Possible involvement of GDI1 protein, a GDP dissociation inhibitor related to vesicle transport, in an amelioration of zinc toxicity in Saccharomyces cerevisiae

The GDI1 protein related vesicle transport system was studied to investigate the possibility that an exclusion of toxic zinc (Zn) from the cytoplasm ameliorates Zn toxicity in Saccharomyces cerevisiae (yeast). A temperature‐sensitive gdi1 mutant (originally called sec19), in which the GDP dissociation inhibitor becomes inactive at the non‐permissive temperature (37 °C), was more sensitive to Zn than its parental GDI1 strain at 32 °C (a moderately non‐permissive temperature). The relative efflux of cytoplasmic Zn in the gdi1 mutant was lower than that in the control strain. Treatment with a vesicle transport‐specific inhibitor, Brefeldin A, caused an increase of Zn sensitivity and a decrease of Zn efflux in these strains. It is therefore suggested that the GDI1‐related vesicle transport system contributes to Zn tolerance in yeast. Furthermore, changes in the number of Zn‐specific fluorescent granules (zincosomes) were observed by zinquin staining in the mutant cells under Zn treatment at 32 °C and 37 °C. We concluded that the GDI1 protein is implicated in control of vesicle numbers. Collectively, the results suggest that the GDI1protein is involved in Zn efflux via small vesicle trafficking and contributes to the control of cytoplasmic Zn content, allowing yeast to survive in the presence of toxic Zn. Copyright © 2011 John Wiley & Sons, Ltd.     

Linear DNA plasmid pPK2 of Pichia kluyveri: distinction between cytoplasmic and mitochondrial linear plasmids in yeasts

The linear plasmids frequently found in plants and filamentous fungi are associated with mitochondria or chloroplasts. In contrast, all the linear plasmids known in yeasts are cytoplasmic elements. From a strain of the yeast Pichia kluyveri, we have isolated a new linear plasmid, pPK2, which was found to be associated with mitochondria. This 7·1 kilobase pairs‐long DNA contained only two genes, which code for DNA and RNA polymerases, as judged from their nucleotide sequences translated by a mitochondrial genetic code. When we examined several recently isolated yeast plasmids for their subcellular localization, we found that two linear plasmids, pPH1 from Pichia heedii, as well as pPK1 from another strain of P. kluyveri, were also localized in mitochondria. These plasmids are the first examples of mitochondria‐associated linear plasmids in yeast. All other linear plasmids we examined were of cytoplasmic origin. Whilst the cytoplasmic type linear plasmids were efficiently eliminated by ultraviolet irradiation of host cells, the mitochondria‐associated plasmids were highly resistant. The mitochondrial pPK2 plasmid was rapidly lost by treatment of the host cells with ethidum bromide. Copyright © 1999 John Wiley & Sons, Ltd.     

Tubulin heterodimers remain functional for one cell cycle after the inactivation of tubulin‐folding cofactor D in fission yeast cells

Tubulin‐folding cofactor D plays a major role in the formation of functional tubulin heterodimers, the subunits of microtubules (MTs) that are essential for cell division. Previous work has suggested that, in Schizosaccharomyces pombe, cofactor D function is required during G₁ or S phases of the cell cycle, and when it fails to function due to the temperature‐sensitive mutation alp1‐t1, cells are unable to segregate their chromosomes in the subsequent mitosis. Here we report that another mutation in the cofactor D gene, alp1‐1315, causes failures in either the first or second mitosis in cells synchronized in G₁ or G₂ phases, respectively. Other results, however, suggest that the kinetics of viability loss in these mutants does not depend on progression through the cell cycle. When cofactor D function is perturbed in cells blocked in G₂, cytoplasmic MTs appear normal for 2–3 h but thereafter they disintegrate quickly, so that only a few short MTs remain. These residual MTs are, however, stably maintained, suggesting that they do not require active cofactor D function. The abrupt disassembly of MT cytoskeleton at restrictive temperature in non‐cycling cofactor D mutant cells strongly suggests that the life‐span of folded tubulin dimers might be downregulated. Indeed, this period is significantly shorter than the previously determined dissociation time of bovine tubulins in vitro. The death of mutant cells occurs inevitably after 2–3 h at restrictive temperature in the following mitosis, and is explained by the idea that MT structures formed in the absence of cofactor D cannot support normal cell division. Copyright © 2009 John Wiley & Sons, Ltd.     

GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria

GABA transaminase (GABA‐T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA‐T from Saccharomyces cerevisiae (ScGABA‐TKG) is an α‐ketoglutarate‐dependent enzyme encoded by the UGA1 gene, while higher plant GABA‐T is a pyruvate/glyoxylate‐dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA‐T). The GABA‐T from A. thaliana is localized in mitochondria and mediated by an 18‐amino acid N‐terminal mitochondrial targeting peptide predicated by both web‐based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA‐TKG and AtGABA‐T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA‐TKG and AtGABA‐T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA‐TKG deletion mutant Δuga1 with AtGABA‐T or ScGABA‐TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat‐induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA‐T is functionally interchangeable with ScGABA‐TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA‐T is about half as efficient in doing so as ScGABA‐TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate‐limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.     

The yeast growth control gene GRC5 is highly homologous to the mammalian putative tumor suppressor gene QM

We isolated the Saccharomyces cerevisiae GRC5 (gr owth control) gene by functional complementation in vivo of a ts (t emperature s ensitive) mutation. Phenotypic analysis suggested involvement of GRC5 in cell growth and proliferation. Mutant cells arrest their cell cycles after one to three cell divisions predominantly as mother cells with a large bud. In the region of the septum, a massive accumulation of cell wall material is observed. The mother and daughter nuclei are well separated and spindles are no longer present, while the cytoskeleton is of aberrant appearance. Arrested cells do not perform protein synthesis and are unable to mate. Furthermore, grc5-1^ts cells rapidly lose viability at the restrictive temperature (37°C) only on full media, but not under nitrogen-starvation conditions, indicating that proper response to this nutrient limitation is still intact in mutant cells after cell cycle arrest. The sequence of GRC5 translates into a basic protein of 221 amino acids with a corresponding M_r of 25·4 kDa. GRC5 is a member of the highly conserved QM gene family, members of which have been reported from plants, invertebrates and vertebrates. The amino acid sequence of GRC5 over its entire length is more than 60% identical to the human QM protein, expression of which is associated with loss of the tumorigenic phenotype in a cell line derived from Wilms' tumor, a malignancy of the embyronic kidney. Here, we show that GRC5 is an essential yeast gene, the function of which as inferred from analysis of the grc5-1^ts mutant is crucial for establishment of proper cytoskeletal structure and regulation of growth in yeast cells.     

Cloning and identification of HEM14, the yeast gene for mitochondrial protoporphyrinogen oxidase

A respiratory-defective mutant (C54) of Saccharomyces cerevisiae was found to have a phenotype consistent with a mutation in either mitochondrial protoporphyrinogen oxidase or ferrochelatase. The mutant is grossly deficient in hemes, accumulates protoporphyrin and is rescued by exogenous heme. The increased levels of protoporphyrin at the expense of heme is indicative of a block in one of the two last steps of the heme biosynthetic pathway. Complementation of C54 by a known ferrochelatase mutant suggested that the defect was most likely in HEM14 encoding protoporphyrinogen oxidase. A plasmid capable of complementing C54 was obtained by transformation with a yeast genomic plasmid library. A partial sequence of the insert identified the gene as reading frame YER014 of yeast chromosome V (GenBank Accession Number U18778). This reading frame codes for a protein homologous to human protoporphyrinogen oxidase. Disruption of this gene elicits a respiratory defect and accumulation of protoporphyrin. The phenotype of the null mutant together with the homology of YER014p to human protoporphyrinogen oxidase provide compelling evidence that YER014 is HEM14.     

