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Caf1 regulates translocation of ribonucleotide reductase by releasing nucleoplasmic Spd1-Suc22 assembly.

Takahashi S, Kontani K, Araki Y, Katada T - Nucleic Acids Res. (2007)

Bottom Line: Here, we show that Caf1, a component of the Ccr4-Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation.DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1-Suc22 assembly.Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

ABSTRACT
Appropriate supply of deoxyribonucleotides by the ribonucleotide reductase (RNR) complex is essential for DNA replication and repair. One recent model for the RNR activation in Schizosaccharomyces pombe is translocation of the regulatory subunit Suc22 from the nucleoplasm to the cytoplasm. The RNR inhibitory protein Spd1, which retains Suc22 in the nucleoplasm, is rapidly degraded upon DNA-replication stress, resulting in release of Suc22 to form the active RNR complex in the cytoplasm. Here, we show that Caf1, a component of the Ccr4-Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation. Caf1 is required not only for the stress-induced translocation of Suc22 from nucleoplasm to cytoplasm but also for the degradation of nucleoplasmic Spd1. DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1-Suc22 assembly. Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

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Caf1 is required for HU-induced degradation of Spd1. (A) Logarithmically growing cells, spd1-13Myc (YSP091) and caf1Δ spd1-13Myc (YSP161) were incubated in the presence and absence of 10 mM HU for 2 h at 30°C. Immunofluorescence-staining images of Spd1 were obtained with an anti-Myc antibody. (B) Logarithmically growing cells, spd1-13Myc (YSP091), ccr4Δ spd1-13Myc (YSP154), caf1Δ spd1-13Myc (YSP161) and not4Δ spd1-13Myc (YSP162), were incubated in the presence (+) and absence (−) of 10 mM HU for 2 h. Extracts from the cells were blotted with an anti-tubulin antibody as an internal control. The extracts were also incubated with anti-Myc antibody, and the immunoprecipitated fractions were subjected to western blot analysis for the detection of Spd1. Three independent experiments were performed, and the HU-induced reduction of the Spd1 bands was quantitated by an imaging analyzer LAS1000. The results of one representative set are also shown in the inset. (C) Logarithmically growing cells, spd1-13Myc pRep1 (YSP226), caf1Δ spd1-13Myc pRep1 (YSP195), caf1Δ spd1-13Myc pRep1-Caf1 (YSP196) and caf1Δ spd1-13Myc pRep1-Caf1/D50A (YSP197), were incubated with the indicated concentrations of HU for 2 h at 30°C, and Spd1 was detected as described in (B).
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Figure 5: Caf1 is required for HU-induced degradation of Spd1. (A) Logarithmically growing cells, spd1-13Myc (YSP091) and caf1Δ spd1-13Myc (YSP161) were incubated in the presence and absence of 10 mM HU for 2 h at 30°C. Immunofluorescence-staining images of Spd1 were obtained with an anti-Myc antibody. (B) Logarithmically growing cells, spd1-13Myc (YSP091), ccr4Δ spd1-13Myc (YSP154), caf1Δ spd1-13Myc (YSP161) and not4Δ spd1-13Myc (YSP162), were incubated in the presence (+) and absence (−) of 10 mM HU for 2 h. Extracts from the cells were blotted with an anti-tubulin antibody as an internal control. The extracts were also incubated with anti-Myc antibody, and the immunoprecipitated fractions were subjected to western blot analysis for the detection of Spd1. Three independent experiments were performed, and the HU-induced reduction of the Spd1 bands was quantitated by an imaging analyzer LAS1000. The results of one representative set are also shown in the inset. (C) Logarithmically growing cells, spd1-13Myc pRep1 (YSP226), caf1Δ spd1-13Myc pRep1 (YSP195), caf1Δ spd1-13Myc pRep1-Caf1 (YSP196) and caf1Δ spd1-13Myc pRep1-Caf1/D50A (YSP197), were incubated with the indicated concentrations of HU for 2 h at 30°C, and Spd1 was detected as described in (B).

