The Saccharomyces cerevisiae enolase‐related regions encode proteins that are active enolases

In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase‐related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2‐phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double‐null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase‐related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes. Copyright © 2012 John Wiley & Sons, Ltd.     

The KlPMR1 gene of Kluyveromyces lactis encodes for a P‐type Ca2+‐ATPase

A novel P‐type Ca²⁺‐ATPase gene has been cloned and sequenced in the yeast Kluyveromyces lactis. The gene has been named KlPMR1 and is localized on chromosome I. The putative gene product contains 936 residues and has a calculated molecular weight of 102 437 Da. Analysis of the deduced amino acid sequence (KlPmr1p) indicated that the encoded protein retains all the highly conserved domains characterizing the P‐type ATPases. KlPmr1p shares 71% amino acid identity with Pmr1p of S. cerevisiae, 62% with HpPmr1p of Hansenula polymorpha, 56% with YlPmr1p of Yarrowia lipolytica and 52% with the Ca²⁺‐ATPase encoded for by the SPCA1 gene of Rattus norvegicus; these similarities place KlPmr1p in the SPCA group (secretory pathway Ca²⁺‐ATPase) of the P‐type ATPases. The K. lactis strain harbouring the Klpmr1 disrupted gene is not able to grow in presence of low calcium concentrations and shows hypersensitivity to high concentrations of EGTA in the medium. These defects are relieved by PMR1 of S. cerevisiae on a centromeric plasmid, demonstrating that KlPMR1 encodes for a functional Pmr1p homologue. The sequence described can be retrieved under EMBL Accession No. AJ001018. Copyright © 1999 John Wiley & Sons, Ltd.     

Establishment of a simple system to analyse the molecular interaction in the agglutination of Saccharomyces cerevisiae

Saccharomyces cerevisiae a-agglutinin, which is involved in mating and covalently anchoring to the cell wall, consists of two components, Aga1p and Aga2p, whose syntheses are individually regulated. To facilitate the analysis of the protein-protein interaction on agglutination between a- and alpha-agglutinins, the construction of a yeast strain (MATa) with the functional protein prepared by genetic fusion of Aga1p- and Aga2p-encoding genes and by the expression system using the UPR-ICL promoter derived from the n-alkane-assimilating yeast, Candida tropicalis, which is functional under the condition of lower glucose concentration was tried and the agglutination ability of the constructed strain was evaluated with a yeast strain (MATa) which expressed AGalpha1 encoding alpha-agglutinin under the control of the same promoter. The genes were integrated into the yeast chromosomes. Cell agglutination between both (MATa) strains was observed microscopically when these two strains were mix-cultured to a glucose-decreased concentration. The agglutination was further confirmed by the sedimentation test and by the quantification using a filter. These results proved that the constructed Aga1p-Aga2p fusion protein was enoughly functional for the interaction with the Agalpha1 protein, and that this phenomenon occurred dependent on glucose concentration, but independent of the peptide pheromones secreted by the cells of the opposite mating types. Using this system, the role of two disulphide linkages between Aga1p and Aga2p on the binding activity between Aga2p and Aga1p was first evaluated. Under the treatment by the SH-compound (dithiothreitol), in which Agalpha2p is easily released into the medium from the intact cell surface, the Aga1p and Aga2p fusion protein was a good tool to make clear the role of the disulphide linkages. As a result, the linkages had a significant effect on not only the assembly but also the binding activity. The novel and simple system described here may further facilitate the study of molecular interaction in agglutination.     

Relationship between growth of food‐spoilage yeast in high‐sugar environments and sensitivity to high‐intensity pulsed UV light irradiation

