Rag−mutations involved in glucose metabolism in yeast: Isolation and genetic characterization

Some natural isolates and many laboratory strains of the yeast Kluyveromyces lactis cannot grow on glucose when respiration is inhibited by antimycin A. The ability or inability to grow on glucose in the absence of mitochondrial respiration has been called Rag⁺ or Rag^? phenotype (resistance to antimycin on glucose, respectively). Rag^? strains, unable to grow on glucose in the presence of the respiratory drug, behave as if they were defective in fermentation. The Rag phenotype was first found to be determined by variant alleles of either of the two nuclear genes, RAG1 and RAG2, which code for a low-affinity glucose transport protein and for phosphoglucose isomerase, respectively. These findings suggested that the Rag^? phenotype can be used to obtain mutations of genes involved in glucose metabolism in K. lactis. We thus looked for other Rag^? mutants. Seventy-four mutants were isolated and genetically characterized. All of the mutations were nuclear recessive alleles, defining 11 new complementation groups, which we designate rag3 through rag13.     

XIII. Yeast sequencing reports. SED6 is identical to ERG6, and encodes a putative methyltransferase required for ergosterol synthesis

Luminal endoplasmic reticulum (ER) proteins carry a sorting signal that allows them to be retrieved from the Golgi apparatus by a specific receptor. In yeast, this receptor is encoded by the ERD2 gene. Although retrieval of ER proteins does not appear to be an essential process, cells lacking ERD2 do not grow. Several multicopy suppressors of this growth defect have been isolated. The sequence of one of these, SED6, is presented here. Its product contains motifs characteristic of methyltransferases, and it is identical to ERG6, the presumed structural gene for S-adenosylmethionine:Δ²⁴-sterol-C-methyltransferase. The gene is located adjacent to PDR4, near the centromere of chromosome XIII.     

ERG1, encoding squalene epoxidase,is located on the right arm of chromosome VII of Saccharomyces cerevisiae

The ERG1 gene of Saccharomyces cerevisiae encodes squalene epoxidase, a key enzyme in the ergosterol pathway. ERG1 is an essential gene. Disruption of the gene with URA3 results in a lethal phenotype when cells are grown under aerobic conditions, even in the presence of ergosterol. However, cells are viable in the presence of ergosterol under anaerobic growth conditions during which ergosterol is taken up by cells. Physical and genetic mapping data reveal that ERG1 is located on the right arm of chromosome VII proximal to QCR9 at a distance of 14·6 cM from ADE3.     

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.     

Effects of deletion of different PP2C protein phosphatase genes on stress responses in Saccharomyces cerevisiae

A key mechanism of signal transduction in eukaryotes is reversible protein phosphorylation, mediated through protein kinases and protein phosphatases (PPases). Modulation of signal transduction by this means regulates many biological processes. Saccharomyces cerevisiae has 40 PPases, including seven protein phosphatase 2C (PP2C PPase) genes (PTC1–PTC7). However, their precise functions remain poorly understood. To elucidate their cellular functions and to identify those that are redundant, we constructed 127 strains with deletions of all possible combinations of the seven PP2C PPase genes. All 127 disruptants were viable under nutrient‐rich conditions, demonstrating that none of the combinations induced synthetic lethality under these conditions. However, several combinations exhibited novel phenotypes, e.g. the Δptc5Δptc7 double disruptant and the Δptc2Δptc3Δptc5Δptc7 quadruple disruptant exhibited low (13°C) and high (37°C) temperature‐sensitive growth, respectively. Interestingly, the septuple disruptant Δptc1Δptc2Δptc3Δptc4Δptc5Δptc6Δptc7 showed an essentially normal growth phenotype at 37°C. The Δptc2Δptc3Δptc5Δptc7 quadruple disruptant was sensitive to LiCl (0.4 m ). Two double disruptants, Δptc1Δptc2 and Δptc1Δptc4, displayed slow growth and Δptc1Δptc2Δptc4 could not grow on medium containing 1.5 m NaCl. The Δptc1Δptc6 double disruptant showed increased sensitivity to caffeine, congo red and calcofluor white compared to each single deletion. Our observations indicate that S. cerevisiae PP2C PPases have a shared and important role in responses to environmental stresses. These disruptants also provide a means for exploring the molecular mechanisms of redundant PTC gene functions under defined conditions. Copyright © 2014 John Wiley & Sons, Ltd.     

Glucose metabolism and ethanol production in adh multiple and null mutants of Kluyveromyces lactis

Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten-fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen-limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants.     

Synthesis of monohydroxylated inositolphosphorylceramide (IPC-C) in Saccharomyces cerevisiae requires Scs7p,a protein with both a cytochrome b5-like domain and a hydroxylase/desaturase domain

Saccharomyces cerevisiae mutants lacking Scs7p fail to accumulate the inositolphosphorylceramide (IPC) species, IPC-C, which is the predominant form found in wild-type cells. Instead scs7 mutants accumulate an IPC-B species believed to be unhydroxylated on the amide-linked C₂₆-fatty acid. Elimination of the SCS7 gene suppresses the Ca²⁺-sensitive phenotype of csg1 and csg2 mutants. The CSG1 and CSG2 genes are required for mannosylation of IPC-C and accumulation of IPC-C by the csg mutants renders them Ca²⁺-sensitive. The SCS7 gene encodes a protein that contains both a cytochrome b₅-like domain and a domain that resembles the family of cytochrome b₅-dependent enzymes that use iron and oxygen to catalyse desaturation or hydroxylation of fatty acids and sterols. Scs7p is therefore likely to be the enzyme that hydroxylates the C₂₆-fatty acid of IPC-C. © 1998 John Wiley & Sons, Ltd.     