Suppression of sdh1 mutations by the SDH1b gene of Saccharomyces cerevisiae

Transformation of the respiratory-defective mutant (E264/U2) of Saccharomyces cerevisiae with a yeast genomic library yielded two different plasmids capable of restoring the ability of the mutant to grow on non-fermentable substrates. One of the plasmids (pG52/T3) contained SDH1 coding for the flavoprotein subunit of mitochondrial succinate dehydrogenase. The absence of detectable succinate dehydrogenase activity in mitochondria of E264/U2 and the lack of complementation of the mutant by an sdh11null strain indicated a mutation in SDH1. The second plasmid (pG52/T8) had an insert with reading frame (YJL045w) of yeast chromosome X coding for a homologue of SDH1. Subclones containing the SDH1 homologue (SDH1b), restored respiration in E264/U2 indicating that the protein encoded by this gene is functional. The expression of the two genes was compared by assaying the β-galactosidase activities of yeast transformed with plasmids containing fusions of lacZ to the upstream regions of SDH1 and SDH1b. The 100–500 times lower activity measured in transformants harbouring the SDH1b-lacZ fusion indicates that the isoenzyme encoded by SDH1b is unlikely to play an important role in mitochondrial respiration. This is also supported by the absence of any obvious phenotype in cells with a disrupted copy of SDH1b. © 1998 John Wiley & Sons, Ltd.     

PER1, GUP1 and CWH43 of methylotrophic yeast Ogataea minuta are involved in cell wall integrity

In eukaryotes, the glycosylphosphatidylinositol (GPI) modification of many glycoproteins on the cell surface is highly conserved. The lipid moieties of GPI‐anchored proteins undergo remodelling processes during their maturation. To date, the products of the PER1, GUP1 and CWH43 genes of the yeast Saccharomyces cerevisiae have been shown to be involved in the lipid remodelling. Here, we focus on the putative GPI remodelling pathway in the methylotrophic yeast Ogataea minuta. We found that the O. minuta homologues of PER1, GUP1 and CWH43 are functionally compatible with those of S. cerevisiae. Disruption of GUP1 or CWH43 in O. minuta caused a growth defect under non‐permissive conditions. The O. minuta per1Δ mutant exhibited a more fragile phenotype than the gup1Δ or cwh43Δ mutants. To address the role of GPI modification in O. minuta, we assessed the effect of these mutations on the processing and localization of the O. minuta homologues of the Gas1 protein; in S. cerevisiae, Gas1p is an abundant and well‐characterized GPI‐anchored protein. We found that O. minuta possesses two copies of the GAS1 gene, which we designate GAS1A and GAS1B. Microscopy and western blotting analysis showed mislocalization and/or lower retention of Gas1Ap and Gas1Bp within the membrane fraction in per1Δ or gup1Δ mutant cells, suggesting the significance of lipid remodelling for GPI‐anchored proteins in O. minuta. Localization behaviour of Gas1Bp differed from that of Gas1Ap. Our data reveals, for the first time (to our knowledge), the existence of genes related to GPI anchor remodelling in O. minuta cells.     

