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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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Impaired Sfp1 localization in BLM10-deleted cells after the diauxic shift. (A) SFP1-GFP (yMS928) and SFP1-GFP blm10Δ (yMS929) cells were grown in synthetic complete media. Sfp1 localization was visualized in log phase via live-cell fluorescence. Differential interference contrast (DIC) images are shown on the right. (B) Sfp1 localization in PDS phase cells was analyzed in SFP1-GFP (yMS928) and SFP1-GFP blm10Δ (yMS929) as in (A). (C) Quantification of cells with nuclear Sfp1 localization in log and PDS in WT (yMS928) and blm10Δ (yMS929) from 10 independent fluorescent micrographs with ∼500 cells each, using ImageJ software 1.42q for visualization.
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Figure 9: Impaired Sfp1 localization in BLM10-deleted cells after the diauxic shift. (A) SFP1-GFP (yMS928) and SFP1-GFP blm10Δ (yMS929) cells were grown in synthetic complete media. Sfp1 localization was visualized in log phase via live-cell fluorescence. Differential interference contrast (DIC) images are shown on the right. (B) Sfp1 localization in PDS phase cells was analyzed in SFP1-GFP (yMS928) and SFP1-GFP blm10Δ (yMS929) as in (A). (C) Quantification of cells with nuclear Sfp1 localization in log and PDS in WT (yMS928) and blm10Δ (yMS929) from 10 independent fluorescent micrographs with ∼500 cells each, using ImageJ software 1.42q for visualization.

Mentions: Sfp1 function appears to be controlled by differential localization. While the protein is localized predominantly to the nucleus if nutrients are abundant, it is found evenly distributed in the cell after the diauxic shift or upon rapamycin treatment (Jorgensen et al., 2004; Marion et al., 2004). A current model for the repression of RP gene transcription under nutrient-limiting conditions involves the dissociation of Sfp1 from RP gene promoters, followed by nuclear export of the protein. We demonstrate here that Sfp1 is degraded after the diauxic shift, and if degradation is abrogated due to loss of Blm10 or upon proteasome inhibition, the protein is stabilized and the repression of RP gene transcription is attenuated. The higher levels of RP mRNA in blm10Δ cells suggest that a significant fraction of Sfp1 must remain functional and bound to RP gene promoters even under repressive conditions. To test Sfp1 localization in BLM10-deleted cells, we tagged the C-terminus of Sfp1 with green fluorescent protein (GFP) and performed live-cell fluorescence microscopy. As reported previously in WT cells (Marion et al., 2004; Lempiainen et al., 2009; Singh and Tyers, 2009), the predominant nuclear localization of Sfp1 during log growth (Figure 9A, top) is lost in PDS (Figure 9B, top). In blm10Δ log phase cells, Sfp1 is also detected predominantly in the nucleus (Figure 9A, bottom). In PDS, however, loss of BLM10 results in a high fraction of Sfp1 retained in the nucleus (Figure 9B, bottom, and 9C). The higher Sfp1 levels in blm10Δ cells after the diauxic shift are also reflected in increased intensity of Sfp1-GFP fluorescence if the cells are imaged with identical exposure times (Supplemental Figure 4).


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)

Impaired Sfp1 localization in BLM10-deleted cells after the diauxic shift. (A) SFP1-GFP (yMS928) and SFP1-GFP blm10Δ (yMS929) cells were grown in synthetic complete media. Sfp1 localization was visualized in log phase via live-cell fluorescence. Differential interference contrast (DIC) images are shown on the right. (B) Sfp1 localization in PDS phase cells was analyzed in SFP1-GFP (yMS928) and SFP1-GFP blm10Δ (yMS929) as in (A). (C) Quantification of cells with nuclear Sfp1 localization in log and PDS in WT (yMS928) and blm10Δ (yMS929) from 10 independent fluorescent micrographs with ∼500 cells each, using ImageJ software 1.42q for visualization.
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Related In: Results  -  Collection

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Figure 9: Impaired Sfp1 localization in BLM10-deleted cells after the diauxic shift. (A) SFP1-GFP (yMS928) and SFP1-GFP blm10Δ (yMS929) cells were grown in synthetic complete media. Sfp1 localization was visualized in log phase via live-cell fluorescence. Differential interference contrast (DIC) images are shown on the right. (B) Sfp1 localization in PDS phase cells was analyzed in SFP1-GFP (yMS928) and SFP1-GFP blm10Δ (yMS929) as in (A). (C) Quantification of cells with nuclear Sfp1 localization in log and PDS in WT (yMS928) and blm10Δ (yMS929) from 10 independent fluorescent micrographs with ∼500 cells each, using ImageJ software 1.42q for visualization.
Mentions: Sfp1 function appears to be controlled by differential localization. While the protein is localized predominantly to the nucleus if nutrients are abundant, it is found evenly distributed in the cell after the diauxic shift or upon rapamycin treatment (Jorgensen et al., 2004; Marion et al., 2004). A current model for the repression of RP gene transcription under nutrient-limiting conditions involves the dissociation of Sfp1 from RP gene promoters, followed by nuclear export of the protein. We demonstrate here that Sfp1 is degraded after the diauxic shift, and if degradation is abrogated due to loss of Blm10 or upon proteasome inhibition, the protein is stabilized and the repression of RP gene transcription is attenuated. The higher levels of RP mRNA in blm10Δ cells suggest that a significant fraction of Sfp1 must remain functional and bound to RP gene promoters even under repressive conditions. To test Sfp1 localization in BLM10-deleted cells, we tagged the C-terminus of Sfp1 with green fluorescent protein (GFP) and performed live-cell fluorescence microscopy. As reported previously in WT cells (Marion et al., 2004; Lempiainen et al., 2009; Singh and Tyers, 2009), the predominant nuclear localization of Sfp1 during log growth (Figure 9A, top) is lost in PDS (Figure 9B, top). In blm10Δ log phase cells, Sfp1 is also detected predominantly in the nucleus (Figure 9A, bottom). In PDS, however, loss of BLM10 results in a high fraction of Sfp1 retained in the nucleus (Figure 9B, bottom, and 9C). The higher Sfp1 levels in blm10Δ cells after the diauxic shift are also reflected in increased intensity of Sfp1-GFP fluorescence if the cells are imaged with identical exposure times (Supplemental Figure 4).

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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