The relationship between prior growth of food‐spoilage yeast in high‐sugar environments and their subsequent survival postpulsed UV (PUV) irradiation was investigated. Test yeast were separately grown to early stationary phase in YPD broth containing increasing concentrations of glucose (1–50% w/v) and were flashed with ≤40 pulses of broad‐spectrum light at lamp discharge energy settings of 3.2, 7.2 and 12.8 J (equivalent to UV doses of 0.53, 1.09 and 3.36 μJ cm^?2, respectively) and their inactivation measured. Findings showed that prior growth in high‐sugar conditions (≥30% glucose w/v) enhanced the sensitivity of all nine representative strains of Zygosaccharomyces bailii, Z. rouxii and Saccharomyces cerevisiae yeast to PUV irradiation. Significant differences in inactivation amongst different yeast types also occurred depending on amount of UV dose applied, where the order of increasing sensitivity of osmotically stressed yeast to PUV irradiation was shown to be Z. rouxii, Z. bailii and >S. cerevisiae. For example, a 1.2‐log order difference in CFU mL^?1 reduction occurred between Z. bailii 11 486 and S. cerevisiae 834 when grown in 50% w/v sugar samples and treated with the uppermost test UV dosage of 3.36 μJ cm^?2, where these two yeast strains were reduced by 3.8 and 5.0 log orders, respectively, after this PUV treatment regime compared to untreated controls. The higher the UV dose applied the greater the reduction in yeast numbers. For example, a 1.0‐, 1.4‐ and 4.0‐log order differences in CFU mL^?1 numbers occurred for S. cerevisiae 834 grown in 15% w/v sugar samples and then treated with PUV dose of 0.53, 1.09 and 3.36 μJ cm^?2, respectively. These findings support the development of PUV for the treatment of high‐sugar foods that are prone to spoilage by osmotolerant yeast.     

Construction of self‐cloning bottom‐fermenting yeast with low vicinal diketone production by the homo‐integration of ILV5

The vicinal diketones (VDK), such as diacetyl and 2,3‐pentandione, impart an unpleasant butter‐like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self‐cloning, bottom‐fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α‐acetolactate and α‐aceto‐α‐hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae‐type ILV5 was inserted upstream of S. cerevisiae‐type ILV2 in bottom‐fermenting yeast to construct self‐cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM‐resistant transformants. The wort fermentation test, using the SC‐ILV5‐homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1‐propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self‐cloning brewer's yeast in which SC‐ILV5 was homo‐inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK‐producing, self‐cloning bottom‐fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. Copyright © 2012 John Wiley & Sons, Ltd.     

Genome‐wide screen of Saccharomyces cerevisiae for killer toxin HM‐1 resistance

We screened a set of Saccharomyces cerevisiae deletion mutants for resistance to killer toxin HM‐1, which kills susceptible yeasts through inhibiting 1,3‐beta‐glucan synthase. By using HM‐1 plate assay, we found that eight gene‐deletion mutants had higher HM‐1‐resistance compared with the wild‐type. Among these eight genes, five—ALG3, CAX4, MNS1, OST6 and YBL083C—were associated with N‐glycan formation and maturation. The ALG3 gene has been shown before to be highly resistant to HM‐1. The YBL083C gene may be a dubious open reading frame that overlaps partially the ALG3 gene. The deletion mutant of the MNS1 gene that encodes 1,2‐alpha‐mannosidase showed with a 13‐fold higher HM‐1 resistance compared with the wild‐type. By HM‐1 binding assay, the yeast plasma membrane fraction of alg3 and mns1 cells had less binding ability compared with wild‐type cells. These results indicate that the presence of the terminal 1,3‐alpha‐linked mannose residue of the B‐chain of the N‐glycan structure is essential for interaction with HM‐1. A deletion mutant of aquaglyceroporin Fps1p also showed increased HM‐1 resistance. A deletion mutant of osmoregulatory mitogen‐activated protein kinase Hog1p was more sensitive to HM‐1, suggesting that high‐osmolarity glycerol pathways plays an important role in the compensatory response to HM‐1 action. Copyright © 2010 John Wiley & Sons, Ltd.     

Yeast Ste23p shares functional similarities with mammalian insulin‐degrading enzymes

