RESUMO
Secreted acid phosphatases (APases) dephosphorylate extracellular organic phosphate compounds to supply inorganic phosphate (Pi) to maintain cellular functions. Here, we show that APases are necessary to maintain a normal replicative lifespan in Saccharomyces cerevisiae. Deletion of all four APase genes shortened the lifespan in yeast strains on synthetic media (irrespective of the concentrations of Pi in the media), but it did not affect the intracellular ortho- and polyphosphate levels. Deletion of inositol-pentakisphosphate 2-kinase (IPK1), which encodes inositol-pentakisphosphate 2-kinase, restored the lifespan in APase-null mutants, and IPK1 overexpression shortened the lifespan in wild-type strains. Overexpression of inositol hexakisphosphate (IP6 ) and heptakisphosphate kinases, KCS1 and VIP1, recovered the lifespan in APase-null mutants. Thus, yeast APases modulate the replicative lifespan, probably through dephosphorylation of intracellular IP6 .
Assuntos
PolifosfatosRESUMO
Nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase elicits nucleophagy degrading nucleolar proteins in budding yeast. After TORC1 inactivation, nucleolar proteins are relocated to sites proximal to the nucleus-vacuole junction (NVJ), where micronucleophagy occurs, whereas ribosomal DNA (rDNA encoding rRNA) escapes from the NVJ. Condensin-mediated rDNA condensation promotes the repositioning and nucleophagic degradation of nucleolar proteins. However, the molecular mechanism of TORC1 inactivation-induced chromosome condensation is still unknown. Here, we show that Cdc14 protein phosphatase and topoisomerase II (Topo II), which are engaged in rDNA condensation in mitosis, facilitate rDNA condensation after TORC1 inactivation. rDNA condensation after rapamycin treatment was compromised in cdc14-1 and top2-4 mutants. In addition, the repositioning of rDNA and nucleolar proteins and nucleophagic degradation of nucleolar proteins were impeded in these mutants. Furthermore, Cdc14 and Topo II were required for the survival of quiescent cells in prolonged nutrient-starved conditions. This study reveals that these factors are critical for starvation responses.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Topoisomerases Tipo II/metabolismo , DNA Fúngico/metabolismo , DNA Ribossômico/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , DNA Topoisomerases Tipo II/genética , DNA Fúngico/genética , DNA Ribossômico/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas Nucleares/genética , Proteínas Tirosina Fosfatases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The rRNA genes (rDNA) in eukaryotes are organized into highly repetitive gene clusters. Each organism maintains a particular number of copies, suggesting that the rDNA is actively stabilized. We previously identified about 700 Saccharomyces cerevisiae genes that could contribute to rDNA maintenance. Here, we further analyzed these deletion mutants with unstable rDNA by measuring the amounts of extrachromosomal rDNA circles (ERCs) that are released as by-products of intrachromosomal recombination. We found that extremely high levels of ERCs were formed in the absence of Pop2 (Caf1), which is a subunit of the CCR4-NOT complex, important for the regulation of all stages of gene expression. In the pop2 mutant, transcripts from the noncoding promoter E-pro in the rDNA accumulated, and the amounts of cohesin and condensin were reduced, which could promote recombination events. Moreover, we discovered that the amount of rRNA was decreased in the pop2 mutant. Similar phenotypes were observed in the absence of subunits Ccr4 and Not4 that, like Pop2, convey enzymatic activity to the complex. These findings indicate that lack of any CCR4-NOT-associated enzymatic activity resulted in a severe unstable rDNA phenotype related to the accumulation of noncoding RNA from E-pro.
Assuntos
RNA Fúngico/genética , RNA Ribossômico/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , DNA Ribossômico/genética , Regulação Fúngica da Expressão Gênica , Genes de RNAr , Instabilidade Genômica , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase induce nucleophagy preferentially degrading only nucleolar components in budding yeast. Nucleolar proteins are relocated to sites proximal to the nucleus-vacuole junction (NVJ), where micronucleophagy occurs, whereas rDNA, which is embedded in the nucleolus under normal conditions, moves to NVJ-distal regions, causing rDNA dissociation from nucleolar proteins after TORC1 inactivation. This repositioning is mediated via chromosome linkage INM protein (CLIP)-cohibin complexes that tether rDNA to the inner nuclear membrane. Here, we show that TORC1 inactivation-induced rDNA condensation promotes the repositioning of rDNA and nucleolar proteins. Defects in condensin, Rpd3-Sin3 histone deacetylase (HDAC), and high-mobility group protein 1 (Hmo1), which are involved in TORC1 inactivation-induced rDNA condensation, compromised the repositioning and nucleophagic degradation of nucleolar proteins, although rDNA still escaped from nucleophagic degradation in these mutants. We propose a model in which rDNA condensation after TORC1 inactivation generates a motive force for the repositioning of rDNA and nucleolar proteins.
Assuntos
Autofagia/imunologia , DNA Ribossômico/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Nucleares/metabolismo , HumanosRESUMO
Nutrient starvation or inactivation of target of rapamycin complex 1 (TORC1) in budding yeast induces nucleophagy, a selective autophagy process that preferentially degrades nucleolar components. DNA, including ribosomal DNA (rDNA), is not degraded by nucleophagy, even though rDNA is embedded in the nucleolus. Here, we show that TORC1 inactivation promotes relocalization of nucleolar proteins and rDNA to different sites. Nucleolar proteins move to sites proximal to the nuclear-vacuolar junction (NVJ), where micronucleophagy (or piecemeal microautophagy of the nucleus) occurs, whereas rDNA dissociates from nucleolar proteins and moves to sites distal to NVJs. CLIP and cohibin, which tether rDNA to the inner nuclear membrane, were required for repositioning of nucleolar proteins and rDNA, as well as effective nucleophagic degradation of the nucleolar proteins. Furthermore, micronucleophagy itself was necessary for the repositioning of rDNA and nucleolar proteins. However, rDNA escaped from nucleophagic degradation in CLIP- or cohibin-deficient cells. This study reveals that rDNA-nucleolar protein separation is important for the nucleophagic degradation of nucleolar proteins.
