Shusuke Kuge

YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides.

The role of the YAP1 transcription factor in the response of Saccharomyces cerevisiae cells to a variety of conditions that induce oxidative stress has been investigated. Cells deficient in YAP1 were found to be hypersensitive to hydroperoxides and thioloxidants, whereas overexpression of YAP1 conferred hyper‐resistance to the same conditions. These treatments resulted in an increase in YAP1‐specific binding to DNA together with an increase in YAP1 dependent transcription. Our results indicate that this increase is not due to an increase in synthesis of YAP1 protein, but rather results from modification of pre‐existing protein. Using a specific genetic screen, the TRX2 gene, one of two genes of S. cerevisiae that encode thioredoxin protein, was identified as being essential for YAP1 dependent resistance to hydroperoxides. Furthermore, efficient expression of TRX2 was dependent on YAP1 and enhanced under conditions of oxidative stress.

Regulation of yAP‐1 nuclear localization in response to oxidative stress

The YAP1 gene of Saccharomyces cerevisiae encodes a bZIP‐containing transcription factor that is essential for the normal response of cells to oxidative stress. Under stress conditions, the activity of yAP‐1 is increased, leading to the induced expression of a number of target genes encoding protective enzymes or molecules. We have examined the mechanism of this activation. Upon imposition of oxidative stress, a small increase in the DNA‐binding capacity of yAP‐1 occurs. However, the major change is at the level of nuclear localization; upon induction the yAP‐1 protein relocalizes from the cytoplasm to the nucleus. This regulated localization is mediated by a cysteine‐rich domain (CRD) at the C‐terminus, its removal resulting in constitutive nuclear localization and high level activity. Furthermore, the CRD of yAP‐1 is sufficient to impose regulated nuclear localization of the GAL4 DNA‐binding domain. Amino acid substitutions indicated that three conserved cysteine residues in the CRD are essential for the regulation. We suggest therefore, that these cysteine residues are important in sensing the redox state of the cell and hence regulating yAP‐1 activity.

The Skn7 response regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae

Deletion of the bacterial two‐component response regulator homologue Skn7 results in sensitivity of yeast to oxidizing agents indicating that Skn7 is involved in the response to this type of stress. Here we demonstrate that following oxidative stress, Skn7 regulates the induction of two genes: TRX2, encoding thioredoxin, and a gene encoding thioredoxin reductase. TRX2 is already known to be induced by oxidative stress dependent on the Yap1 protein, an AP1‐like transcription factor responsible for the induction of gene expression in response to various stresses. The thioredoxin reductase gene has not previously been shown to be activated by oxidative stress and, significantly, we find that it too is regulated by Yap1. The control of at least TRX2 by Skn7 is a direct mechanism as Skn7 binds to the TRX2 gene promoter in vitro. This shows Skn7 to be a transcription factor, at present the only such eukaryotic two‐component signalling protein. Our data further suggest that Skn7 and Yap1 co‐operate on the TRX2 promoter, to induce transcription in response to oxidative stress.

Regulation of the Yeast Yap1p Nuclear Export Signal Is Mediated by Redox Signal-Induced Reversible Disulfide Bond Formation

ABSTRACT Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae, is transported in and out of the nucleus under nonstress conditions. The nuclear export step is specifically inhibited by H2O2 or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H2O2 and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES). The H2O2 or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization. Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected. The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H2O2 and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H2O2-induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys598) and second (Cys620) cysteine residues; this linkage could be reduced by thioredoxin. In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response. Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.

Crm1 (XpoI) dependent nuclear export of the budding yeast transcription factor yAP-1 is sensitive to oxidative stress

The yAP‐1 transcription factor is crucial for the oxidative stress response of the budding yeast Saccharomyces cerevisiae; its activity is induced in response to oxidative stress, and as a consequence the expression of a number of target genes is enhanced. We have shown previously that yAP‐1 is mainly found in the cytoplasm, but that upon the imposition of oxidative stress it localizes to the nucleus. In this study, we addressed the mechanism through which yAP‐1 nuclear localization is regulated.

Receptor (CD155)-Dependent Endocytosis of Poliovirus and Retrograde Axonal Transport of the Endosome

ABSTRACT Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic mice carrying the human PV receptor (hPVR/CD155) gene. Here, we demonstrated by using an immunoelectron microscope that PV particles exist on vesicle structures in nerve terminals of neuromuscular junctions. We also demonstrated in glutathione S-transferase pull-down experiments that the dynein light chain, Tctex-1, interacts directly with the cytoplasmic domain of hPVR. In the axons of differentiated rat PC12 cells transfected with expression vectors for hPVRs, vesicles composed of PV and hPVRα, as well as a mutant hPVRα (hPVRMα) that had a reduced ability to bind Tctex-1, colocalized with Tctex-1. However, vesicles containing PV, dextran, and hPVRα had only retrograde motion, while those containing PV, dextran, and hPVRMα had anterograde or retrograde motion. Topical application of the antimicrotubule agent vinblastine to the sciatic nerve reduced the amount of virus transported from the calf to the spinal cord. These results suggest that direct efficient interaction between the cytoplasmic domain and Tctex-1 is essential for the efficient retrograde transport of PV-containing vesicles along microtubules in vivo.