Characterization of the SAC3 gene of Saccharomyces cerevisiae

A temperature-sensitive mutation (act1-1) in the essential actin gene of Saccharomyces cerevisiae can be suppressed by mutations in the SAC3 gene. A DNA fragment containing the SAC3 gene was sequenced. SAC3 codes for a 150 kDa hydrophillic protein which does not show any significant similarities with other proteins in the databases. Sac3 therefore is a novel yeast protein. A nuclear localization of Sac3 is suggested by the presence of a putative nuclear localization signal in the Sac3 sequence. A SAC3 disruption mutation was constructed. SAC3 disruption mutants were viable but grew more slowly and were larger than wild-type cells. In contrast to the sac3-1 mutation, the SAC3 disruption was not able to suppress the temperature sensitivity and the osmosensitivity of the act1-1 mutant. This demonstrates that act1-1 suppression by sac3-1 is not the result of a simple loss of SAC3 function. Furthermore, we examined the act1-1 and the sac3 mutants for defects in polarized cell growth by FITC-Concanavalin A (Con A)-labelling. The sac3 mutants showed a normal ConA-labelling pattern. In the act1-1 mutant, however, upon shift to non-permissive temperature, newly synthesized cell wall material, instead of being directed towards the bud, was deposited at discrete spots in the mother cell.     

GUT2, a gene for mitochondrial glycerol 3-phosphate dehydrogenase of Saccharomyces cerevisiae

A gut2 mutant of Saccharomyces cerevisiae is deficient in the mitochondrial glycerol 3-phosphate dehydrogenase and hence cannot utilize glycerol. Upon transformation of a gut2 mutant strain with a low-copy yeast genomic library, hybrid plasmids were isolated which complemented the gut2 mutation. The nucleotide sequence of a 3·2 kb PstI-XhoI fragment complementing a gut2 mutant strain is presented. The fragment reveals an open reading frame (ORF) encoding a polypeptide with a predicted molecular weight of 68·8 kDa. Disruption of the ORF leads to a glycerol non-utilizing phenotype. A putative flavin-binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins. The cloned gene has been mapped both physically and genetically to the left arm of chromosome IX, where the original gut2 mutation also maps. We conclude that the presented ORF is the GUT2 gene and propose that it is the structural gene for the mitochondrial glycerol 3-phosphate dehydrogenase.     

Measurement of nuclear DNA content in fission yeast by flow cytometry

Cell division cycle (cdc) mutants of Schizosaccharomyces pombe are arrested at specific points in the cell cycle when grown at restrictive temperature. Flow cytometry of such cells reveals an anomalous increase in the DNA fluorescence signal, which represents a problem in experiments designed to determine the cell cycle arrest point. The increased fluorescence signal is due to cytoplasmic constituents and has been attributed to mitochondrial DNA synthesis (S. Sazer and S. W. Sherwood, J. Cell Sci.97: 509–516, 1990). Here we have studied the cdc10 mutant by flow cytometry using different DNA-binding fluorochromes and found no evidence that the increased fluorescence signal was caused by mitochondrial DNA synthesis. To determine more accurately the nuclear DNA content we have developed a novel method to remove most of the cytoplasmic material by exposing the cells to Triton X-100 and hypotonic conditions after cell wall digestion. The DNA fluorescence from cells treated in this way was more constant with time of incubation at restrictive temperature in spite of a considerable increase in cell size. With this method we could determine that the recently isolated temperature sensitive orp1 mutant is arrested with a 1C DNA content. Premature and abnormal mitosis (‘cut’) could be observed for the orp1 mutant after only 4 h at restrictive temperature. © 1997 John Wiley & Sons, Ltd.     

Isolation and expression of the gene encoding mitochondrial ADP/ATP carrier (AAC) from the pathogenic yeast Candida parapsilosis

A gene homologous to Saccharomyces cerevisiae AAC genes coding for mitochondrial ADP/ATP carriers has been cloned from the pathogenic yeast Candida parapsilosis. A probe obtained by PCR amplification from C. parapsilosis DNA, using primers derived from the conserved transmembrane region of yeast ADP/ATP carriers, was used for screening of the C. parapsilosis genomic library. The cloned gene was sequenced and found to encode a polypeptide of 303 amino acids that shows homology with other yeast and fungal mitochondrial ADP/ATP carriers. The gene was designated CpAAC1 and was able to complement the growth phenotypes of S. cerevisiae double deletion mutant (Δaac2; Δaac3). The expression of the CpAAC1 gene was reduced under semi‐anaerobic conditions and it was affected at normal aerobic conditions by the nature of carbon sources used for growth. Hybridization experiments indicate that C. parapsilosis possesses a single gene encoding a mitochondrial ADP/ATP carrier. The GenBank Accession No. for the C. parapsilosis CpAAC1 gene is AF085429. Copyright © 1999 John Wiley & Sons, Ltd.     