Mentions: The data presented above predict that Caf1 is required for the cytoplasmic translocation of Suc22 in response to the replication stress by regulating the localization or the degradation of Spd1. To examine these possibilities, we investigated subcellular localization of Spd1 after HU treatment in wild-type and caf1Δ cells (Figure 5A). Under no-stress conditions, Spd1 was localized in the nucleoplasm of both cells. In response to HU, the signal of Spd1 disappeared from the nucleoplasm in wild-type cells, but was still present in caf1Δ cells. Thus, the stress-induced Spd1 degradation is impaired in caf1Δ cells. To confirm the role of Caf1 in the nucleoplasmic reduction of Spd1, we determined the quantity of Spd1 in various cells before and after HU treatment. The expression level of spd1 mRNA was almost unchanged among these cells (data not shown). As shown in Figure 5B, Spd1 was detected as multiple bands in untreated wild-type cells (lane 1), and these bands disappeared almost completely after incubation of the cells with 10 mM HU (lane 2). Figure 5C (first panel) shows the concentration-dependent effect of HU: there was a progressive decrease in the amount of Spd1, as the concentration of HU was increased (0–5 mM). The HU treatment also reduced the amount of Spd1 in not4Δ cells (Figure 5B, lane 8). However, the HU-induced reduction of Spd1 was markedly inhibited in caf1Δ cells (lane 6). Such inhibition was also observed in ccr4Δ cells to a lesser extent (lane 4), and this may be related to the observation that ccr4Δ cells were less sensitive to HU (Figure 1A). The HU-induced reduction of Spd1 in wild-type or not4Δ cells and its diminishment in caf1Δ or ccr4Δ cells were still apparently observed in the presence of a protein synthesis inhibitor (50 μg/ml cycloheximide, data not shown). This excludes the possibility that the reduction of Spd1 might be due to changes in de novo protein synthesis of Spd1.Figure 5.


Caf1 regulates translocation of ribonucleotide reductase by releasing nucleoplasmic Spd1-Suc22 assembly.

Takahashi S, Kontani K, Araki Y, Katada T - Nucleic Acids Res. (2007)

