Improved tools for efficient mapping of fission yeast genes: identification of microtubule nucleation modifier mod22‐1 as an allele of chromatin‐ remodelling factor gene swr1

Fission yeast genes identified in genetic screens are usually cloned by transformation of mutants with plasmid libraries. However, for some genes this can be difficult, and positional cloning approaches are required. The mutation swi5‐39 reduces recombination frequency in homozygous crosses and has been used as a tool in mapping gene position (Schmidt, 1993 ). However, strain construction in swi5‐39‐based mapping is significantly more laborious than is desirable. Here we describe a set of strains designed to make swi5‐based mapping more efficient and more powerful. The first improvement is the use of a swi5Δ strain marked with kanamycin (G418) resistance, which greatly facilitates identification of swi5 mutants. The second improvement, which follows directly from the first, is the introduction of a large number of auxotrophic markers into mapping strains, increasing the likelihood of finding close linkage between a marker and the mutation of interest. We combine these new mapping strains with a rec12Δ‐based approach for initial mapping of a mutation to an individual chromosome. Together, the two methods allow an approximate determination of map position in only a small number of crosses. We used these to determine that mod22‐1, a modifier of microtubule nucleation phenotypes, encodes a truncation allele of Swr1, a chromatin‐remodelling factor involved in nucleosomal deposition of H2A.Z histone variant Pht1. Expression microarray analysis of mod22‐1, swr1Δ and pht1Δ cells suggests that the modifier phenotype of mod22‐1 mutants may be due to small changes in expression of one or more genes involved in tubulin function. Copyright © 2009 John Wiley & Sons, Ltd.     

FEN2: A gene implicated in the catabolite repression-mediated regulation of ergosterol biosynthesis in yeast

We have isolated and characterized a pleiotropic recessive mutation, fen2-1, that causes resistance to fenpropimorph and a low level of ergosterol in Saccharomyces cerevisiae. Ergosterol synthesis in the mutant strain was 5·5-fold slower than in the wild type; however, in vitro assays of the enzymes involved in ergosterol biosynthesis could not account for this low rate in the mutant. The mutant phenotype was expressed only in media exerting both carbon and nitrogen catabolite repression. To our knowledge, this is the first locus in yeast that reveals a concerted regulation between different pathways (carbon and nitrogen catabolite repression and/or general control of amino acid biosynthesis and ergosterol biosynthesis). The yeast gene FEN2 has been isolated and contains an open reading frame (ORF) of 512 codons. This ORF was found to be identical to YCR28C of chromosome III. A possible function of the FEN2 gene product in yeast is discussed.     

Effect of low temperature upon vitality of Saccharomyces cerevisiae phospholipid mutants

The phospholipid metabolism of Saccharomyces cerevisiae plays a central role in its adaptation to low temperatures. In order to detect the key genes in this adaptation, various phospholipid mutants from the EUROSCARF collection of Saccharomyces cerevisiae BY4742 were tested to ascertain whether the suppression of some genes could improve the fermentation vitality of the cells at low temperature. The cell vitality and phospholipid composition of these mutants were analysed. Some knockouts improved (hmn1Δ) or impaired (cho2Δ and psd1Δ) their vitality at low temperature (13 °C) but were not affected at optimum temperature (25 °C). A common trait of the mutants that had some defect in vitality was a lower concentration of phosphatidylcholine and/or phosphatidylethanolamine. The supplementation with choline allowed them to recover viability, probably by synthesis through the Kennedy pathway. Hmn1Δ showed a lower concentration of phosphatidylcholine, which explains the dominant role of the de novo pathway in cellular phosphatidylethanolamine and phosphatidylcholine vs the Kennedy pathway. The absence of such genes as CRD1 or OPI3 produced important changes in phospholipid composition. Cardiolipin was not detected in crd1Δ but phosphatidylglycerol circumvents most of the functions assigned to CL. The considerable reduction in PC diminished the cell vitality of opi3Δ at both temperatures, although the decrease at 13 °C was more marked. Copyright © 2012 John Wiley & Sons, Ltd.     

