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Proteasomal degradation of Sfp1 contributes to the repression of ribosome biogenesis during starvation and is mediated by the proteasome activator Blm10.

Lopez AD, Tar K, Krügel U, Dange T, Ros IG, Schmidt M - Mol. Biol. Cell (2011)

Bottom Line: Repression of RP gene transcription appears to be regulated predominantly by posttranslational modification and cellular localization of transcriptional activators.We report here that one of these factors, Sfp1, is degraded by the proteasome and that the proteasome activator Blm10 is required for regulated Sfp1 degradation.Thus we conclude that proteasomal degradation of Sfp1 is mediated by Blm10 and contributes to the repression of ribosome biogenesis under nutrient depletion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
The regulation of ribosomal protein (RP) gene transcription is tightly linked to the nutrient status of the cell and is under the control of metabolic signaling pathways. In Saccharomyces cerevisiae several transcriptional activators mediate efficient RP gene transcription during logarithmic growth and dissociate from RP gene promoters upon nutrient limitation. Repression of RP gene transcription appears to be regulated predominantly by posttranslational modification and cellular localization of transcriptional activators. We report here that one of these factors, Sfp1, is degraded by the proteasome and that the proteasome activator Blm10 is required for regulated Sfp1 degradation. Loss of Blm10 results in the stabilization and increased nuclear abundance of Sfp1 during nutrient limitation, increased transcription of RP genes, increased levels of RPs, and decreased rapamycin-induced repression of RP genes. Thus we conclude that proteasomal degradation of Sfp1 is mediated by Blm10 and contributes to the repression of ribosome biogenesis under nutrient depletion.

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Proteasomal degradation of Sfp1 requires the activator Blm10. Logarithmically growing SFP1-HA pdr5Δ (yMS957) or SFP1-HA blm10Δ pdr5Δ (yMS958) cells were grown in the absence (A and C) or presence of the proteasome inhibitor MG132 (B and D) for 3 h at 30°C. Subsequently, translation was blocked with 200 μg/ml CHX, and aliquots were harvested and processed at the time points indicated. Equal amounts of protein were subjected to SDS–PAGE, followed by immunodetection with an HA-specific antibody to detect Sfp1 protein levels (left). Pgk1 immunodetection was used as a loading control (bottom). A densitometric analysis of the Sfp1 protein levels is depicted on the right. Sfp1 half-life (t½) was calculated from an exponential decay curve with SigmaPlot 11.0.
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Figure 6: Proteasomal degradation of Sfp1 requires the activator Blm10. Logarithmically growing SFP1-HA pdr5Δ (yMS957) or SFP1-HA blm10Δ pdr5Δ (yMS958) cells were grown in the absence (A and C) or presence of the proteasome inhibitor MG132 (B and D) for 3 h at 30°C. Subsequently, translation was blocked with 200 μg/ml CHX, and aliquots were harvested and processed at the time points indicated. Equal amounts of protein were subjected to SDS–PAGE, followed by immunodetection with an HA-specific antibody to detect Sfp1 protein levels (left). Pgk1 immunodetection was used as a loading control (bottom). A densitometric analysis of the Sfp1 protein levels is depicted on the right. Sfp1 half-life (t½) was calculated from an exponential decay curve with SigmaPlot 11.0.

Mentions: To corroborate that Sfp1 turnover is mediated by proteasomes and is dependent on Blm10, we performed CHX chase experiments (Kornitzer, 2002) in rapidly growing cells. The experimental approach involves lethal doses of CHX, which blocks translation and cell cycle and results in a growth arrest (McCusker and Haber, 1988; Supplemental Figure 2). In the absence of new synthesis, the half-life of a protein can be determined. We found that in WT cells Sfp1 has a short half-life of ∼35 min, indicating continuous turnover of Sfp1 (Figure 6A). In the presence of MG132, a proteasome inhibitor, Sfp1 half-life increased to ∼108 min (Figure 6B). Thus Sfp1 degradation is mediated by the proteasome. A similar increase in Sfp1 half-life was detected in blm10Δ cells, demonstrating that Sfp1 degradation is executed most likely by Blm10-proteasomes (Figure 6C). Because the addition of MG132 did not increase Sfp1 half-life further in BLM10-deleted cells (Figure 6D), our data argue for a model in which Sfp1 degradation might be specifically mediated by Blm10-proteasomes, but not by other proteasome complexes. Although MG132 is readily taken up by yeast cells, it is also rapidly exported from the cell by specific transporters. To investigate the effect of MG132, a PDR5 deletion strain, a gene that codes for an export pump had to be used (Fleming et al., 2002). To ascertain that the lethal CHX dose used for the chase experiment indeed inhibits cell growth in a pdr5Δ strain background, we tested the strains used in Figure 6, A–D, for sensitivity to CHX. The pdr5Δ strains are exquisitely sensitive to CHX, independent of the presence or absence of BLM10 (Supplemental Figure 3).