Isolation of the Pichia pastoris PYC1 gene encoding pyruvate carboxylase and identification of a suppressor of the pyc phenotype

We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris. Disruption of this gene produced inability to grow in minimal medium with glucose as carbon source and ammonium as nitrogen source. Growth was possible with aspartate or glutamate as nitrogen source. The gene PpPYC1 expressd from its own promoter was able to rescue the phenotype of Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. In a P. pastoris strain carrying a disrupted PpPYC1 gene we have isolated spontaneous mutants able to grow in non-permissive conditions. In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepressed. The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions. The sequence of the PpPYC1 gene has been entered in the EMBL nucleotide sequence databank: Accession Number Y11106. © 1998 John Wiley & Sons, Ltd.     

Isolation and characterization of Schizosaccharomyces pombe fragile mutants

Three Schizosaccharomyces pombe fragile mutants requiring the presence of an osmotic stabilizer to grow, that lyse when transferred into hypotonic solutions and that secrete to the extracellular medium more protein than the parental strain were isolated. In the three mutants, the fragile phenotype segregated in a Mendelian fashion, indicating a single chromosomal gene mutation, and behaved as a recessive character. By complementation analysis, the three fragile mutants fell in a single complementation group, defining the same gene (SRB1). Mutations of this gene are responsible for alterations in the cells such as fragile character, increase in the cell wall porosity, changes in the cell morphology and floc-forming ability. The study of the three srb1 alleles indicated that the degree of these alterations is proportional to a significant decrease in the galactomannan fraction of the mutants cell wall. The data presented in this report suggest that the product of the SRB1 gene is critical for the maintenance of the integrity and structure of Sz. pombe cell wall.     

QCR9, the nuclear gene encoding a small subunit of the mitochondrial cytochrome bc1 complex,maps to the right arm of chromosome VII in Saccharomyces cerevisiae

We present here mapping data for QCR9, a nuclear gene encoding a subunit of the ubiquinol-cytochrome c oxidoreductase complex. Deletion of QCR9 results in the inability of cells to grow on non-fermentable carbon sources at 37°C. Thus, qcr9 mutants can be scored by growing cells on YPE/G at 37°C, or followed by the URA3 marker, which was inserted when making the qcr9 deletion strain, JDP1. The location of QCR9 on the right arm of chromosome VII with respect to the previously mapped genes ADE3, SER2 and PET54 is given.     

基于尿嘧啶磷酸核糖转移酶为负向筛选标记的生酮古龙酸杆菌基因组无痕修饰系统的构建

通过构建尿嘧啶磷酸核糖转移酶基因(upp)敲除打靶质粒,采用同源重组的方法获得了生酮古龙酸杆菌(Ketogulonigenium vulgare)的upp基因缺失突变株K.vulgareΔupp,可作为基因组无痕修饰系统底盘细胞的upp基因表达载体,转化突变株K.vulgareΔupp,获得upp基因回补菌株PBB-upp,并验证了upp作为负向筛选标记的可行性。实验结果表明:突变株Δupp在1 mg/m L的5-氟尿嘧啶培养基上可生长,野生型和回补菌株PBB-upp不能生长,3株菌对5-氟尿嘧啶的抗性存在显著差异,说明可以将upp基因作为负向筛选标记,与正向筛选标记相结合,在upp基因缺失的底盘细胞基础上通过两次同源重组,实现基因组的无痕修饰与改造。     

Hansenula polymorpha Pex1p and Pex6p are peroxisome‐associated AAA proteins that functionally and physically interact

We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional complementation of the corresponding peroxisome‐deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the AAA protein family. Cells deleted for either gene (Δpex1 or Δpex6) were characterized by the presence of small peroxisomal remnants which contained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell fractionation studies HpPex1p and HpPex6p co‐sedimented with the peroxisomal membrane protein HpPex3p in both wild‐type cells and in Δpex4, Δpex8 or Δpex14 cells. Both proteins are loosely membrane‐bound and face the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisomal matrix protein import. By contrast, overexpression of PEX1 was not detrimental to the cells. Interestingly, co‐overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex1p and HpPex6p remained predominantly membrane‐bound, but only partially co‐localized with the peroxisomal membrane protein HpPex3p. Our data indicate that HpPex1p and HpPex6p function in a protein complex associated with the peroxisomal membrane and that overproduced, mislocalized HpPex6p prevents HpPex1p from reaching its site of activity. Copyright © 1999 John Wiley & Sons, Ltd.     

