RESUMEN
Targeted protein degradation systems developed for eukaryotes employ cytoplasmic machineries to perform proteolysis. This has prevented mitochondria-specific analysis of proteins that localize to multiple locations, for example, the mitochondria and the nucleus. Here, we present an inducible mitochondria-specific protein degradation system in Saccharomyces cerevisiae based on the Mesoplasma florum Lon (mf-Lon) protease and its corresponding ssrA tag (called PDT). We show that mitochondrially targeted mf-Lon protease efficiently and selectively degrades a PDT-tagged reporter protein localized to the mitochondrial matrix. The degradation can be induced by depleting adenine from the medium, and tuned by altering the promoter strength of the MF-LON gene. We furthermore demonstrate that mf-Lon specifically degrades endogenous, PDT-tagged mitochondrial proteins. Finally, we show that mf-Lon-dependent PDT degradation can also be achieved in human mitochondria. In summary, this system provides an efficient tool to selectively analyze the mitochondrial function of dually localized proteins.
Asunto(s)
Mitocondrias , Proteasa La , Humanos , Proteolisis , Mitocondrias/metabolismo , Proteasa La/genética , Proteasa La/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The canonical role of cohesin is to mediate sister chromatid cohesion. In addition, cohesin plays important roles in processes such as DNA repair and regulation of gene expression. Mounting evidence suggests that various post-translational modifications, including phosphorylation, acetylation and sumoylation regulate cohesin functions. Our mass spectrometry analysis of cohesin purified from Schizosaccharomyces pombe cells revealed that the cohesin subunit Psm1 is methylated on two evolutionarily conserved lysine residues, K536 and K1200. We found that mutations that prevent methylation of Psm1 K536 and K1200 render sensitivity to DNA-damaging agents and show positive genetic interactions with mutations in genes encoding the Mus81-Eme1 endonuclease. Yeast two-hybrid and co-immunoprecipitation assays showed that there were interactions between subunits of the cohesin and Mus81-Eme1 complexes. We conclude that cohesin is methylated and that mutations that prevent methylation of Psm1 K536 and K1200 show synthetic phenotypes with mutants defective in the homologous recombination DNA repair pathway.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Secuencias de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Metilación , Mutación , Unión Proteica , Schizosaccharomyces/química , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , CohesinasRESUMEN
One of the key features of meiosis is that shugoshin in complex with protein phosphatase 2A (PP2A) protects centromeric cohesin during meiosis I, but not during meiosis II. A new model suggests that a PP2A inhibitor mediates deprotection of centromeric cohesin during meiosis II.