Proteasomal degradation of Sfp1 contributes to the repression of ribosome biogenesis during starvation and is mediated by the proteasome activator Blm10.

Lopez AD, Tar K, Krügel U, Dange T, Ros IG, Schmidt M - Mol. Biol. Cell (2011)

Proteasomal degradation of Sfp1 requires the activator Blm10. Logarithmically growing SFP1-HA pdr5Δ (yMS957) or SFP1-HA blm10Δ pdr5Δ (yMS958) cells were grown in the absence (A and C) or presence of the proteasome inhibitor MG132 (B and D) for 3 h at 30°C. Subsequently, translation was blocked with 200 μg/ml CHX, and aliquots were harvested and processed at the time points indicated. Equal amounts of protein were subjected to SDS–PAGE, followed by immunodetection with an HA-specific antibody to detect Sfp1 protein levels (left). Pgk1 immunodetection was used as a loading control (bottom). A densitometric analysis of the Sfp1 protein levels is depicted on the right. Sfp1 half-life (t½) was calculated from an exponential decay curve with SigmaPlot 11.0.
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Related In: Results  -  Collection

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Figure 6: Proteasomal degradation of Sfp1 requires the activator Blm10. Logarithmically growing SFP1-HA pdr5Δ (yMS957) or SFP1-HA blm10Δ pdr5Δ (yMS958) cells were grown in the absence (A and C) or presence of the proteasome inhibitor MG132 (B and D) for 3 h at 30°C. Subsequently, translation was blocked with 200 μg/ml CHX, and aliquots were harvested and processed at the time points indicated. Equal amounts of protein were subjected to SDS–PAGE, followed by immunodetection with an HA-specific antibody to detect Sfp1 protein levels (left). Pgk1 immunodetection was used as a loading control (bottom). A densitometric analysis of the Sfp1 protein levels is depicted on the right. Sfp1 half-life (t½) was calculated from an exponential decay curve with SigmaPlot 11.0.
Mentions: To corroborate that Sfp1 turnover is mediated by proteasomes and is dependent on Blm10, we performed CHX chase experiments (Kornitzer, 2002) in rapidly growing cells. The experimental approach involves lethal doses of CHX, which blocks translation and cell cycle and results in a growth arrest (McCusker and Haber, 1988; Supplemental Figure 2). In the absence of new synthesis, the half-life of a protein can be determined. We found that in WT cells Sfp1 has a short half-life of ∼35 min, indicating continuous turnover of Sfp1 (Figure 6A). In the presence of MG132, a proteasome inhibitor, Sfp1 half-life increased to ∼108 min (Figure 6B). Thus Sfp1 degradation is mediated by the proteasome. A similar increase in Sfp1 half-life was detected in blm10Δ cells, demonstrating that Sfp1 degradation is executed most likely by Blm10-proteasomes (Figure 6C). Because the addition of MG132 did not increase Sfp1 half-life further in BLM10-deleted cells (Figure 6D), our data argue for a model in which Sfp1 degradation might be specifically mediated by Blm10-proteasomes, but not by other proteasome complexes. Although MG132 is readily taken up by yeast cells, it is also rapidly exported from the cell by specific transporters. To investigate the effect of MG132, a PDR5 deletion strain, a gene that codes for an export pump had to be used (Fleming et al., 2002). To ascertain that the lethal CHX dose used for the chase experiment indeed inhibits cell growth in a pdr5Δ strain background, we tested the strains used in Figure 6, A–D, for sensitivity to CHX. The pdr5Δ strains are exquisitely sensitive to CHX, independent of the presence or absence of BLM10 (Supplemental Figure 3).

Bottom Line: Repression of RP gene transcription appears to be regulated predominantly by posttranslational modification and cellular localization of transcriptional activators.We report here that one of these factors, Sfp1, is degraded by the proteasome and that the proteasome activator Blm10 is required for regulated Sfp1 degradation.Thus we conclude that proteasomal degradation of Sfp1 is mediated by Blm10 and contributes to the repression of ribosome biogenesis under nutrient depletion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
The regulation of ribosomal protein (RP) gene transcription is tightly linked to the nutrient status of the cell and is under the control of metabolic signaling pathways. In Saccharomyces cerevisiae several transcriptional activators mediate efficient RP gene transcription during logarithmic growth and dissociate from RP gene promoters upon nutrient limitation. Repression of RP gene transcription appears to be regulated predominantly by posttranslational modification and cellular localization of transcriptional activators. We report here that one of these factors, Sfp1, is degraded by the proteasome and that the proteasome activator Blm10 is required for regulated Sfp1 degradation. Loss of Blm10 results in the stabilization and increased nuclear abundance of Sfp1 during nutrient limitation, increased transcription of RP genes, increased levels of RPs, and decreased rapamycin-induced repression of RP genes. Thus we conclude that proteasomal degradation of Sfp1 is mediated by Blm10 and contributes to the repression of ribosome biogenesis under nutrient depletion.

Show MeSH
Related in: MedlinePlus