Pir1p mediates translocation of the yeast Apn1p endonuclease into the mitochondria to maintain genomic stability.
The mitochondrial genome is continuously subject to attack by reactive oxygen species generated through aerobic metabolism. This leads to the formation of a variety of highly genotoxic DNA lesions, including abasic sites. Yeast Apn1p is localized to the nucleus, where it functions to cleave abasic sites, and apn1 Delta mutants are hypersensitive to agents such as methyl methanesulfonate (MMS) that induce abasic sites. Here we demonstrate for the first time that yeast Apn1p is also localized to the mitochondria. We found that Pir1p, initially isolated as a cell wall constituent of unknown function, interacts with the C-terminal end of Apn1p, which bears a bipartite nuclear localization signal. Further analysis revealed that Pir1p is required to cause Apn1p mitochondrial localization, presumably by competing with the nuclear transport machinery. pir1 Delta mutants displayed a striking (approximately 3-fold) increase of Apn1p in the nucleus, which coincided with drastically reduced levels in the mitochondria. To explore the functional consequences of the Apn1p-Pir1p interaction, we measured the rate of mitochondrial mutations in the wild type and pir1 Delta and apn1 Delta mutants. pir1 Delta and apn1 Delta mutants exposed to MMS exhibited 3.6- and 5.8-fold increases, respectively, in the rate of mitochondrial mutations, underscoring the importance of Apn1p in repair of the mitochondrial genome. We conclude that Pir1p interacts with Apn1p, at the level of either the cytoplasm or nucleus, and facilitates Apn1p transport into the mitochondria to repair damaged DNA.
Vongsamphanh
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Guy-Bernier Research Centre, University of Montreal, Montreal, Quebec, Canada H1T 2M4.
Fortier
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Ramotar
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D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D045643:DNA Repair Enzymes; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D016681:Genome, Fungal; D005982:Glutathione Transferase / Q000378:metabolism; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008741:Methyl Methanesulfonate; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009153:Mutagens; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions; D013997:Time Factors; D020798:Two-Hybrid System Techniques
D005656:Fungal Proteins; D006023:Glycoproteins; D008164:Luminescent Proteins; D009153:Mutagens; C423367:PIR1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D008741:Methyl Methanesulfonate; D005982:Glutathione Transferase; D004706:Endodeoxyribonucleases; C083274:Apn1 protein, S cerevisiae; D045643:DNA Repair Enzymes