins, respectively. The C. albicans CCT8 sequence has been assigned the Accession Number U37371 in the GenBank/EMBL database.     

Isolation of a Schizosaccharomyces pombe Gene Which in High Copy Confers Resistance to the Nucleoside Analogue 5-Azacytidine

Treatment of Schizosaccharomyces pombe with the C5 DNA methyltransferase (C5Mtase) inhibitor 5-azacytidine (5-azaC) has previously been shown to induce G2 checkpoint-dependent cell cycle arrest. S. pombe strains defective in both the checkpoint control pathways and in DNA repair processes are sensitive to 5-azaC. Here we describe the isolation of azr1as a multi-copy suppressor of the 5-azaC sensitivity of G2 checkpoint and DNA repair-deficient strains. azr1⁺ encodes a putative 25 kDa protein with limited homology to a Saccharomyces cerevisiae open reading frame of unknown function. The azr1⁺ gene is not essential and the null mutant shows no alteration in either DNA repair or checkpoint properties. We also report the sequence of the putative fission yeast cytidine deaminase gene, designated pcd1⁺, which lies immediately adjacent to azr1⁺ but which plays only a moderate role in suppression of 5-azaC sensitivity. These data have been deposited with EMBL nucleotide sequence database, Accession Number X98329. © 1997 John Wiley & Sons, Ltd.     

Fermentation Characteristics and Aromatic Profile of Plum Wines Produced with Indigenous Microbiota and Pure Cultures of Selected Yeast

The aim of this study was to assess and compare fermentation characteristics and aromatic profile of plum wines produced with indigenous microbiota and pure cultures of different selected yeast. Experiments were carried out with plum (Prunus domestica L.) varieties of different fruit ripening times (?a?anska rana, ?a?anska lepotica, and Po?ega?a). Wine fermentations were conducted by the activity of indigenous microbiota, commercially available Saccharomyces cerevisiae and Saccharomyces bayanus yeast strains and joint activity of Schizosaccharomyces pombe and S. cerevisiae (sequential inoculation). Statistically significant differences in fermentative characteristics and the content of certain volatile compounds were observed as a result of metabolic activity of various indigenous and/or selected yeasts during fermentation of plum pomace. Minimal duration of fermentation (4 to 5 d) and fastest ethanol production rate (from 12.3 to 15.5 g/L/d) were the characteristics of the studied S. cerevisiae strains. Isobutanol, 3‐methyl‐1‐butanol, 1‐heptanol, and 1‐octanol were the most prevalent higher alcohols in the tested plum wine samples. The predominant ester in plum wines was ethyl acetate, ethyl lactate, amyl acetate, isoamyl acetate, and ethyl palmitate, esters responsible for the floral and fruity olfactory tones, were also present in large amounts. Also, the use of S. cerevisiae strains resulted in the production of plum wines with better sensory characteristics than ones produced with other investigated yeasts. Obtained results are significant since there is limited data on the compounds responsible for the unique flavor of plum wine, as well as on the impact of different yeast starter cultures application on the overall quality of fruit wines.     

MPR1 as a novel selection marker in Saccharomyces cerevisiae

L ‐Azetidine‐2‐carboxylic acid (AZC) is a toxic four‐membered ring analogue of L ‐proline that is transported into cells by proline transporters. AZC and L ‐proline in the cells are competitively incorporated into nascent proteins. When AZC is present in a minimum medium, misfolded proteins are synthesized in the cells, thereby inhibiting cell growth. The MPR1 gene has been isolated from the budding yeast Saccharomyces cerevisiae Σ1278b as a multicopy suppressor of AZC‐induced growth inhibition. MPR1 encodes a novel acetyltransferase that detoxifies AZC via N‐acetylation. Since MPR1 is absent in the laboratory strain of S. cerevisiae S288C, it could be a positive selection marker that confers AZC resistance in the S288C background strains. To examine the usefulness of MPR1, we constructed some plasmid vectors that harboured MPR1 under the control of various promoters and introduced them into the S288C‐derived strains. The expression of MPR1 conferred AZC resistance that was largely dependent on the expression level of MPR1. In an additional experiment, the galactose‐inducible MPR1 and ppr1⁺, the fission yeast Schizosaccharomyces pombe homologue of MPR1, were used for gene disruption by homologous recombination, and here AZC‐resistant colonies were also successfully selected. We concluded that our MPR1–AZC system provides a powerful tool for yeast transformation. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of YHR019 in Saccharomyces cerevisiae chromosome VIII as the gene for the cytosolic asparaginyl-tRNA synthetase

Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae mutant strain 8556a, we have isolated the gene for the cytosolic asparaginyl-tRNA synthetase (AsnRS) of S. cerevisiae, by functional complementation of the mutation affecting this strain. The isolated gene could be identified to the open reading frame YHR019, called DED81, located on chromosome VIII. The mutant gene from the 8556a strain, asnrs⁻-1, was amplified from genomic DNA by PCR. This gene contains a point mutation, leading to the replacement of a glycine residue by a serine in a region of the protein probably important for the asparaginyl-adenylate recognition. The protein encoded by YHR019 is very similar to cytosolic AsnRS from other eukaryotic sources. In a phylogenetic analysis based on AsnRS sequences from various organisms, the eukaryotic sequences were clustered. Expression of YHR019 in Escherichia coli demonstrated that a yeast AsnRS activity was produced. The recombinant enzyme was purified to homogeneity in three chromatography steps. We showed that the recombinant S. cerevisiae AsnRS was able to charge unfractionated yeast tRNA, but not E. coli tRNA, with asparagine. © 1998 John Wiley & Sons, Ltd.