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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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Deletion of BLM10 results in elevated Sfp1 levels. (A) Sfp1 steady-state levels are increased in BLM10-deleted cells. SFP1-HA3 (yMS908) and SFP1-HA3 blm10Δ (yMS909) strains were grown to the different metabolic phases in YPD and lysed. Equal protein amounts were separated by SDS–PAGE and subjected to immunodetection with an anti-HA antibody to detect SFP1-HA3 (top). Pgk1 protein levels were used as a loading control (bottom). (B) SFP1 transcription is not significantly altered upon loss of BLM10. WT (yMS524) and BLM10-deleted cells (yMS63) were grown to the different metabolic phases, and SFP1 mRNA levels were determined via qRT-PCR. CT values were normalized to ACT1 expression levels. SFP1 mRNA levels in blm10Δ were normalized to the WT levels. Data are reported as mean ± SEM. A single asterisk indicates a P-value < 0.05; a double asterisk indicates P < 0.01. (C) Sfp1 protein levels are increased in rpt3R mutants. WT (yMS1092) and rpt3R (yMS1093) were grown to the different metabolic phases and analyzed as in Figure 2B. (D) Epistatic genetic interaction between BLM10 and SFP1. Log phase WT (yMS268), blm10Δ (yMS131), sfp1Δ (yMS1011), and sfp1Δ blm10Δ (yMS1012) strains were serially diluted and spotted onto YPD media in the absence (left) or presence of 0.2 μg/ml CHX (right).
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Figure 5: Deletion of BLM10 results in elevated Sfp1 levels. (A) Sfp1 steady-state levels are increased in BLM10-deleted cells. SFP1-HA3 (yMS908) and SFP1-HA3 blm10Δ (yMS909) strains were grown to the different metabolic phases in YPD and lysed. Equal protein amounts were separated by SDS–PAGE and subjected to immunodetection with an anti-HA antibody to detect SFP1-HA3 (top). Pgk1 protein levels were used as a loading control (bottom). (B) SFP1 transcription is not significantly altered upon loss of BLM10. WT (yMS524) and BLM10-deleted cells (yMS63) were grown to the different metabolic phases, and SFP1 mRNA levels were determined via qRT-PCR. CT values were normalized to ACT1 expression levels. SFP1 mRNA levels in blm10Δ were normalized to the WT levels. Data are reported as mean ± SEM. A single asterisk indicates a P-value < 0.05; a double asterisk indicates P < 0.01. (C) Sfp1 protein levels are increased in rpt3R mutants. WT (yMS1092) and rpt3R (yMS1093) were grown to the different metabolic phases and analyzed as in Figure 2B. (D) Epistatic genetic interaction between BLM10 and SFP1. Log phase WT (yMS268), blm10Δ (yMS131), sfp1Δ (yMS1011), and sfp1Δ blm10Δ (yMS1012) strains were serially diluted and spotted onto YPD media in the absence (left) or presence of 0.2 μg/ml CHX (right).

Mentions: We reasoned that the elevated RP mRNA levels upon nutrient depletion in blm10Δ cells might originate from inefficient proteasome-mediated elimination of transcriptional activators during nutrient deprivation. Such a model is supported by reports that demonstrate that proteasomes participate in the regulation of transcription through the degradation of transcriptional activators (Lipford and Deshaies, 2003; Collins and Tansey, 2006). An important transcriptional activator for sustained RP gene transcription during logarithmic growth is Sfp1 (Marion et al., 2004; Lempiainen et al., 2009; Singh and Tyers, 2009). We tested the steady-state levels of Sfp1 in the different metabolic phases in WT cells. A strong reduction of Sfp1 protein levels was apparent in WT cells after the diauxic shift and in stat phase (Figure 5A), indicating that Sfp1 function might be regulated at the protein level. In blm10Δ cells increased protein levels of Sfp1 were found in all metabolic phases analyzed as compared with WT (log, PDS, and stat) (Figure 5A), suggesting a function for Blm10 in proteasome-mediated turnover of Sfp1. To rule out that BLM10 deletion results in elevated SFP1 transcription, which could explain an increase in Sfp1 abundance, we tested SFP1 mRNA levels and found them largely unaffected or even reduced by BLM10 deletion (Figure 5B). Thus the elevated Sfp1 protein levels observed upon BLM10 deletion are likely caused by a defect in protein turnover. To investigate whether Sfp1 stabilization is a general consequence of proteasome dysfunction, we tested the steady-state level of Sfp1 in the proteasomal ATPase mutant (rpt3R; Rubin et al., 1998). As with BLM10 deletion, loss of the ATPase activity of Rpt3 results in elevated Sfp1 levels (Figure 5C).