Dual functions of Mdt1 in genome maintenance and cell integrity pathways in Saccharomyces cerevisiae

Recent evidence indicates considerable cross‐talk between genome maintenance and cell integrity control pathways. The RNA recognition motif (RRM)‐ and SQ/TQ cluster domain (SCD)‐containing protein Mdt1 is required for repair of 3′‐blocked DNA double‐strand breaks (DSBs) and efficient recombinational maintenance of telomeres in budding yeast. Here we show that deletion of MDT1 (PIN4/YBL051C) leads to severe synthetic sickness in the absence of the genes for the central cell integrity MAP kinases Bck1 and Slt2/Mpk1. Consistent with a cell integrity function, mdt1Δ cells are hypersensitive to the cell wall toxin calcofluor white and the Bck1–Slt2 pathway activator caffeine. An RRM‐deficient mdt1‐RRM0 allele shares the severe bleomycin hypersensitivity, inefficient recombinational telomere maintenance and slt2 synthetic sickness phenotypes, but not the cell wall toxin hypersensitivity with mdt1Δ. However, the mdt1‐RRM(3A) allele, where only the RNA‐binding site is mutated, behaves similarly to the wild‐type, suggesting that the Mdt1 RRM functions as a protein–protein interaction rather than a nucleic acid‐binding module. Surprisingly, in a strain background where double mutants are sick but still viable, bck1Δmdt1Δ and slt2Δmdt1Δ mutants differ in some of their phenotypes, consistent with the emerging concept of flexible signal entry and exit points in the Bck1–Mkk1/2–Slt2 pathway. Overall, the results indicate that Mdt1 has partially separable functions in both cell wall and genome integrity pathways. Copyright © 2009 John Wiley & Sons, Ltd.     

ERG6 gene deletion modifies Kluyveromyces lactis susceptibility to various growth inhibitors

The ERG6 gene encodes an S‐adenosylmethionine dependent sterol C‐24 methyltransferase in the ergosterol biosynthetic pathway. In this work we report the results of functional analysis of the Kluyveromyces lactis ERG6 gene. We cloned the KlERG6 gene, which was able to complement the erg6Δ mutation in both K. lactis and Saccharomyces cerevisiae. The lack of ergosterol in the Klerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthesis pathway as well as the KlPDR5 gene encoding an ABC transporter. The Klerg6Δ mutation resulted in reduced cell susceptibility to amphotericin B, nystatin and pimaricin and increased susceptibility to azole antifungals, fluphenazine, terbinafine, brefeldin A and caffeine. The susceptibility phenotype was suppressed by the KlPDR16 gene encoding one of the phosphatidylinositol transfer proteins belonging to the Sec14 family. Decreased activity of KlPdr5p in Klerg6Δ mutant (measured as the ability to efflux rhodamine 6G) together with increased amount of KlPDR5 mRNA suggest that the zymosterol which accumulates in the Klerg6Δ mutant may not fully compensate for ergosterol in the membrane targeting of efflux pumps. These results point to the fact that defects in sterol transmethylation appear to cause a multitude of physiological effects in K. lactis cells. Copyright © 2016 John Wiley & Sons, Ltd.     

Direct Evidence That Maltose Transport Activity Is Affected by the Lipid Composition of Brewer's Yeast

A brewer's yeast strain was grown with maltose as sole carbon source under strictly anaerobic conditions with and without ergosterol and/or unsaturated fatty acid (Tween 80) supplements. Under all these conditions the MALx1 genes for maltose transporters were strongly expressed during growth. The fatty acid unsaturation indices of growing and stationary phase yeast were increased from about 20% to 56–69% by supplementation with Tween 80. Ergosterol contents were increased up to at least 4‐fold by supplementation with ergosterol and Tween 80. Maltose transport activity measured at 20°C was not increased by supplementation with Tween 80 alone, but was increased 2‐fold and 3‐fold, respectively, in growing and stationary phase yeast by supplementation with ergosterol together with Tween 80. The stimulation of maltose transport by ergosterol was greater when the transport was measured at temperatures (10°C and 0°C) lower than 20°C. The results show that proper function of maltose transporters requires adequate amounts of ergosterol in the yeast. This effect may partly explain the low maltose (and maltotriose) uptake rates both in the second half of brewery fermentations, when the sterol content of yeast has fallen, and when fresh wort is pitched with sterol‐deficient cropped yeast.     

