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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 or disruption of its ability to bind to the proteasome results in cycloheximide (CHX) resistance. (A) Overnight cultures of WT (BY4741), blm10Δ (yMS63), and ubp6Δ (yMS222) cells (top) or WT (SUB62), rpt1S (DY106), rpt2RF (DY62), and rpt3R (DY93) (bottom) were serially diluted and spotted onto YPD in the absence or presence of 0.3 μg/ml (top) or 0.5 μg/ml (bottom) CHX or 60 μg/ml hygromycin B and incubated at 30ºC for 2 d (YPD) or 4 d (CHX, hygromycin B). (B) C-terminal Blm10 mutants exhibit a loss-of-function phenotype. Fivefold serial dilutions of overnight cultures of wild-type (WT) yeast strains, strains deleted for BLM10 (Δblm10), strains with genomically integrated C-terminal deletion mutants (BLM10ΔC1, BLM10ΔC2, and BLM10ΔC3), and a chimeric Blm10 protein where the last seven residues were exchanged against the corresponding residues of PA26 (BLM10PA26C) were spotted on YPD in the absence (left) or in the presence of 0.3 μg CHX (right). The C-terminal sequences are indicated to the right. (C) Purified WT Blm10–CP and complexes with C-terminal Blm10 mutants as indicated in (B) were purified and subjected to native gel electrophoresis, followed by an in-gel activity assay with the fluorogenic proteasome substrate Suc-LLVY-AMC (top). Subsequently the gel was stained with silver nitrate (bottom). The positions of Blm10–CP and CP are indicated.
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Figure 1: Loss of BLM10 or disruption of its ability to bind to the proteasome results in cycloheximide (CHX) resistance. (A) Overnight cultures of WT (BY4741), blm10Δ (yMS63), and ubp6Δ (yMS222) cells (top) or WT (SUB62), rpt1S (DY106), rpt2RF (DY62), and rpt3R (DY93) (bottom) were serially diluted and spotted onto YPD in the absence or presence of 0.3 μg/ml (top) or 0.5 μg/ml (bottom) CHX or 60 μg/ml hygromycin B and incubated at 30ºC for 2 d (YPD) or 4 d (CHX, hygromycin B). (B) C-terminal Blm10 mutants exhibit a loss-of-function phenotype. Fivefold serial dilutions of overnight cultures of wild-type (WT) yeast strains, strains deleted for BLM10 (Δblm10), strains with genomically integrated C-terminal deletion mutants (BLM10ΔC1, BLM10ΔC2, and BLM10ΔC3), and a chimeric Blm10 protein where the last seven residues were exchanged against the corresponding residues of PA26 (BLM10PA26C) were spotted on YPD in the absence (left) or in the presence of 0.3 μg CHX (right). The C-terminal sequences are indicated to the right. (C) Purified WT Blm10–CP and complexes with C-terminal Blm10 mutants as indicated in (B) were purified and subjected to native gel electrophoresis, followed by an in-gel activity assay with the fluorogenic proteasome substrate Suc-LLVY-AMC (top). Subsequently the gel was stained with silver nitrate (bottom). The positions of Blm10–CP and CP are indicated.

Mentions: To gain insight into the cellular functions of Blm10, we performed a screen for loss-of-function phenotypes of cells deleted for BLM10. We found that blm10Δ cells exhibit resistance to sublethal doses of the translational inhibitor cycloheximide (CHX) (Figure 1A). The same phenotype has been associated with proteasome CP and regulatory particle mutants (crl mutants) defective in the turnover of ubiquitin conjugates (Gerlinger et al., 1997), derived from a screen for CHX-resistant yeast mutants (McCusker and Haber, 1988). Similar observations have been made in Arabidopsis thaliana (Kurepa et al., 2010). To corroborate that CHX resistance is a general phenotype of proteasome loss-of-function mutants, we tested the growth of the proteasomal ATPase mutants rpt1S, rpt2RF, and rpt3R (Rubin et al., 1998) under the same conditions. The respective point mutations prevent nucleotide binding to the ATPase subunits due to a point mutation within the Walker A motif, yet still allow cell growth. The ATPase mutants showed similar CHX resistance as loss of BLM10 (Figure 1A, bottom).