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)

Deletion of BLM10 results in elevated Sfp1 levels. (A) Sfp1 steady-state levels are increased in BLM10-deleted cells. SFP1-HA3 (yMS908) and SFP1-HA3 blm10Δ (yMS909) strains were grown to the different metabolic phases in YPD and lysed. Equal protein amounts were separated by SDS–PAGE and subjected to immunodetection with an anti-HA antibody to detect SFP1-HA3 (top). Pgk1 protein levels were used as a loading control (bottom). (B) SFP1 transcription is not significantly altered upon loss of BLM10. WT (yMS524) and BLM10-deleted cells (yMS63) were grown to the different metabolic phases, and SFP1 mRNA levels were determined via qRT-PCR. CT values were normalized to ACT1 expression levels. SFP1 mRNA levels in blm10Δ were normalized to the WT levels. Data are reported as mean ± SEM. A single asterisk indicates a P-value < 0.05; a double asterisk indicates P < 0.01. (C) Sfp1 protein levels are increased in rpt3R mutants. WT (yMS1092) and rpt3R (yMS1093) were grown to the different metabolic phases and analyzed as in Figure 2B. (D) Epistatic genetic interaction between BLM10 and SFP1. Log phase WT (yMS268), blm10Δ (yMS131), sfp1Δ (yMS1011), and sfp1Δ blm10Δ (yMS1012) strains were serially diluted and spotted onto YPD media in the absence (left) or presence of 0.2 μg/ml CHX (right).
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Figure 5: Deletion of BLM10 results in elevated Sfp1 levels. (A) Sfp1 steady-state levels are increased in BLM10-deleted cells. SFP1-HA3 (yMS908) and SFP1-HA3 blm10Δ (yMS909) strains were grown to the different metabolic phases in YPD and lysed. Equal protein amounts were separated by SDS–PAGE and subjected to immunodetection with an anti-HA antibody to detect SFP1-HA3 (top). Pgk1 protein levels were used as a loading control (bottom). (B) SFP1 transcription is not significantly altered upon loss of BLM10. WT (yMS524) and BLM10-deleted cells (yMS63) were grown to the different metabolic phases, and SFP1 mRNA levels were determined via qRT-PCR. CT values were normalized to ACT1 expression levels. SFP1 mRNA levels in blm10Δ were normalized to the WT levels. Data are reported as mean ± SEM. A single asterisk indicates a P-value < 0.05; a double asterisk indicates P < 0.01. (C) Sfp1 protein levels are increased in rpt3R mutants. WT (yMS1092) and rpt3R (yMS1093) were grown to the different metabolic phases and analyzed as in Figure 2B. (D) Epistatic genetic interaction between BLM10 and SFP1. Log phase WT (yMS268), blm10Δ (yMS131), sfp1Δ (yMS1011), and sfp1Δ blm10Δ (yMS1012) strains were serially diluted and spotted onto YPD media in the absence (left) or presence of 0.2 μg/ml CHX (right).
Mentions: We reasoned that the elevated RP mRNA levels upon nutrient depletion in blm10Δ cells might originate from inefficient proteasome-mediated elimination of transcriptional activators during nutrient deprivation. Such a model is supported by reports that demonstrate that proteasomes participate in the regulation of transcription through the degradation of transcriptional activators (Lipford and Deshaies, 2003; Collins and Tansey, 2006). An important transcriptional activator for sustained RP gene transcription during logarithmic growth is Sfp1 (Marion et al., 2004; Lempiainen et al., 2009; Singh and Tyers, 2009). We tested the steady-state levels of Sfp1 in the different metabolic phases in WT cells. A strong reduction of Sfp1 protein levels was apparent in WT cells after the diauxic shift and in stat phase (Figure 5A), indicating that Sfp1 function might be regulated at the protein level. In blm10Δ cells increased protein levels of Sfp1 were found in all metabolic phases analyzed as compared with WT (log, PDS, and stat) (Figure 5A), suggesting a function for Blm10 in proteasome-mediated turnover of Sfp1. To rule out that BLM10 deletion results in elevated SFP1 transcription, which could explain an increase in Sfp1 abundance, we tested SFP1 mRNA levels and found them largely unaffected or even reduced by BLM10 deletion (Figure 5B). Thus the elevated Sfp1 protein levels observed upon BLM10 deletion are likely caused by a defect in protein turnover. To investigate whether Sfp1 stabilization is a general consequence of proteasome dysfunction, we tested the steady-state level of Sfp1 in the proteasomal ATPase mutant (rpt3R; Rubin et al., 1998). As with BLM10 deletion, loss of the ATPase activity of Rpt3 results in elevated Sfp1 levels (Figure 5C).

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