Sterols of seed oils of Vernonia galamensis,Amaranthus cruentus,Amaranthus caudatus,Amaranthus hybridus and Amaranthus hypochondriacus grown in the humid tropics

Analysis of sterols of seed oils from Vernonia galamensis, Amaranthus cruentus, A caudatus, A hybridus and A hypochondriacus, the last four being exotic breeds planted in the humid tropics of Africa, is presented in this report. Identifiable sterols in all the seed oil samples include campesterol, stigmasterol, ß-sitosterol, Δ⁵-avenasterol and Δ⁷-avenasterol except for Vernonia galamensis where cholesterol was detected (63.61 mg per 100 g oil).     

A short-chain dehydrogenase gene from Pichia stipitis having D-arabinitol dehydrogenase activity

An NAD⁺-dependent D-arabinitol dehydrogenase (polyol dehydrogenase) gene was isolated from Pichia stipitis CBS 6054 and cloned in Saccharomyces cerevisiae. The gene was isolated by screening of a λ-cDNA library with a zymogram technique. D -Arabinitol, xylitol, D -glucitol and galactitol are substrates for the recominant protein. With D -arabinitol as substrate the reaction product is D -ribulose. The molecular weight of the native tetramer enzyme is 110 000 Da and the monomer is 30 000 Da. The amino acid sequence is homologous to the short-chain dehydrogenase family. It is 85·5% identical to a D -arabinitol dehydrogenase from Candida albicans. The gene in P. stipitis was induced by D -arabinitol and P. stipitis was able to grow on D -arabinitol. The physiological role of D -rabinitol metabolism is discussed.     

Phytosterol content in seven oat cultivars grown at three locations in Sweden

The total and individual sterol content in 21 oat samples (seven cultivars grown at three different locations in Sweden) were analysed by gas chromatography after acid hydrolysis. The total sterol content in these oat cultivars varied between 350–491 µg g⁻¹ of dry weight of kernel. The most abundant phytosterol was β‐sitosterol (237–321 µg g⁻¹) followed by campesterol (32–46 µg g⁻¹), Δ⁵‐avenasterol (15–47 µg g⁻¹) and stigmasterol (11–21 µg g⁻¹). There was a statistically significant difference in total sterol content between cultivars (p < 0.05) but no effect was found for cultivation location. Furthermore when contents of Δ⁵‐avenasterol in hexane‐extracted oat oil and acid‐hydrolysed oat samples were compared, it was noticed that the content of Δ⁵‐avenasterol was lowered due to acid hydrolysis. © 1999 Society of Chemical Industry     

Identification of peroxisomal membrane ghosts with an epitope-tagged integral membrane protein in yeast mutants lacking peroxisomes

Many yeast peroxisome biogenesis mutants have been isolate in which peroxisomes appear to be completely absent. Introduction of a wild-type copy of the defective gene causes the reappearance of peroxisomes, despite the fact that new peroxisomes are thought to form only from pre-existing peroxisomes. This apparent paradox has been explained for similar human mutant cell lines (from patients with Zellweger syndrome) by the discovery of peroxisomal membrane ghosts in the mutant cells (Santos, M. J., T. Imanaka, H. Shio, G. M. Small and P. B. Lazarow. 1988. Science 239 , 1536–1538). Introduction of a wild-type gene is thought to restore to the ghosts the ability to import matrix proteins, and thus lead to the refilling of the peroxisomes. It is vitally important to our understanding of peroxisome biogenesis to determine whether the yeast mutants contain ghosts. We have solved this problem by introducing an epitope-tagged version of Pas3p, a peroxisome integral membrane protein (that is essential for peroxisome biogenesis). Nucleotides encoding a nine amino acid HA epitope were added to the PAS3 gene immediately before the stop codon. The tagged gene (PAS3_HA) was inserted in the genome, replacing the wild-type gene at its normal locus. It was fully functional (the cells assembled peroxisomes normally and grew on oleic acid) but the expression level was too low to detect the protein with monoclonal antibody 12CA5. PAS3_HA was expressed in greater quantity from an episomal plasmid with the CUP1 promoter. The gene product, Pas3p_HA, was detected by immunogold labelling on the membranes of individual and clustered peroxisomes; the clusters appeared as large spots in immunofluorescence. PAS3_HA was similarly expressed in peroxisome biogenesis mutants peb2 and peb4, which lack morphologically recognizable peroxisomes. Gold-labelled membranes were clearly visible in both mutants: in peb2 the labelled membrane vesicles were generally much smaller than those in peb4, which resembled normal peroxisomes in size.     

Effect of plant sterols on the rate of deterioration of heated oils

Three different plant sterol fractions were added to refined cottonseed oil. The first fraction was isolated from olive oil, the second fraction Δ⁵-avenasterol was extracted from the green algae (Ulva lactuca) and the third fraction was a sterol mixture, made up chiefly of β-sitosterol. Cottonseed oil with the different sterols added was heated at 180±5°C and the rate of oxidation followed by changes in the composition and in physical constants. Olive oil sterol mixtures containing Δ⁵-avenasterol and Δ⁵-avenasterol alone reduced the extent of oxidation. β-sitosterol was initially ineffective and became slightly prooxidant after prolonged heating.