Role of the sulphate assimilation pathway in utilization of glutathione as a sulphur source bySaccharomyces cerevisiae

Glutathione (␥-L-glutamyl-L-cysteinylglycine) is an important antioxidant molecule, helping to buffer the cell against free radicals and toxic electrophiles. Expression of the yeast GSH1 gene, encoding the first enzyme involved in glutathione biosynthesis, ␥-glutamylcysteine synthetase, is regulated by oxidants and the heavy metal cadmium at the level of transcription. We present evidence that the transcription factors involved in controlling the network of sulfur amino acid metabolism genes are also responsible for regulating GSH1 expression in response to cadmium. In particular the transcription factors Met-4, Met-31, and Met-32 are essential for cadmium-mediated regulation of gene expression, whereas the DNA-binding protein Cbf1 appears to play a negative role in controlling GSH1 expression.Glutathione (␥-L-glutamyl-L-cysteinylglycine) is an important molecule that plays a major role in protecting cells against damage caused by oxidants, heavy metals, and pesticides (1). Glutathione can act as as a free radical scavenger, with the redox-active sulfhydryl group reacting with oxidants to produce oxidized glutathione (GSSG). In response to oxidative stress caused by compounds such as H 2 O 2 and superoxide aniongenerating agents (for example menadione), the yeast Saccharomyces cerevisiae induces stress responses that result in protection against subsequent toxic levels of oxidants (2-6). We and others (7-10) have shown that glutathione can protect S. cerevisiae against oxidative stress and is therefore an important antioxidant molecule in this organism. In addition to its role as an antioxidant, there is also evidence that glutathione can act as a storage compound for both sulfur and nitrogen (11,12).It is also apparent that much of the regulation of the oxidative adaptive stress responses in yeast appears to be at the level of transcription (4, 13-15). We have previously demonstrated (13) that exposure of yeast cells to oxidants results in an increase in the steady state level of GSH1 mRNA, which encodes ␥-glutamylcysteine synthetase, the first enzyme in the GSH biosynthetic pathway. Furthermore, expression of the GSH1 gene has also been shown to be dependent upon the transcription factor Yap1 (13, 16). The expression of the gene encoding yeast glutathione reductase (GLR1) has also been shown to be regulated by oxidants and dependent upon the Yap1 protein (17). The pattern of gene expression controlled by the Yap1 protein has also been investigated using microarrays and high resolution two-dimensional gel electrophoresis (18,19). We reported that GSH1 gene expression was also inducible by the heavy metal cadmium (20). Glutathione is known to be important in countering cadmium toxicity; indeed, gsh1 mutants are hypersensitive to cadmium (21, 22). Moreover, yap1 mutants are defective in regulation of GSH1 expression and are also sensitive to cadmium (16, 23). There is also clear evidence for a direct link between metal ions and oxidant resistance/sensitivity. Mutations in a number of genes give rise to either res...

Section: Discussionmentioning

confidence: 99%

Cadmium-inducible Expression of the Yeast GSH1 Gene Requires a Functional Sulfur-Amino Acid Regulatory Network

Dormer¹,

Westwater²,

McLaren³

et al. 2000

“…We observed enrichment of the cytoplasmic protein O-acetylhomoserine sulfhydrolase (MET17, Fig. 2B, panel 5), a protein central for sulfur metabolism and glutathione synthesis (37). Glutathione is a known antioxidant in yeast (13,38).…”

Section: Identified Proteins Differentially Found In Mitochondrial Exmentioning

confidence: 99%

Crucial Mitochondrial Impairment upon CDC48 Mutation in Apoptotic Yeast

Braun¹,

Zischka

Madeo

et al. 2006

Mutation in CDC48 (cdc48 S565G ), a gene essential in the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, led to the discovery of apoptosis as a mechanism of cell death in the unicellular organism Saccharomyces cerevisiae. Elucidating Cdc48p-mediated apoptosis in yeast is of particular interest, because Cdc48p is the highly conserved yeast orthologue of human valosin-containing protein (VCP), a pathological effector for polyglutamine disorders and myopathies. Here we show distinct proteomic alterations in mitochondria in the cdc48 S565G yeast strain. These observed molecular alterations can be related to functional impairment of these organelles as suggested by respiratory deficiency of cdc48 S565G cells. Mitochondrial dysfunction in the cdc48 S565G strain is accompanied by structural damage of mitochondria indicated by the accumulation of cytochrome c in the cytosol and mitochondrial enlargement. We demonstrate accumulation of reactive oxygen species produced predominantly by the cytochrome bc 1 complex of the mitochondrial respiratory chain as suggested by the use of inhibitors of this complex. Concomitantly, emergence of caspase-like enzymatic activity occurs suggesting a role for caspases in the cell death process. These data strongly point for the first time to a mitochondrial involvement in Cdc48p/VCPdependent apoptosis.Fundamental cellular processes, such as the formation of organelles (ER,3 Golgi apparatus, and the nuclear envelope), or ubiquitin-dependent ER-associated protein degradation (ERAD) have been linked to the yeast protein Cdc48p and its highly conserved mammalian orthologue VCP (1-4). Mutations in VCP have been associated with "inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia" (IBMPFD), a dominant human disorder (5, 6). A genetic screening of a Drosophila model for human polyglutamine diseases, a class of inherited neurodegenerative disorders, identified the Drosophila homologue of Cdc48p/VCP as a modulator of apoptotic cell death (7), leading these authors to propose VCP as a pathological effector for polyglutamine-induced neurodegeneration. However, the cellular mechanisms underlying VCP-mediated cell death in these human disorders remain largely unknown. Apoptotic phenotypes in cells expressing mutated Cdc48p/ VCP have originally been described in budding yeast (8) and were thereafter confirmed in mammalian cell cultures (9, 10), in trypanosomes (11), and in zebrafish (12). Notably, Cdc48p was the first apoptotic mediator found in Saccharomyces cerevisiae (8). The expression of a point-mutated CDC48 gene (cdc48 S565G ) leads to a characteristic apoptotic phenotype: phosphatidylserine externalization, DNA fragmentation, chromatin condensation, nuclear fragmentation, and vacuolization (8, 13). These results obtained in the cdc48 S565G strain initiated the establishment of yeast as a model to study evolutionary conserved mechanisms of apoptotic regulation (14 -16).Mitochondria play a crucial role in many apoptotic pathways in ...

“…Under conditions of sulfur starvation, glutathione is the last of various sulfur-containing compounds to decrease in the cell. 1 It is also known that, under conditions of sulfur starvation, Saccharomyces cerevisiae utilizes sulfate, produced by the consumption of glutathione, as a sulfur source (16). Therefore, it appears that maintenance of the intracellular level of glutathione is of great importance for viability in terms of sustaining sulfur metabolism and protecting the cell from oxidative stress.…”

mentioning

confidence: 99%

Involvement of the VDE Homing Endonuclease and Rapamycin in Regulation of the Saccharomyces cerevisiae GSH11 Gene Encoding the High Affinity Glutathione Transporter

Miyake

Hiraishi

Sammoto

et al. 2003

Self Cite

The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004 -1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.