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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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Loss of BLM10 does not lead to a general impairment of proteasome function (A and B). Turnover of the proteasome substrate Ubc6 was determined in WT (UBC6-HA3 pdr5Δ [yMS792]) or in blm10Δ (UBC6-HA3 blm10Δ pdr5Δ [yMS1089]). (C) Ubc6 is stabilized in rpn4Δ cells. Turnover of the proteasome substrate Ubc6 in rpn4Δ (UBC6-HA3 rpn4Δ pdr5Δ [yMS1364]) strain. Pgk1 immunodetection was used as a loading control (bottom). (D) Sfp1 interacts with Blm10-proteasomes. For CP pull-down cells from yMS1189 and from yMS1190 containing protein A–tagged Pre1 and carrying SFP1–HA3 in the presence (yMS1189) or in the absence (yMS1190) of BLM10 were harvested in logarithmic (log) phase, lysed, and subjected to immune precipitation. The samples were separated by SDS–PAGE and probed with anti-HA (top) or anti-CP (bottom) antibodies.
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Figure 7: Loss of BLM10 does not lead to a general impairment of proteasome function (A and B). Turnover of the proteasome substrate Ubc6 was determined in WT (UBC6-HA3 pdr5Δ [yMS792]) or in blm10Δ (UBC6-HA3 blm10Δ pdr5Δ [yMS1089]). (C) Ubc6 is stabilized in rpn4Δ cells. Turnover of the proteasome substrate Ubc6 in rpn4Δ (UBC6-HA3 rpn4Δ pdr5Δ [yMS1364]) strain. Pgk1 immunodetection was used as a loading control (bottom). (D) Sfp1 interacts with Blm10-proteasomes. For CP pull-down cells from yMS1189 and from yMS1190 containing protein A–tagged Pre1 and carrying SFP1–HA3 in the presence (yMS1189) or in the absence (yMS1190) of BLM10 were harvested in logarithmic (log) phase, lysed, and subjected to immune precipitation. The samples were separated by SDS–PAGE and probed with anti-HA (top) or anti-CP (bottom) antibodies.

Mentions: Blm10 has been implicated in proteasome assembly (Fehlker et al., 2003; Li et al., 2007; Marques et al., 2007). In consequence, impaired Sfp1 turnover could potentially be explained by a general impairment of proteasome structural integrity upon loss of BLM10. To compare the proteolytic capacity of proteasomes in WT and blm10Δ cells, we tested the turnover of a general proteasome substrate, Ubc6 (Walter et al., 2001; Ravid et al., 2006). Loss of BLM10 did not influence the turnover of Ubc6 (Figure 7, A and B), whereas deletion of the proteasome-related transcription factor Rpn4, which results in reduced proteasome abundance (Xie and Varshavsky, 2001; Schmidt et al., 2005), effectively inhibited Ubc4 turnover. Thus, in the absence of Blm10, proteasomes are fully functional, arguing against impaired proteasome assembly in blm10Δ cells. The result additionally corroborates a model in which Blm10-proteasomes might target a subgroup of proteasome substrates (Schmidt et al., 2005). To further investigate the interaction between Sfp1 and Blm10-proteasomes, we mapped their physical interaction by CP immunoprecipitation (Figure 7D, right) in the absence or presence of BLM10. We found that Sfp1 copurifies with the proteasome in both WT and blm10Δ cells, indicating that Sfp1 interaction with the proteasome is not mediated by direct interaction of Sfp1 with Blm10. However, in blm10Δ cells the Sfp1 protein level, which copurified with the CP, was elevated, demonstrating that Blm10 positively affects the regulated turnover of Sfp1 at the proteasome.


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)

Loss of BLM10 does not lead to a general impairment of proteasome function (A and B). Turnover of the proteasome substrate Ubc6 was determined in WT (UBC6-HA3 pdr5Δ [yMS792]) or in blm10Δ (UBC6-HA3 blm10Δ pdr5Δ [yMS1089]). (C) Ubc6 is stabilized in rpn4Δ cells. Turnover of the proteasome substrate Ubc6 in rpn4Δ (UBC6-HA3 rpn4Δ pdr5Δ [yMS1364]) strain. Pgk1 immunodetection was used as a loading control (bottom). (D) Sfp1 interacts with Blm10-proteasomes. For CP pull-down cells from yMS1189 and from yMS1190 containing protein A–tagged Pre1 and carrying SFP1–HA3 in the presence (yMS1189) or in the absence (yMS1190) of BLM10 were harvested in logarithmic (log) phase, lysed, and subjected to immune precipitation. The samples were separated by SDS–PAGE and probed with anti-HA (top) or anti-CP (bottom) antibodies.
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Related In: Results  -  Collection

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Figure 7: Loss of BLM10 does not lead to a general impairment of proteasome function (A and B). Turnover of the proteasome substrate Ubc6 was determined in WT (UBC6-HA3 pdr5Δ [yMS792]) or in blm10Δ (UBC6-HA3 blm10Δ pdr5Δ [yMS1089]). (C) Ubc6 is stabilized in rpn4Δ cells. Turnover of the proteasome substrate Ubc6 in rpn4Δ (UBC6-HA3 rpn4Δ pdr5Δ [yMS1364]) strain. Pgk1 immunodetection was used as a loading control (bottom). (D) Sfp1 interacts with Blm10-proteasomes. For CP pull-down cells from yMS1189 and from yMS1190 containing protein A–tagged Pre1 and carrying SFP1–HA3 in the presence (yMS1189) or in the absence (yMS1190) of BLM10 were harvested in logarithmic (log) phase, lysed, and subjected to immune precipitation. The samples were separated by SDS–PAGE and probed with anti-HA (top) or anti-CP (bottom) antibodies.
Mentions: Blm10 has been implicated in proteasome assembly (Fehlker et al., 2003; Li et al., 2007; Marques et al., 2007). In consequence, impaired Sfp1 turnover could potentially be explained by a general impairment of proteasome structural integrity upon loss of BLM10. To compare the proteolytic capacity of proteasomes in WT and blm10Δ cells, we tested the turnover of a general proteasome substrate, Ubc6 (Walter et al., 2001; Ravid et al., 2006). Loss of BLM10 did not influence the turnover of Ubc6 (Figure 7, A and B), whereas deletion of the proteasome-related transcription factor Rpn4, which results in reduced proteasome abundance (Xie and Varshavsky, 2001; Schmidt et al., 2005), effectively inhibited Ubc4 turnover. Thus, in the absence of Blm10, proteasomes are fully functional, arguing against impaired proteasome assembly in blm10Δ cells. The result additionally corroborates a model in which Blm10-proteasomes might target a subgroup of proteasome substrates (Schmidt et al., 2005). To further investigate the interaction between Sfp1 and Blm10-proteasomes, we mapped their physical interaction by CP immunoprecipitation (Figure 7D, right) in the absence or presence of BLM10. We found that Sfp1 copurifies with the proteasome in both WT and blm10Δ cells, indicating that Sfp1 interaction with the proteasome is not mediated by direct interaction of Sfp1 with Blm10. However, in blm10Δ cells the Sfp1 protein level, which copurified with the CP, was elevated, demonstrating that Blm10 positively affects the regulated turnover of Sfp1 at the proteasome.

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