Toxic and mutagenic effects of carcinogens on the mitochondria of Saccharomyces cerevisiae.

Summary Nineteen haploid yeast (Saccharomyces cerevisiae) strains were used to assess the relative growth inhibitory potencies on fermentable vs. non-fermentable media of a collection of carcinogenic and noncarcinogenic chemicals. The majority of carcinogens were distinctly more potent on the non-fermentable (glycerol) medium, where mitochondrial function is required for growth, than on the fermentable medium, where it is not. The anti-mitochondrial selectivity indicated by these growth tests was much slighter for the non-carcinogens. Similarly most carcinogens induced the cytoplasmic petite mutation whereas the non-carcinogens did not.Five carcinogens which were tested impaired the development of cytochromes aa ₃ and b in glucose cultures.Six carcinogens, when tested, inhibited growth on three fermentable sugars, the utilisation of which requires mitochondrial function.Out of five carcinogens which were examined, four suppressed the surface-dependent phenomenon of flocculence in a flocculating strain of yeast, at concentrations primarily affecting the mitochondrial system; the fifth had a similar but less pronounced effect.     

Functional analysis of phosphorylation on Saccharomyces cerevisiae syntaxin 1 homologues Sso1p and Sso2p

Background

The Saccharomyces cerevisiae syntaxin1 homologues Sso1p and Sso2p perform an essential function in membrane fusion in exocytosis. While deletion of either SSO1 or SSO2 causes no obvious phenotype in vegetatively grown cells, deletion of both genes is lethal. In sporulating diploid S. cerevisiae cells only Sso1p, but not Sso2p, is needed for membrane fusion during prospore membrane formation. Mass spectrometry and in vivo labeling data suggest that serines 23, 24, and 79 in Sso1p and serines 31 and 34 in Sso2p can be phosphorylated in vivo. Here we set out to assess the contribution of phosphorylation on Sso protein in vivo function.

Principal Findings

Different mutant versions of SSO1 and SSO2 were generated to target the phosphorylation sites in Sso1p and Sso2p. Basal or overexpression of phospho-mimicking or putative non-phosphorylated Sso1p or Sso2p mutants resulted in no obvious growth phenotype. However, S79A and S79E mutations caused a mild defect in the ability of Sso1p to complement the temperature-sensitive growth phenotype of sso2-1 sso1Δ cells. Combination of all mutations did not additionally compromise Sso1p in vivo function. When compared to the wild type SSO1 and SSO2, the phosphoamino acid mutants displayed similar genetic interactions with late acting sec mutants. Furthermore, diploid cells expressing only the mutant versions of Sso1p had no detectable sporulation defects. In addition to sporulation, also pseudohyphal and invasive growth modes are regulated by the availability of nutrients. In contrast to sporulating diploid cells, deletion of SSO1 or SSO2, or expression of the phospho-mutant versions of SSO1 or SSO2 as the sole copies of SSO genes caused no defects in haploid or diploid pseudohyphal and invasive growth.

Conclusions

The identified phosphorylation sites do not significantly contribute to the in vivo functionality of Sso1p and Sso2p in S. cerevisiae.     

An RNA Transport System in Candida albicans Regulates Hyphal Morphology and Invasive Growth

Localization of specific mRNAs is an important mechanism through which cells achieve polarity and direct asymmetric growth. Based on a framework established in Saccharomyces cerevisiae, we describe a She3-dependent RNA transport system in Candida albicans, a fungal pathogen of humans that grows as both budding (yeast) and filamentous (hyphal and pseudohyphal) forms. We identify a set of 40 mRNAs that are selectively transported to the buds of yeast-form cells and to the tips of hyphae, and we show that many of the genes encoded by these mRNAs contribute to hyphal development, as does the transport system itself. Although the basic system of mRNA transport is conserved between S. cerevisiae and C. albicans, we find that the cargo mRNAs have diverged considerably, implying that specific mRNAs can easily move in and out of transport control over evolutionary timescales. The differences in mRNA cargos likely reflect the distinct selective pressures acting on the two species.     

