title: The yeast phosphotyrosyl phosphatase activator protein, yPtpa1/Rrd1, interacts with Sit4 phosphatase to mediate resistance to 4-nitroquinoline-1-oxide and UVA.
abstract: We previously reported the isolation of mutants hypersensitive to the genotoxic agent 4-nitroquinoline-1-oxide, a potent inducer of oxidative stress. One of the mutants was defective in a gene designated yPTPA1, encoding a protein related to the human phosphotyrosyl phosphatase activator hPTPA, which is believed to play a role in activating the serine/threonine phosphatase PP2A. Yeast yptpa1Delta mutants are also sensitive to the UVA component of sunlight known to produce reactive oxygen species, suggesting a role for yPtpa1 in oxidative stress response. We now report the characterization of another 4-nitroquinoline-1-oxide-sensitive mutant, EBY20. We show that this mutant is defective in the SIT4 gene encoding a catalytic subunit of the PP2A phosphatases and that sit4Delta mutants exhibit hypersensitivity to 4-nitroquinoline-1-oxide and UVA, but not to UVC at 254 nm. Like the yptpa1Delta mutants, sit4Delta mutants are also defective in the repair of 4-nitroquinoline-1-oxide-induced DNA lesions. Genetic analysis revealed that both yPtpa1 and Sit4 function in the same pathway to protect cells against the lethal effects of 4-nitroquinoline-1-oxide and UVA. Moreover, we demonstrate that yPtpa1-affinity columns specifically retain Sit4, confirming a previous report that these two proteins indeed belong to a complex. Cellular localization studies using GFP-tagged proteins reveals that yPtpa1 is localized to the cytoplasm and the nucleus, while the Sit4 protein shows an intense staining spot in the cytoplasm and diffused staining in this organelle. We suggest that the yPtpa1-Sit4 complex may participate in a novel mechanism that mediates repair of oxidative DNA damage caused by 4-nitroquinoline-1-oxide and UVA.
authors.affiliation: Centre de Recherche, Hopital Maisonneuve-Rosemont, Université de Montréal, 5415 Boulevard de l' Assomption, Montréal, Quebec, H1T 2M4, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Douville
authors.forename: J
authors.initials: J
authors.lastname: David
authors.forename: P-K
authors.initials: PK
authors.lastname: Fortier
authors.forename: Dindial
authors.initials: D
authors.lastname: Ramotar
mesh_terms: D015112:4-Nitroquinoline-1-oxide / Q000633:toxicity*; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics*; D019696:Peptidylprolyl Isomerase; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D054648:Protein Phosphatase 2; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012685:Sepharose / Q000031:analogs & derivatives*; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014170:Transformation, Genetic; D014466:Ultraviolet Rays
chemical_list: D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D015112:4-Nitroquinoline-1-oxide; C020927:phenyl-sepharose; D012685:Sepharose; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C104343:SIT4 protein, S cerevisiae; D019696:Peptidylprolyl Isomerase; C490203:RRD1 protein, S cerevisiae