Mutations sensitizing yeast cells to the start inhibitor nalidixic acid

The regulatory step Start in the cell cycle of the budding yeast Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal). To study this inhibition, mutations were identified that alter the sensitivity of yeast cells to Nal. Nal-sensitive mutations were sought because the inhibitory effects of Nal on wild-type cells are only transient, and wild-type cells naturally become refractory to Nal. Three complementation groups of Nal-sensitive mutations were found. Mutations in the first complementation group were shown to reside in the ARO7 gene, encoding chorismate mutase; tyrosine and phenylalanine synthesis was inhibited by Nal in these aro7 mutants, whereas wild-type chorismate mutase was unaffected. These aro7 alleles demonstrate ‘recruitment’, by mutation, of an innately indifferent protein to an inhibitor-sensitive form. The Nal-sensitive aro7 mutant cells were used to show that the resumption of Nal-inhibited nuclear activity and cell proliferation takes place while cytoplasmic Nal persists at concentrations inhibitory for the mutant chorismate mutase. Mutations in the second complementation group, nss2 (Nal-supersensitive), increased intracellular Nal concentrations, and may simply alter the permeability of cells to Nal. The third complementation group was found to be the ERG6 gene, previously suggested to encode the ergosterol biosynthetic enzyme sterol methyltransferase. Mutation or deletion of the ERG6 gene had little effect on the inhibition of Start by Nal, but prevented recovery from this inhibition. Mutation of ERG3, encoding another ergosterol biosynthetic enzyme, also caused Nal sensitivity, suggesting that plasma membrane sterol composition, and plasma membrane function, mediates recovery from Nal-mediated inhibition of Start.     

The sequence of 55 kb on the left arm of yeast chromosome XVI identifies a small nuclear RNA,a new putative protein kinase and two new putative regulators

We have sequenced and analysed a 55 786 bp fragment located on the left arm of chromosome XVI of Saccharomyces cerevisiae. The sequence contains 29 non-overlapping open reading frames (ORFs) longer than 300 bp, among which 12 genes have previously been sequenced: OYE3, REV3, SVS1, BEM4, CDC60, KIP2, PEP4, SPK1, PAL1, KES1, SNR17B and RPL37A. Three new ORFs, P2591, P2594 and P2597 are highly homologous to the human phosphotyrosyl phosphatase activator PTPA, to the pleiotropic regulator PRL1 of PP1 and PP2a protein phosphatases in plants and to the protein kinase PAR-1 in Caenorhabditis elegans, respectively. Three other ORFs, P2545, P2567 and P2578 have significant homology with ORFs of unknown function located on yeast chromosomes VIII, XVI and IV respectively. The sequences in nucleotides and in amino acids have been deposited in the EMBL data library under the Accession Number X96770.     

Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid‐induced oxidative stress in saccharomyces cerevisiae

Mutations in adenine biosynthesis pathway genes ADE1 and ADE2 have been conventionally used to score for prion [PSI⁺] in yeast. If ade1‐14 mutant allele is present, which contains a premature stop codon, [psi^?] yeast appear red on YPD medium owing to accumulation of a red intermediate compound in vacuoles. In [PSI⁺] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing to its amyloid aggregation allows for read‐through of the ade1‐14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red colour development in ade1 and ade2 mutant yeast requires reduced‐glutathione, which helps in transport of the intermediate metabolite P‐ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid‐induced oxidative stress would deplete reduced‐glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid‐forming human proteins TDP‐43, Aβ‐42 and Poly‐Gln‐103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour eventually reverted back to red upon turning off the amyloid protein's expression. Also, the aggregate‐bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2‐1, ade2Δ, and ade1Δ mutant yeast and also in an ade1‐14 mutant with erg6 gene deletion that increases cell‐wall permeability. This model would be useful tool for drug‐screening against general amyloid‐induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd.     

Mutations in the nucleotide-binding domain of putative sterol importers Aus1 and Pdr11 selectively affect utilization of exogenous sterol species in yeast

Sterol uptake in the yeast Saccharomyces cerevisiae is mediated by two plasma membrane ATP-binding cassette transporters, Aus1 and Pdr11. Their expression is regulated by oxygen and is triggered by anaerobic growth conditions. Under these conditions, internal ergosterol synthesis is arrested and utilization of exogenous sterol is vital for yeast cells. Here, we demonstrate that Aus1 is the major importer of non–yeast sterols, mammalian cholesterol, and plant sterols under anaerobic conditions. In contrast, uptake of yeast native sterol, ergosterol, is relatively low. This uptake could not be enhanced by overexpression of either of the transporters. Interestingly, overexpression of the minor importer Pdr11 resulted in a substantial import of non–yeast sterols. We show that mutation of the conserved residue in one of the ABC characteristic motifs—the H-loop in Aus1 and Pdr11—lowered their ATPase activity. The residual activity was sufficient to import exogenous sterols and to preserve cell viability. Importantly, the reduction of sterol import was dramatic for mammalian cholesterol and plant sterols, whereas import of yeast ergosterol was decreased only slightly indicating substrate selectivity of the sterol utilization process.     

