Abstract
Toxic metals are ubiquitous in the environment and all organisms possess systems to evade toxicity and acquire tolerance. The Saccharomyces cerevisiae AP-1-like protein Yap8p (systematic name YPR199c; also known as Acr1p and Arr1p) confers arsenic tolerance by stimulating enhanced transcription of the arsenic-specific detoxification genes ACR2 and ACR3. Here, we report that Yap8p is regulated at the level of degradation. We show that Yap8p is stabilized in arsenite-exposed cells in a time- and dose-dependent manner. Yap8p degradation proceeds through the ubiquitin-proteasome pathway and is dependent on the ubiquitin-conjugating enzyme Ubc4p. Further, we show that mutants that are defective in the ubiquitin-proteasome pathway display increased Yap8p levels and elevated expression of the Yap8p gene-target ACR3. Yap8p forms homodimers in vivo but dimerization is not regulated by arsenite. Instead, arsenite-stimulated Yap8p stabilization and transcriptional activation of ACR3 requires critical cysteine residues within Yap8p. Collectively, our data is consistent with a model where Yap8p is degraded by the ubiquitin-proteasome pathway in untreated cells, whereas arsenite-exposure results in Yap8p stabilization and gene activation. In this way, regulated degradation contributes to Yap8p control.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Alanine / metabolism
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Amino Acid Substitution
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Arsenic / pharmacology*
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Arsenites / pharmacology
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Basic-Leucine Zipper Transcription Factors / chemistry
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Basic-Leucine Zipper Transcription Factors / genetics
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Basic-Leucine Zipper Transcription Factors / metabolism
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Basic-Leucine Zipper Transcription Factors / physiology*
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Blotting, Western
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Cysteine / chemistry
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Dimerization
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Dose-Response Relationship, Drug
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Gene Expression Regulation, Fungal*
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Genes, Fungal
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Plasmids
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Precipitin Tests
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Proteasome Endopeptidase Complex / metabolism*
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Protein Structure, Tertiary
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / growth & development
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
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Saccharomyces cerevisiae Proteins / physiology*
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Time Factors
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Transcriptional Activation
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Ubiquitin / metabolism*
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Ubiquitin-Conjugating Enzymes / genetics
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Ubiquitin-Conjugating Enzymes / metabolism
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beta-Galactosidase / analysis
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beta-Galactosidase / metabolism
Substances
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ARR1 protein, S cerevisiae
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Arsenites
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Basic-Leucine Zipper Transcription Factors
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Saccharomyces cerevisiae Proteins
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Ubiquitin
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Ubc4 protein, S cerevisiae
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Ubiquitin-Conjugating Enzymes
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beta-Galactosidase
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Proteasome Endopeptidase Complex
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Cysteine
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arsenite
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Arsenic
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Alanine