The S. cerevisiae genome encodes two M16A enzymes: Axl1p and Ste23p. Of the two, Ste23p shares significantly higher sequence identity with M16A enzymes from other species, including mammalian insulin‐degrading enzymes (IDEs). In this study, recombinant Ste23p and R. norvegicus IDE (RnIDE) were isolated from E. coli, and their enzymatic properties compared. Ste23p was found to cleave established RnIDE substrates, including the amyloid‐β peptide (Aβ1–40) and insulin B‐chain. A novel internally quenched fluorogenic substrate (Abz–SEKKDNYIIKGV–nitroY‐OH) based on the polypeptide sequence of the yeast P2 a‐factor mating propheromone was determined to be a suitable substrate for both Ste23p and RnIDE, and was used to conduct comparative enzymological studies. Both enzymes were most active at 37 °C, in alkaline buffers and in high salt environments. In addition, the proteolytic activities of both enzymes towards the fluorogenic substrate were inhibited by metal chelators, thiol modifiers, inhibitors of cysteine protease activity and insulin. Characteristics of STE23 expression were also evaluated. Our analysis indicates that the 5′ terminus of the STE23 gene has been mischaracterized, with the physiologically relevant initiator corresponding to residue M53 of the publicly annotated protein sequence. Finally, we demonstrate that, unlike haploid‐specific Axl1p, Ste23p is expressed in both haploid and diploid cell types. Our study presents the first comprehensive biochemical analysis of a yeast M16A enzyme, and provides evidence that S. cerevisiae Ste23p has enzymatic properties that are highly consistent with mammalian IDEs and other M16A enzymes. Copyright © 2009 John Wiley & Sons, Ltd.     

Plasmids with E2 epitope tags: tagging modules for N‐ and C‐terminal PCR‐based gene targeting in both budding and fission yeast,and inducible expression vectors for fission yeast

A single‐step PCR‐based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10‐amino acids (epitope E2a: SSTSSDFRDR)‐ and 12 amino acids (epitope E2b: GVSSTSSDFRDR)‐long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C‐terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM‐series plasmid. The N‐terminal E2a and E2b tagging modules were based on pOM‐series plasmid. The pOM‐series plasmids were selected for this study because of their use of the Cre–loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N‐terminal or C‐terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti‐E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2‐tagged strains. Copyright © 2009 John Wiley & Sons, Ltd.     

Isolation of a Histoplasma capsulatum cDNA that complements a mitochondrial NAD(+)‐isocitrate dehydrogenase subunit I‐deficient mutant of Saccharomyces cerevisiae

A cDNA library was prepared from Histoplasma capsulatum strain G‐217B yeast cells and an apparently full‐length cDNA for a subunit of the citric acid cycle enzyme NAD(+)‐isocitrate dehydrogenase was identified by sequence analysis. Its predicted amino acid sequence is more similar to the IDH1 regulatory subunit of S. cerevisiae NAD(+)‐isocitrate dehydrogenase than to the IDH2 catalytic subunit. After expression in S. cerevisiae from an S. cerevisiae promoter, it was shown to functionally complement an S. cerevisiae idh1 mutant, but not an idh2 mutant, for growth on acetate as a carbon source and for production of NAD(+)‐isocitrate dehydrogenase enzyme activity. These results confirm that the H. capsulatum cDNA encodes a homologue of subunit I of the S. cerevisiae mitochondrial isocitrate dehydrogenase isozyme that functions in the citric acid cycle. The HcIDH1 cDNA sequence is available in GenBank with Accession No. AF009036. Copyright © 1999 John Wiley & Sons, Ltd.     

Effects of Lipid‐Transfer Protein from Malting Barley Grain on Brewers Yeast Fermentation

A lipid‐transfer protein (LTP), which belongs to a family of pathogenesis‐related (PR) proteins, was isolated from malting barley grain. This LTP significantly decreased fermentation and respiration of brewers yeast (Saccharomyces cerevisiae) and caused the leakage of cell constituents. These effects were dose dependent tending to saturation at higher concentrations (～ 200 μg/mg yeast dry weight cells). It was found that LTP survives the thermal treatment during the mashing process. Despite yeast fermentation inhibition in vitro, this LTP did not appear to cause impairment of yeast fermentation capability in the brewing process.     