Yeast sequencing reports. Sequence of the AFG3 gene encoding a new member of the FtsH/Yme1/Tma subfamily of the AAA-protein family

A nuclear gene from Saccharomyces cerevisiae was cloned by genetic complementation of a temperature-sensitive respiratory-deficient mutant. DNA sequence analysis reveals that it encodes a protein with homology to Yme1, FtsH and Tma, proteins which belong to the AAA-protein family (ÃPases associated with diverse cellular activities). The members of this family are involved in very different biological processes. Yme1p, a yeast mitochondrial protein, affects the rate of DNA escape from mitochondria to the nucleus and the Escherichia coli FtsH protein is apparently involved in the post-translational processing of PBP3, a protein necessary for septation during cell division. This newly sequenced gene, which we have designated AFG3 for ÃPase family gene 3, encodes a putative mitochondrial protein of 760 amino acid residues that is closely related to FtsH, Tma (protein from Lactococcus lactis) and Yme1p with 58, 55 and 46% identity respectively. The sequence has been deposited in the EMBL data library under Accession Number X76643.     

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.     

Mitochondrial activity of sake brewery yeast affects malic and succinic acid production during alcoholic fermentation

The mitochondrial states and activities of production yeasts used in the fermentation industry vary according to the availability of oxygen, size of the fermentation tank and temperature of the raw material. However, the involvement of the mitochondrial states of these yeasts in the production profile of organic acids during alcoholic fermentation has not been investigated in detail. In this study, the effects of the mitochondrial state of a sake brewing yeast on the organic acid production profile during an alcoholic fermentation process were investigated. It was elucidated that the mitochondrial state during the propagation stage significantly affected the mitochondrial morphology and the organic acid production profile during the alcoholic fermentation. When yeast mitochondria were active, they were highly branched in the propagation stage, and the yeast cells produced significantly more succinate and less malate. In contrast, when the yeast mitochondria were inactive, they were long and filamentous in appearance, and the yeast produced significantly less succinate and more malate. The change in malic acid content was reversed when an uncoupler of mitochondrial membrane potential, carbonylcyanide p‐trifluoromethoxyphenylhydrazone, was added to the culture, indicating that the change in the organic acid production profile could be attributed to mitochondrial activity. Furthermore, the content of malic acid and succinic acid could be converted from a respirative to a fermentative profile by exposing the yeast to a mitochondrion‐inactivating environment for 12 or 24 h. Taken together, it was shown that the mitochondrial status of the yeast affects malic acid production during alcoholic fermentation. Copyright © 2012 The Institute of Brewing & Distilling     

Absence of cell wall chitin in Saccharomyces cerevisiae leads to resistance to Kluyveromyces lactis killer toxin

Kluyveromyces lactis killer toxin causes sensitive strains of a variety of yeasts to arrest at the G1 stage of the cell cycle, and to lose viability. We describe here the isolation and characterization of a class of recessive mutations in Saccharomyces cerevisiae that leads to toxin resistance and a temperature-sensitive phenotype. These mutant cells arrest growth at 37°C with a characteristic phenotype of elongated buds. Cloning of the gene complementing these defects revealed it to be CAL1, coding for chitin synthase 3 activity. Calcofluor staining of the mutant cells indicated that chitin is absent both at 23°C and 37°C. Given that the CAL1 activity is responsible for the synthesis of most of chitin in yeast cells, and that in its absence the cells are viable but resistant to the killer toxin, our results strongly suggest that chitin might represent the receptor for this killer toxin.