Caf1 is required for HU-induced degradation of Spd1. (A) Logarithmically growing cells, spd1-13Myc (YSP091) and caf1Δ spd1-13Myc (YSP161) were incubated in the presence and absence of 10 mM HU for 2 h at 30°C. Immunofluorescence-staining images of Spd1 were obtained with an anti-Myc antibody. (B) Logarithmically growing cells, spd1-13Myc (YSP091), ccr4Δ spd1-13Myc (YSP154), caf1Δ spd1-13Myc (YSP161) and not4Δ spd1-13Myc (YSP162), were incubated in the presence (+) and absence (−) of 10 mM HU for 2 h. Extracts from the cells were blotted with an anti-tubulin antibody as an internal control. The extracts were also incubated with anti-Myc antibody, and the immunoprecipitated fractions were subjected to western blot analysis for the detection of Spd1. Three independent experiments were performed, and the HU-induced reduction of the Spd1 bands was quantitated by an imaging analyzer LAS1000. The results of one representative set are also shown in the inset. (C) Logarithmically growing cells, spd1-13Myc pRep1 (YSP226), caf1Δ spd1-13Myc pRep1 (YSP195), caf1Δ spd1-13Myc pRep1-Caf1 (YSP196) and caf1Δ spd1-13Myc pRep1-Caf1/D50A (YSP197), were incubated with the indicated concentrations of HU for 2 h at 30°C, and Spd1 was detected as described in (B).
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Figure 5: Caf1 is required for HU-induced degradation of Spd1. (A) Logarithmically growing cells, spd1-13Myc (YSP091) and caf1Δ spd1-13Myc (YSP161) were incubated in the presence and absence of 10 mM HU for 2 h at 30°C. Immunofluorescence-staining images of Spd1 were obtained with an anti-Myc antibody. (B) Logarithmically growing cells, spd1-13Myc (YSP091), ccr4Δ spd1-13Myc (YSP154), caf1Δ spd1-13Myc (YSP161) and not4Δ spd1-13Myc (YSP162), were incubated in the presence (+) and absence (−) of 10 mM HU for 2 h. Extracts from the cells were blotted with an anti-tubulin antibody as an internal control. The extracts were also incubated with anti-Myc antibody, and the immunoprecipitated fractions were subjected to western blot analysis for the detection of Spd1. Three independent experiments were performed, and the HU-induced reduction of the Spd1 bands was quantitated by an imaging analyzer LAS1000. The results of one representative set are also shown in the inset. (C) Logarithmically growing cells, spd1-13Myc pRep1 (YSP226), caf1Δ spd1-13Myc pRep1 (YSP195), caf1Δ spd1-13Myc pRep1-Caf1 (YSP196) and caf1Δ spd1-13Myc pRep1-Caf1/D50A (YSP197), were incubated with the indicated concentrations of HU for 2 h at 30°C, and Spd1 was detected as described in (B).
Mentions: The data presented above predict that Caf1 is required for the cytoplasmic translocation of Suc22 in response to the replication stress by regulating the localization or the degradation of Spd1. To examine these possibilities, we investigated subcellular localization of Spd1 after HU treatment in wild-type and caf1Δ cells (Figure 5A). Under no-stress conditions, Spd1 was localized in the nucleoplasm of both cells. In response to HU, the signal of Spd1 disappeared from the nucleoplasm in wild-type cells, but was still present in caf1Δ cells. Thus, the stress-induced Spd1 degradation is impaired in caf1Δ cells. To confirm the role of Caf1 in the nucleoplasmic reduction of Spd1, we determined the quantity of Spd1 in various cells before and after HU treatment. The expression level of spd1 mRNA was almost unchanged among these cells (data not shown). As shown in Figure 5B, Spd1 was detected as multiple bands in untreated wild-type cells (lane 1), and these bands disappeared almost completely after incubation of the cells with 10 mM HU (lane 2). Figure 5C (first panel) shows the concentration-dependent effect of HU: there was a progressive decrease in the amount of Spd1, as the concentration of HU was increased (0–5 mM). The HU treatment also reduced the amount of Spd1 in not4Δ cells (Figure 5B, lane 8). However, the HU-induced reduction of Spd1 was markedly inhibited in caf1Δ cells (lane 6). Such inhibition was also observed in ccr4Δ cells to a lesser extent (lane 4), and this may be related to the observation that ccr4Δ cells were less sensitive to HU (Figure 1A). The HU-induced reduction of Spd1 in wild-type or not4Δ cells and its diminishment in caf1Δ or ccr4Δ cells were still apparently observed in the presence of a protein synthesis inhibitor (50 μg/ml cycloheximide, data not shown). This excludes the possibility that the reduction of Spd1 might be due to changes in de novo protein synthesis of Spd1.Figure 5.

Bottom Line: Here, we show that Caf1, a component of the Ccr4-Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation.DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1-Suc22 assembly.Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

ABSTRACT
Appropriate supply of deoxyribonucleotides by the ribonucleotide reductase (RNR) complex is essential for DNA replication and repair. One recent model for the RNR activation in Schizosaccharomyces pombe is translocation of the regulatory subunit Suc22 from the nucleoplasm to the cytoplasm. The RNR inhibitory protein Spd1, which retains Suc22 in the nucleoplasm, is rapidly degraded upon DNA-replication stress, resulting in release of Suc22 to form the active RNR complex in the cytoplasm. Here, we show that Caf1, a component of the Ccr4-Not complex, is responsible for resistance of the replication stress and control of the Suc22 translocation. Caf1 is required not only for the stress-induced translocation of Suc22 from nucleoplasm to cytoplasm but also for the degradation of nucleoplasmic Spd1. DNA-replication stress appears to allow Caf1 to interact with Suc22, resulting in release of the nucleoplasmic Spd1-Suc22 assembly. Taken together, these results suggest a novel function of Caf1 as a key regulator in the stress-induced RNR activation.

Show MeSH
Related in: MedlinePlus