Pooled Segregant Sequencing Reveals Genetic Determinants of Yeast Pseudohyphal Growth

The pseudohyphal growth response is a dramatic morphological transition and presumed foraging mechanism wherein yeast cells form invasive and surface-spread multicellular filaments. Pseudohyphal growth has been studied extensively as a model of conserved signaling pathways controlling stress responses, cell morphogenesis, and fungal virulence in pathogenic fungi. The genetic contribution to pseudohyphal growth is extensive, with at least 500 genes required for filamentation; as such, pseudohyphal growth is a complex trait, and linkage analysis is a classical means to dissect the genetic basis of a complex phenotype. Here, we implemented linkage analysis by crossing each of two filamentous strains of Saccharomyces cerevisiae (Σ1278b and SK1) with an S288C-derived non-filamentous strain. We then assayed meiotic progeny for filamentation and mapped allelic linkage in pooled segregants by whole-genome sequencing. This analysis identified linkage in a cohort of genes, including the negative regulator SFL1, which we find contains a premature stop codon in the invasive SK1 background. The S288C allele of the polarity gene PEA2, encoding Leu409 rather than Met, is linked with non-invasion. In Σ1278b, the pea2-M409L mutation results in decreased invasive filamentation and elongation, diminished activity of a Kss1p MAPK pathway reporter, decreased unipolar budding, and diminished binding of the polarisome protein Spa2p. Variation between SK1 and S288C in the mitochondrial inner membrane protein Mdm32p at residues 182 and 262 impacts invasive growth and mitochondrial network structure. Collectively, this work identifies new determinants of pseudohyphal growth, while highlighting the coevolution of protein complexes and organelle structures within a given genome in specifying complex phenotypes.     

Isolation and Characterization of EPD1, an Essential Gene for Pseudohyphal Growth of a Dimorphic Yeast, Candida maltosa

Additional copies of the centromeric DNA (CEN) region induce pseudohyphal growth in a dimorphic yeast, Candida maltosa (T. Nakazawa, T. Motoyama, H. Horiuchi, A. Ohta, and M. Takagi, J. Bacteriol. 179:5030–5036, 1997). To understand the mechanism of this transition, we screened the gene library of C. maltosa for sequences which could suppress this morphological change. As a result, we isolated the 5′ end of a new gene, EPD1 (for essential for pseudohyphal development), and then cloned the entire gene. The predicted amino acid sequence of Epd1p was highly homologous to those of Ggp1/Gas1/Cwh52p, a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae, and Phr1p and Phr2p of Candida albicans. The expression of EPD1 was moderately regulated by environmental pH. A homozygous EPD1 null mutant showed some morphological defects and reduction in growth rate and reduced levels of both alkali-soluble and alkali-insoluble β-glucans. Moreover, the mutant could not undergo the transition from yeast form to pseudohyphal form induced by additional copies of the CEN sequence at pH 4 or by n-hexadecane at pH 4 or pH 7, suggesting that EPD1 is not essential for yeast form growth but is essential for transition to the pseudohyphal form. Overexpression of the amino-terminal part of Epd1p under the control of the GAL promoter suppressed the pseudohyphal development induced by additional copies of the CEN sequence, whereas overexpression of the full-length EPD1 did not. This result and the initial isolation of the 5′ end of EPD1 as a suppressor of the pseudohyphal growth induced by the CEN sequence suggest that the amino-terminal part of Epd1p may have a dominant-negative effect on the functions of Epd1p in the pseudohyphal growth induced by the CEN sequence.     

Regulation of cell-cycle initiation in yeast by nutrients and protein synthesis.

Arrested Saccharomyces cerevisiae cells initiate the cell cycle in an asynchronous mode. The asynchronous manner of cycle initiation generates variability in cell-cycle times of individual cells. Limiting concentrations of adenine, methionine or histidine regulate the rate of cycle initiation in auxotrophs. A sigmoidal curve of rate vs. concentration is obtained for each of the three substances. Moreover, the three curves have similar Hill coefficients of 2.4, suggesting that a common intermediate requiring adenine, methionine and histidine regulates cell-cycle initiation in yeast. Low concentrations of cycloheximide reduce the rate of cycle initiation of arrested cells that are released from the block in a similar way as limiting nutrients. It thus appears that the common intermediate that requires the limiting nutrients depends upon protein synthesis. The rate of cycle initiation is more sensitive to cycloheximide or nutrient limitation than is protein synthesis. It is also affected by limiting nutrients to a much greater extent than is the overall rate of protein accumulation (i.e., net protein synthesis). Hence the mechanism that controls cycle initiation does not depend on the overall synthesis or accumulation of proteins in the cell. It may depend on synthesis of particular proteins whose production or function requires the limiting nutrients. The high sensitivity of cycle initiation to a decrease in the rate of protein synthesis could explain the ability of yeast cells to complete the cycle and arrest at stationary phase upon depletion of medium components. The cells cannot initiate the cycle although their protein synthesis capacity remains sufficiently high to allow traversal of the rest of the cell cycle.     