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 or disruption of its ability to bind to the proteasome results in cycloheximide (CHX) resistance. (A) Overnight cultures of WT (BY4741), blm10Δ (yMS63), and ubp6Δ (yMS222) cells (top) or WT (SUB62), rpt1S (DY106), rpt2RF (DY62), and rpt3R (DY93) (bottom) were serially diluted and spotted onto YPD in the absence or presence of 0.3 μg/ml (top) or 0.5 μg/ml (bottom) CHX or 60 μg/ml hygromycin B and incubated at 30ºC for 2 d (YPD) or 4 d (CHX, hygromycin B). (B) C-terminal Blm10 mutants exhibit a loss-of-function phenotype. Fivefold serial dilutions of overnight cultures of wild-type (WT) yeast strains, strains deleted for BLM10 (Δblm10), strains with genomically integrated C-terminal deletion mutants (BLM10ΔC1, BLM10ΔC2, and BLM10ΔC3), and a chimeric Blm10 protein where the last seven residues were exchanged against the corresponding residues of PA26 (BLM10PA26C) were spotted on YPD in the absence (left) or in the presence of 0.3 μg CHX (right). The C-terminal sequences are indicated to the right. (C) Purified WT Blm10–CP and complexes with C-terminal Blm10 mutants as indicated in (B) were purified and subjected to native gel electrophoresis, followed by an in-gel activity assay with the fluorogenic proteasome substrate Suc-LLVY-AMC (top). Subsequently the gel was stained with silver nitrate (bottom). The positions of Blm10–CP and CP are indicated.
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Figure 1: Loss of BLM10 or disruption of its ability to bind to the proteasome results in cycloheximide (CHX) resistance. (A) Overnight cultures of WT (BY4741), blm10Δ (yMS63), and ubp6Δ (yMS222) cells (top) or WT (SUB62), rpt1S (DY106), rpt2RF (DY62), and rpt3R (DY93) (bottom) were serially diluted and spotted onto YPD in the absence or presence of 0.3 μg/ml (top) or 0.5 μg/ml (bottom) CHX or 60 μg/ml hygromycin B and incubated at 30ºC for 2 d (YPD) or 4 d (CHX, hygromycin B). (B) C-terminal Blm10 mutants exhibit a loss-of-function phenotype. Fivefold serial dilutions of overnight cultures of wild-type (WT) yeast strains, strains deleted for BLM10 (Δblm10), strains with genomically integrated C-terminal deletion mutants (BLM10ΔC1, BLM10ΔC2, and BLM10ΔC3), and a chimeric Blm10 protein where the last seven residues were exchanged against the corresponding residues of PA26 (BLM10PA26C) were spotted on YPD in the absence (left) or in the presence of 0.3 μg CHX (right). The C-terminal sequences are indicated to the right. (C) Purified WT Blm10–CP and complexes with C-terminal Blm10 mutants as indicated in (B) were purified and subjected to native gel electrophoresis, followed by an in-gel activity assay with the fluorogenic proteasome substrate Suc-LLVY-AMC (top). Subsequently the gel was stained with silver nitrate (bottom). The positions of Blm10–CP and CP are indicated.
Mentions: To gain insight into the cellular functions of Blm10, we performed a screen for loss-of-function phenotypes of cells deleted for BLM10. We found that blm10Δ cells exhibit resistance to sublethal doses of the translational inhibitor cycloheximide (CHX) (Figure 1A). The same phenotype has been associated with proteasome CP and regulatory particle mutants (crl mutants) defective in the turnover of ubiquitin conjugates (Gerlinger et al., 1997), derived from a screen for CHX-resistant yeast mutants (McCusker and Haber, 1988). Similar observations have been made in Arabidopsis thaliana (Kurepa et al., 2010). To corroborate that CHX resistance is a general phenotype of proteasome loss-of-function mutants, we tested the growth of the proteasomal ATPase mutants rpt1S, rpt2RF, and rpt3R (Rubin et al., 1998) under the same conditions. The respective point mutations prevent nucleotide binding to the ATPase subunits due to a point mutation within the Walker A motif, yet still allow cell growth. The ATPase mutants showed similar CHX resistance as loss of BLM10 (Figure 1A, bottom).

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