Characterization of a branched-chain amino-acid aminotransferase from Schizosaccharomyces pombe

The Saccharomyces cerevisiae genes for the cytosolic and mitochondrial branched-chain amino-acid aminotransferases (BCAT) were isolated recently. These genes show significant homology to mammalian ECA39, originally isolated as a gene regulated by the c-myc oncogene. We now report the isolation of the Schizosaccharomyces pombe eca39/BCAT gene. The S. pombe protein shows 47–52% identity to other eukaryotic BCAT proteins isolated from S. cerevisiae, nematode, mouse and man. A genetic growth assay for BCAT activity was established using an S. cerevisiae strain disrupted in both BCAT isoenzymes. Consequently, the activity of the S. pombe BCAT was demonstrated by genetic and biochemical means. Possible applications of BCAT-encoding genes as selection markers in yeast transformation are proposed. The sequence has been deposited in the GenBank data library under Accession Number U88029. © 1998 John Wiley & Sons, Ltd.     

Cloning,sequencing and disruption of the ARG8 gene encoding acetylornithine aminotransferase in the petite-negative yeast Kluyveromyces lactis

A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68·1% and 35·0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine. The nucleotide sequence of KlARG8 has been submitted to GenBank under Accession Number U93209.     

Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4⁺ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe. © 1998 John Wiley & Sons, Ltd.     

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.     

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.     

Heterologous HIS3 Marker and GFP Reporter Modules for PCR-Targeting in Saccharomyces cerevisiae

We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosaccharomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1·4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His⁺ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0·9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2·4 kb-long double modules flanked by 40–45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter. © 1997 by John Wiley & Sons, Ltd.     

Schizosaccharomyces pombe RNase MRP RNA is homologous to metazoan RNase MRP RNAs and may provide clues to interrelationships between RNase MRP and RNase P

RNase MRP and RNase P ribonucleoproteins are structurally and functionally similar across a large evolutionary distance. To better characterize possible complex interrelationships between these two enzymes, we have employed the fission yeast Schizosaccharomyces pombe. Unlike Saccharomyces cerevisiae, S. pombe is believed to harbour only one genetic locus for the RNA component of RNase P and does not contain a known mitochondrially encoded RNase P RNA. We have identified the single nuclear gene for the RNA component of RNase MRP in S. pombe, mrp-1, by homology to vertebrate RNase MRP RNAs. The mrp-1 gene encodes an RNA of maximum mature length 400 nucleotides that shares a high degree of identity, in evolutionarily conserved regions, to both vertebrate RNase MRP RNAs and S. pombe RNase P RNA. Disruption of mrp-1 in the diploid strain SP826 and sporulation of tetrads resulted in a 2 dead:2 viable segregation, consistent with the gene being essential. Lethality is rescued by a plasmid-borne copy of mrp-1. Partially purified ribonucleoprotein RNase MRP activity correctly and efficiently processed all previously characterized heterologous mitochondrial RNA substrates. The compact mitochondrial genome of S. pombe contains sequence elements with >50% identity to mammalian D-loop CSBI and CSBII elements. The identification of mrp-1 in S. pombe should facilitate not only comparisons between the related ribonucleoproteins RNase MRP and RNase P, but should also provide an opportunity for genetic elucidation of RNase MRP function in a situation reflective of the animal kingdom.     

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.     

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.     

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated,sorted and matured in the fission yeast Schizosaccharomyces pombe

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPY^sc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPY^sc produced in the fission yeast has a higher molecular mass than mature CPY^sc produced by the budding yeast. CPY^sc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPY^sc produced by S. cerevisiae. CPY^sc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPY^sc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.     

Inhibitory effect of arginine on proline utilization in Saccharomyces cerevisiae

Proline is a predominant amino acid in grape must, but it is poorly utilized by the yeast Saccharomyces cerevisiae in wine-making processes. This sometimes leads to a nitrogen deficiency during fermentation and proline accumulation in wine. Although the presence of other nitrogen sources under fermentation conditions is likely to interfere with proline utilization, the inhibitory mechanisms of proline utilization remain unclear. In this study, we examined the effect of arginine on proline utilization in S. cerevisiae. We first constructed a proline auxotrophic yeast strain and identified an inhibitory factor by observing the growth of cells when proline was present as a sole nitrogen source. Intriguingly, we found that arginine, and not ammonium ion, clearly inhibited the growth of proline auxotrophic cells. In addition, arginine prevented the proline consumption of wild-type and proline auxotrophic cells, indicating that arginine is an inhibitory factor of proline utilization in yeast. Next, quantitative polymerase chain reaction (PCR) analysis showed that arginine partially repressed the expression of genes involved in proline degradation and uptake. We then observed that arginine induced the endocytosis of the proline transporters Put4 and Gap1, whereas ammonium induced the endocytosis of only Gap1. Hence, our results may involve an important mechanism for arginine-mediated inhibition of proline utilization in yeast. The breeding of yeast that utilizes proline efficiently could be promising for the improvement of wine quality.     

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.     

The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids

The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein is similar to models proposed for other integral mambrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1·5 kb. The mae1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5′ to the MFm1 gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid. The sequence of the S. pombe mae1 gene is available in GenBank under Accession Number U21002.     

SpOPT1, a member of the oligopeptide family (OPT) of the fission yeast Schizosaccharomyces pombe,is involved in the transport of glutathione through the outer membrane of the cell

A protein involved in the transport of glutathione has been identified, cloned and characterized from the fission yeast Schizosaccharomyces pombe. Database searches revealed the Sz. pombe ORF SPAC29B12.10c as a close homologue to several members of the OPT family, including the Saccharomyces cerevisiae high‐affinity glutathione transporter Hgt1p. The gene product of SPAC29B12.10c has been identified as a protein, named SpOPT1, localized within the plasma membrane, transporting the tripeptide glutathione. Disruption of SPAC29B12.10c led to strains inable to grow on media containing glutathione as a sole source of sulphur, due to the inability to internalize the tripeptide. Disruptants contained significantly less glutathione than wild‐type cells. Furthermore, ΔSpopt1 strains were non‐viable in a glutathione biosynthesis‐defective (Δgsh2) background. However, it was possible to complement the disruption of Spopt1 by overexpressing the intact ORF in the disrupted strain. Copyright © 2009 John Wiley & Sons, Ltd.