Role of Oxidative Phosphorylation in Histatin 5-Induced Cell Death in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The succeptibility of Saccharomyces cerevisiae to the anti-microbial peptide, histatin 5, was tested after pre-growth in fermentable and non-fermentable carbon sources and in the absence or presence of the uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP). S. cerevisiae was more resistant to histatin 5 when grown on a fermentable carbon source compared to growth on a non-fermentable carbon source, indicating an important role for oxidative phosphorylation in histatin 5-induced cell death. Oxidative phosphorylation is a pre-requisite for histatin 5-induced cell death in Candida albicans but this is not the case in S. cerevisiae. Incubation of CCCP-treated S. cerevisiae cells with histatin 5 still resulted in cell death. These results suggest that histatin 5-induced cell death in S. cerevisiae differs from that in C. albicans.Revisions received 28 September 2004     

Paddy straw as substrate for ethanol production

Pretreatment of paddy straw with 2% sodium hydroxide at 15 psi for 1 h resulted in 83% delignification. The hydrolysis of alkali treated paddy straw with a commercial preparation of cellulase for 2 h at 50°C resulted in release of 65% total reducing sugars. Maximum sugars were released at enzyme loading of 1.5% (v/v). The fermentation of hydrolysate supplemented with nutrients by S. cerevisiae resulted in the production of 20–30 g L⁻¹ ethanol after 48 h incubation which was further improved with addition of yeast nitrogen base and inoculated with 1% (w/v) yeast cells.     

Asc1p,a ribosomal protein,plays a pivotal role in cellular adhesion and virulence in <Emphasis Type="Italic">Candida albicans</Emphasis>

Candida albicans, the common human fungal pathogen, can switch morphology from yeast to pseudohyphal or hyphal form upon various environmental cues. It is well-known that the ability of morphological conversion and adhesive growth renders C. albicans virulent. It is noteworthy that every factor involved in the morphogenesis is known to be important for the virulence of this pathogen. To examine a functional relevance of Asc1p, a ribosomal protein, in morphogenesis and virulence, an asc1 homozygous null mutant was generated. Although a normal morphological transition of the asc1 deletion strain in liquid media was found, it did not change its morphology on solid media. Moreover, the adhesion activity and hyphal-specific gene expression were defective due to ASC1 deletion. Finally, it was found that the asc1 null mutant was avirulent in a mouse model. These results strongly suggested that Asc1p a component of the 40S ribosomal subunit and a signal transducer, plays a pivotal role in cellular adhesion and virulence through regulation of specific gene expression in C. albicans.     

Expression of a tomato sugar transporter is increased in leaves of mycorrhizal or Phytophthora parasitica-infected plants

A full-length cDNA clone (LeST3), encoding a putative tomato sugar transporter, was isolated from mycorrhizal roots by using a PCR-based approach. Based on sequence similarity, conserved motifs and predicted membrane topology, LeST3 was classified as a putative monosaccharide transporter of the sugar transporter subgroup of the major facilitator superfamily. Southern blot analysis showed that LeST3 represents a single-copy gene in tomato. To investigate its function, LeST3 was expressed in a hexose transport-deficient mutant of Saccharomyces cerevisiae. Although LeST3 was correctly transcribed in yeast, it did not restore growth on hexoses of the S. cerevisiae mutant. LeST3 gene expression was increased in the leaves of plants colonised by the arbuscular mycorrhizal (AM) fungi Glomus mosseae or Glomus intraradices and in those of plants infected with the root pathogen Phytophthora parasitica. These data suggest that LeST3 plays a role in the transport of sugars into the sink tissues and responds to the increased demand for carbohydrates exerted by two AM fungi and by a root pathogen to cope with the increased metabolic activity of the colonised/infected tissues or to supply carbohydrates to the AM fungus.     

Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling

Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein β and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth state with very few genetic and regulatory changes.     

Molecular cloning and analysis of CDC28 and cyclin homologues from the human fungal pathogen Candida albicans

In the budding yeast Saccharomyces cerevisiae, progress of the cell cycle beyond the major control point in G1 phase, termed START, requires activation of the evolutionarily conserved Cdc28 protein kinase by direct association with GI cyclins. We have used a conditional lethal mutation in CDC28 of S. cerevisiae to clone a functional homologue from the human fungal pathogen Candida albicans. The protein sequence, deduced from the nucleotide sequence, is 79% identical to that of S. cerevisiae Cdc28 and as such is the most closely related protein yet identified. We have also isolated from C. albicans two genes encoding putative G1 cyclins, by their ability to rescue a conditional GI cyclin defect in S. cerevisiae; one of these genes encodes a protein of 697 amino acids and is identical to the product of the previously described CCN1 gene. The second gene codes for a protein of 465 residues, which has significant homology to S. cerevisiae Cln3. These data suggest that the events and regulatory mechanisms operating at START are highly conserved between these two organisms.     

The effects of vanadate on the yeast Saccharomyces cerevisiae

Growth of Saccharomyces cerevisiae on non-fermentable medium was more sensitive to inhibition by vanadate than growth of fermentable medium. The frequency of petite mutants increased in cultures grown for 18 hours in fermentable medium containing vanadate. However, oxygen uptake markedly increased in yeast cultures grown in the presence of vanadate, a similar effect being produced by phosphate. It was also found that oligomycin toxicity was relieved by vanadate. These results suggest that vanadate may interact with the mitochondria of S. cerevisiae.     

Functional characterization of the ferroxidase, permease high-affinity iron transport complex from Candida albicans

Saccharomyces cerevisiae expresses two proteins that together support high‐affinity Fe‐uptake. These are a multicopper oxidase, Fet3p, with specificity towards Fe²⁺ and a ferric iron permease, Ftr1p, which supports Fe‐accumulation. Homologues of the genes encoding these two proteins are found in all fungal genomes including those for the pathogens, Candida albicans and Cryptococcus neoformans. At least one of these loci represents a virulence factor for each pathogen suggesting that this complex would be an appropriate pharmacologic target. However, the mechanism by which this protein pair supports Fe‐uptake in any fungal pathogen has not been elucidated. Taking advantage of the robust molecular genetics available in S. cerevisiae, we identify the two of five candidate ferroxidases likely involved in high‐affinity Fe‐uptake in C. albicans, Fet31 and Fet34. Both localize to the yeast plasma membrane and both support Fe‐uptake along with an Ftr1 protein, either from C. albicans or from S. cerevisiae. We express and characterize Fet34, demonstrating that it is functionally homologous to ScFet3p. Using S. cerevisiae as host for the functional expression of the C. albicans Fe‐uptake proteins, we demonstrate that they support a mechanism of Fe‐trafficking that involves channelling of the CaFet34‐generated Fe³⁺ directly to CaFtr1 for transport into the cytoplasm.     

Evolution of ecological dominance of yeast species in high‐sugar environments

In budding yeasts, fermentation in the presence of oxygen evolved around the time of a whole genome duplication (WGD) and is thought to confer dominance in high‐sugar environments because ethanol is toxic to many species. Although there are many fermentative yeast species, only Saccharomyces cerevisiae consistently dominates wine fermentations. In this study, we use coculture experiments and intrinsic growth rate assays to examine the relative fitness of non‐WGD and WGD yeast species across environments to assess when S. cerevisiae’s ability to dominate high‐sugar environments arose. We show that S. cerevisiae dominates nearly all other non‐WGD and WGD species except for its sibling species S. paradoxus in both grape juice and a high‐sugar rich medium. Of the species we tested, S. cerevisiae and S. paradoxus have evolved the highest ethanol tolerance and intrinsic growth rate in grape juice. However, the ability of S. cerevisiae and S. paradoxus to dominate certain species depends on the temperature and the type of high‐sugar environment. Our results indicate that dominance of high‐sugar environments evolved much more recently than the WGD, most likely just prior to or during the differentiation of Saccharomyces species, and that evolution of multiple traits contributes to S. cerevisiae's ability to dominate wine fermentations.