A Novel Fluorescent Sensor Protein for Visualization of Redox States in the Cytoplasm and in Peroxisomes

ABSTRACT Reactive oxygen species are generated within peroxisomes during peroxisomal metabolism. However, due to technological difficulties, the intraperoxisomal redox state remain elusive, and the effect of peroxisome deficiency on the intracellular redox state is controversial. A newly developed, genetically encoded fluorescence resonance energy transfer (FRET) probe, Redoxfluor, senses the physiological redox state via its internal disulfide bonds, resulting in a change in the conformation of the protein leading to a FRET response. We made use of Redoxfluor to measure the redox states at the subcellular level in yeast and Chinese hamster ovary (CHO) cells. In wild-type peroxisomes harboring an intact fatty acid β-oxidation system, the redox state within the peroxisomes was more reductive than that in the cytosol, despite the fact that reactive oxygen species were generated within the peroxisomes. Interestingly, we observed that the redox state of the cytosol of cell mutants for peroxisome assembly, regarded as models for a neurological metabolic disorder, was more reductive than that of the wild-type cells in yeast and CHO cells. Furthermore, Redoxfluor was utilized to develop an efficient system for the screening of drugs that moderate the abnormal cytosolic redox state in the mutant CHO cell lines for peroxisome assembly without affecting the redox state of normal cells.

Activity of the Yap1 transcription factor in Saccharomyces cerevisiae is modulated by methylglyoxal, a metabolite derived from glycolysis.

ABSTRACT Methylglyoxal (MG) is synthesized during glycolysis, although it inhibits cell growth in all types of organisms. Hence, it has long been asked why such a toxic metabolite is synthesized in vivo. Glyoxalase I is a major enzyme detoxifying MG. Here we show that the Yap1 transcription factor, which is critical for the oxidative-stress response in Saccharomyces cerevisiae, is constitutively concentrated in the nucleus and activates the expression of its target genes in a glyoxalase I-deficient mutant. Yap1 contains six cysteine residues in two cysteine-rich domains (CRDs), i.e., three cysteine residues clustering near the N terminus (n-CRD) and the remaining three cysteine residues near the C terminus (c-CRD). We reveal that any of the three cysteine residues in the c-CRD is sufficient for MG to allow Yap1 to translocate into the nucleus and to activate the expression of its target gene. A Yap1 mutant possessing only one cysteine residue in the c-CRD but no cysteine in the n-CRD and deletion of the basic leucine zipper domain can concentrate in the nucleus with MG treatment. However, substitution of all the cysteine residues in Yap1 abolishes the ability of this transcription factor to concentrate in the nucleus following MG treatment. The redox status of Yap1 is substantially unchanged, and protein(s) interaction with Yap1 through disulfide bond is hardly detected in cells treated with MG. Collectively, neither intermolecular nor intramolecular disulfide bond formation seems to be involved in Yap1 activation by MG. Moreover, we show that nucleocytoplasmic localization of Yap1 closely correlates with growth phase and intracellular MG level. We propose a novel regulatory pathway underlying Yap1 activation by a natural metabolite in the cell.

A Major Peroxiredoxin-induced Activation of Yap1 Transcription Factor Is Mediated by Reduction-sensitive Disulfide Bonds and Reveals a Low Level of Transcriptional Activation

Redox reactions involving cysteine thiol-disulfide exchange are crucial for the intracellular monitoring of hydrogen peroxide (H2O2). Yap1, the master transcription factor for the oxidative stress response in budding yeast, is activated by the formation of disulfide bonds in response to H2O2. Gpx3 (glutathione peroxidase-like protein 3) acts as a receptor for H2O2, and Ybp1 (Yap1-binding protein 1) is crucial for Gpx3-dependent disulfide bond formation in Yap1. We previously reported that Tsa1, a major peroxiredoxin in yeast cells, is required for activation of Yap1 in a widely used yeast strain, W303-1b, carrying the ybp1-1 mutant allele encoding a truncated Ybp1 protein. In the present study, we show that Tsa1 can interact with Yap1 via disulfide linkages and induce the formation of intramolecular disulfide bonds in Yap1 in ybp1-1 cells. The results provide evidence that Prx can have intrinsic activity as an H2O2 receptor and can relay H2O2 as a signal to the Prx target proteins in terms of formation of disulfide linkage. Furthermore, our data reveal that there is more of the reduction-resistant active form of Yap1 (i.e. Yap1 (oxII)) when it is partnered with Gpx3 than with Tsa1. These data support our hypothesis that changes in the redox status of Yap1 to reduction-resistant forms by multiple disulfide bond formation are important for determining the level and duration of Yap1 activity in the dynamic equilibrium of redox reactions in cells exposed to H2O2.

GFAT as a target molecule of methylmercury toxicity in Saccharomyces cerevisiae

Using a genomic library constructed from Saccharomyces cerevisiae, we have identified a gene GFA1 that confers resistance to methylmercury toxicity. GFA1 encodes L‐glutamine:D‐fructose‐6‐phosphate amidotransferase (GFAT) and catalyzes synthesis of glucosamine‐6‐phosphate. Transformed yeast cells expressing GFA1 demonstrated resistance to methylmercury but no resistance to />‐chloromer‐curibenzoate, a GFAT inhibitor. The cytotoxicity of methylmercury was inhibited by loading excess glu‐cosamine 6‐phosphate into yeast. Considering that GFAT is an essential cellular enzyme, our findings suggest that GFAT is the major target molecule of methylmercury in yeasts. This report is the first to identify the target molecule of methylmercury toxic‐ity in eukaryotic cells.—Naganuma, A., Miura, N., Kaneko, S., Mishina, T., Hosoya, S., Miyairi, S., Furuchi, T., Kuge, S. GFAT as a target molecule of methylmercury toxicity in Saccharomyces cerevisiae. FASEBJ. 14, 968–972 (2000)