ARH1 of Saccharomyces cerevisiae: A new essential gene that codes for a protein homologous to the human adrenodoxin reductase

A yeast gene was found in which the derived protein sequence has similarity to human and bovine adrenodoxin reductase (Nobrega, F. G., Nobrega, M. P. and Tzagoloff, A. (1992). EMBO J. 11, 3821–3829; Lacour, T. and Dumas, B. (1996). Gene 174, 289–292), an enzyme in the mitochondrial electron transfer chain that catalyses in mammals the conversion of cholesterol into pregnenolone, the first step in the synthesis of all steroid hormones. It was named ARH1 (Adrenodoxin Reductase Homologue 1) and here we show that it is essential. Rescue was possible by the yeast gene, but failed with the human gene. Supplementation was tried without success with various sterols, ruling out its involvement in the biosynthesis of ergosterol. Immunodetection with a specific polyclonal antibody located the gene product in the mitochondrial fraction. Consequently ARH1p joins the small group of gene products that affect essential functions carried out by the organelle and not linked to oxidative phosphorylation. © 1998 John Wiley & Sons, Ltd.     

Catalytic isoforms Tpk1 and Tpk2 of Candida albicans PKA have non‐redundant roles in stress response and glycogen storage

Candida albicans cAMP‐dependent protein kinase (PKA) is coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1). In this organism the cAMP/PKA signalling pathway mediates basic cellular processes, such as the yeast‐to‐hyphae transition and cell cycle regulation. In the present study, we investigated the role of C. albicans PKA in response to saline, heat and oxidative stresses as well as in glycogen storage. To fine‐tune the analysis, we performed the studies on several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in a different BCY1 genetic background. We observed that tpk1Δ/tpk1Δ strains developed a lower tolerance to saline exposure, heat shock and oxidative stress, while wild‐type and tpk2Δ/tpk2Δ mutants were resistant to these stresses, indicating that both isoforms play different roles in the stress response pathway. We also found that regardless of the TPK background, heterozygous and homozygous BCY1 mutants were highly sensitive to heat treatment. Surprisingly, we observed that those strains devoid of one or both TPK1 alleles were defective in glycogen storage, while strains lacking Tpk2 accumulated higher levels of the polysaccharide, indicating that Tpk1 and Tpk2 have opposite roles in carbohydrate metabolism. Copyright © 2009 John Wiley & Sons, Ltd.     

Targeting of heterologous membrane proteins into proliferated internal membranes in Saccharomyces cerevisiae

Overproduction of chimeric proteins containing the HMG2/1 peptide, which comprises the seven transmembrane domains of Saccharomyces cerevisiae 3-hydroxy-3-methylglutaryl-CoA reductase isozymes 1 and 2, has previously been observed to induce the proliferation of internal endoplasmic reticulum-like membranes. In order to exploit this amplified membrane surface area for the accommodation of heterologous microsomal proteins, we fused sequences coding for human cytochrome P4501A1 (CYP1A1) to sequence encoding the HMG2/1 peptide and expressed the hybrid genes in yeast. The heterologous hybrid proteins were targeted into strongly proliferated membranes, as shown by electron microscopic and immunofluorescent analysis. Fusion proteins comprising the whole CYP1A1 polypeptide (HMG2/1-CYP1A1) exhibited 7-ethoxyresorufin-O-deethylase activity, whereas fusion proteins lacking the N-terminal 56 amino acids of CYP1A1 (HMG2/1-ΔCYP1A1) were inactive and appeared to be unable to incorporate protoheme. Similar amounts of heterologous protein were detected in cells expressing HMG2/1-CYP1A1, HMG2/1-ΔCYP1A1 and CYP1A1, respectively. Replacement of the N-terminal membrane anchor domain of human NADPH-cytochrome P450 oxidoreductase by the HMG2/1 peptide also resulted in a functional fusion enzyme, which was able to interact with HMG2/1-CYP1A1 and the yeast endogenous P450 enzyme lanosterol-14α-demethylase.     

PAD1 and FDC1 are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae

The volatile phenols, to which Saccharomyces cerevisiae converts from phenylacrylic acids including ferulic acid, p-coumaric acid, and cinnamic acid, generate off-flavors in alcoholic beverages such as beer and wine. Using gene disruptants, transformants and cell-free extracts of these strains, we have verified that the adjacent PAD1 (phenylacrylic acid decarboxylase, YDR538W) and FDC1 (ferulic acid decarboxylase, YDR539W) genes are essential for the decarboxylation of phenylacrylic acids in S. cerevisiae. Pad1p and Fdc1p are homologous with UbiX and UbiD, respectively, in the ubiquinone synthetic pathway of Escherichia coli. However, ubiquinone was detected quantitatively in all of the yeast single-deletion mutants, Δpad1, Δfdc1, and double-deletion mutant, Δpad1Δfdc1.