Identification of a putative response regulator two‐component phosphorelay gene ( CaSSK1) from Candida albicans

We have identified and analysed a putative response regulator two‐component gene (CaSSK1) from Candida albicans and its encoding protein (CaSsk1p). CaSSK1 has an open reading frame of 2022 bp. In the promotor region of CaSSK1 a short sequence is found that matches the consensus sequence of the stress response elements (STRE) from Saccharomyces cerevisiae. CaSSK1 is located on chromosome 1 and is expressed in either yeast or mycelial phases of C. albicans. CaSSK1 encodes a 674 amino acid protein (CaSsk1p) with an estimated molecular mass of 73·5 kDa and a basic isoelectric point (pI 9·5). It has a tripeptide (NKA) located in its C‐terminus, which resembles the peroxisomal signalling target type 1 sequence (PST1) of most of the peroxisomal matrix proteins. A homology search of CaSsk1p with other proteins in databases showed that the C‐terminus of CaSsk1p exhibits the greatest similarity with the C‐terminus of Ssk1p and Mcs4 from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The response regulator domain of CaSsk1p contains the motifs that are characteristic of all response regulators, including the conserved aspartate and lysine residues as well as the putative aspartate, which is phosphorylated by a phosphohistidine residue. Finally, in spite of the structural similarities among CaSsk1p, Ssk1p and Mcs4, CaSsk1p does not seem to exhibit functional homology with these proteins. The Accession No. for the described sequence is AF084608, as filed in the EMBL/GenBank/DDBJ database. Copyright © 1999 John Wiley & Sons, Ltd.     

Isolation and characterization of the carbon catabolite‐derepressing protein kinase Snf1 from the stress tolerant yeast Torulaspora delbrueckii

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae snf1Δ mutant strain. DNA sequence analysis revealed that the fragment contained a complete open reading frame (ORF), which shares a high similarity with the S. cerevisiae energy sensor protein kinase Snf1. The cloned TdSNF1 gene was able to restore growth of the S. cerevisiae snf1Δ mutant strain on media containing nonfermentable carbon sources. Furthermore, cells of the Tdsnf1Δ mutant were unable to proliferate under nonfermenting conditions. Finally, protein domain analysis showed that TdSnf1p contains a typical catalytic protein kinase domain (positions 41–293), which is also present in other Snf1p homologues. Within this region we identified a protein kinase ATP‐binding region (positions 48–71) and a consensus Ser/Thr protein kinase active site (positions 160–172). The GenBank Accession No. for the sequenced DNA fragment is HM131845. Copyright © 2010 John Wiley & Sons, Ltd.     

Human NKCC2 cation–Cl– co‐transporter complements lack of Vhc1 transporter in yeast vacuolar membranes

Cation–chloride co‐transporters serve to transport Cl^– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co‐transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co‐transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt‐sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma‐membrane alkali–metal cation exporters Nha1 and Ena1‐5 and the vacuolar cation–chloride co‐transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild‐type and mutated cation–chloride co‐transporters. Copyright © 2013 John Wiley & Sons, Ltd.     

Next‐generation biofuels: a new challenge for yeast

Economic growth depends strongly on the availability and price of fuels. There are various reasons in different parts of the world for efforts to decrease the consumption of fossil fuels, but biofuels are one of the main solutions considered towards achieving this aim globally. As the major bioethanol producer, the yeast Saccharomyces cerevisiae has a central position among biofuel‐producing organisms. However, unprecedented challenges for yeast biotechnology lie ahead, as future biofuels will have to be produced on a large scale from sustainable feedstocks that do not interfere with food production, and which are generally not the traditional carbon source for S. cerevisiae. Additionally, the current trend in the development of biofuels is to synthesize molecules that can be used as drop‐in fuels for existing engines. Their properties should therefore be more similar to those of oil‐derived fuels than those of ethanol. Recent developments and challenges lying ahead for cost‐effective production of such designed biofuels, using S. cerevisiae‐based cell factories, are presented in this review. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification and characterization of a drug‐sensitive strain enables puromycin‐based translational assays in Saccharomyces cerevisiae

Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C‐terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6?, pdr1? and pdr3? (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin‐based assays in yeast, which will enable new avenues of inquiry into protein production and maturation. Copyright © 2014 John Wiley & Sons, Ltd.     

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen‐specific but non‐neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen‐specific antibody responses in mice. When used as a model antigen, parenteral immunization of mice with surface‐expressing GFP yeast induced a strong anti‐GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection. Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti‐JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238–398, 373–399 and 373–500 in front of a Gly–Ser linker were detected by anti‐JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface‐expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants. Copyright © 2009 John Wiley & Sons, Ltd.