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Sch9 regulates intracellular protein ubiquitination by controlling stress responses.

Qie B, Lyu Z, Lyu L, Liu J, Gao X, Liu Y, Duan W, Zhang N, Du L, Liu K - Redox Biol (2015)

Bottom Line: In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase.Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function.Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu, Sichuan 610064, China.

No MeSH data available.


Related in: MedlinePlus

Autophagy is not responsible for the decreased level of ubiquitinated proteins in sch9Δ cells. (A) The mRNA levels of ATG8 were tested in WT cells (DBY746) transformed with empty vector (WT+vector) and SCH9 deletion mutant cells (PF102(1-1)) transformed with empty vector (sch9Δ+vector) or pRS416−SCH9 (sch9Δ+Sch9) at log phase (OD600 nm=0.5). (B) GFP-Atg8 processing is enhanced by the deletion of SCH9. WT (DBY746), atg1Δ, sch9Δ and sch9Δ/atg1Δ strains were transformed with pUG36-Ura/ATG8 and the autophagy activities were detected in log phase cells (OD600 nm=0.5). (C) WT (DBY746), atg1Δ, sch9Δ and sch9Δ/atg1Δ were harvested at OD600 nm of 0.5 and the levels of ubiquitinated protein and Sch9 were tested by western blotting with actin as the loading control. (D) The quantifications of three repeats from panel C (*P<0.05, ***P < 0.001, “ns” no significance).
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f0015: Autophagy is not responsible for the decreased level of ubiquitinated proteins in sch9Δ cells. (A) The mRNA levels of ATG8 were tested in WT cells (DBY746) transformed with empty vector (WT+vector) and SCH9 deletion mutant cells (PF102(1-1)) transformed with empty vector (sch9Δ+vector) or pRS416−SCH9 (sch9Δ+Sch9) at log phase (OD600 nm=0.5). (B) GFP-Atg8 processing is enhanced by the deletion of SCH9. WT (DBY746), atg1Δ, sch9Δ and sch9Δ/atg1Δ strains were transformed with pUG36-Ura/ATG8 and the autophagy activities were detected in log phase cells (OD600 nm=0.5). (C) WT (DBY746), atg1Δ, sch9Δ and sch9Δ/atg1Δ were harvested at OD600 nm of 0.5 and the levels of ubiquitinated protein and Sch9 were tested by western blotting with actin as the loading control. (D) The quantifications of three repeats from panel C (*P<0.05, ***P < 0.001, “ns” no significance).

Mentions: Besides proteasome, autophagy is another major intracellular proteolytic system which degrades ubiquitinated proteins [6]. Genome-wide studies have suggested that the deletion of SCH9 enhances the expression of autophagy related genes [36]. We also found that the transcription of Atg8 gene, the homolog of mammalian LC3 which is a key factor of autophagy [44], was upregulated to 3 folds upon SCH9 deletion (Fig. 3A). It has been demonstrated that PKA and Sch9 cooperatively regulate the induction of autophagy [45]. By using the Atg8-GFP protein as an autophagy activity reporter [46], we found that sch9Δ cells generated more degradation product which diminished when autophagy was inhibited by the deletion of Atg1, confirming that sch9Δ cells have higher autophagy activity than WT cells (Fig. 3B, compare lane 3 to lane 1). If the enhanced autophagy contributes to the decreased ubiquitination in sch9Δ cell, shutting down autophagy should be able to restore the ubiquitination level. However, the depletion of ATG1 did not elevate the level of ubiquitinated proteins in sch9Δ cells despite its autophagy blocking effect (Fig. 3C and D), suggesting that the enhanced autophagy in sch9Δ cells was not the direct mechanism by which the ubiquitinated proteins are decreased.


Sch9 regulates intracellular protein ubiquitination by controlling stress responses.

Qie B, Lyu Z, Lyu L, Liu J, Gao X, Liu Y, Duan W, Zhang N, Du L, Liu K - Redox Biol (2015)

Autophagy is not responsible for the decreased level of ubiquitinated proteins in sch9Δ cells. (A) The mRNA levels of ATG8 were tested in WT cells (DBY746) transformed with empty vector (WT+vector) and SCH9 deletion mutant cells (PF102(1-1)) transformed with empty vector (sch9Δ+vector) or pRS416−SCH9 (sch9Δ+Sch9) at log phase (OD600 nm=0.5). (B) GFP-Atg8 processing is enhanced by the deletion of SCH9. WT (DBY746), atg1Δ, sch9Δ and sch9Δ/atg1Δ strains were transformed with pUG36-Ura/ATG8 and the autophagy activities were detected in log phase cells (OD600 nm=0.5). (C) WT (DBY746), atg1Δ, sch9Δ and sch9Δ/atg1Δ were harvested at OD600 nm of 0.5 and the levels of ubiquitinated protein and Sch9 were tested by western blotting with actin as the loading control. (D) The quantifications of three repeats from panel C (*P<0.05, ***P < 0.001, “ns” no significance).
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Related In: Results  -  Collection

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f0015: Autophagy is not responsible for the decreased level of ubiquitinated proteins in sch9Δ cells. (A) The mRNA levels of ATG8 were tested in WT cells (DBY746) transformed with empty vector (WT+vector) and SCH9 deletion mutant cells (PF102(1-1)) transformed with empty vector (sch9Δ+vector) or pRS416−SCH9 (sch9Δ+Sch9) at log phase (OD600 nm=0.5). (B) GFP-Atg8 processing is enhanced by the deletion of SCH9. WT (DBY746), atg1Δ, sch9Δ and sch9Δ/atg1Δ strains were transformed with pUG36-Ura/ATG8 and the autophagy activities were detected in log phase cells (OD600 nm=0.5). (C) WT (DBY746), atg1Δ, sch9Δ and sch9Δ/atg1Δ were harvested at OD600 nm of 0.5 and the levels of ubiquitinated protein and Sch9 were tested by western blotting with actin as the loading control. (D) The quantifications of three repeats from panel C (*P<0.05, ***P < 0.001, “ns” no significance).
Mentions: Besides proteasome, autophagy is another major intracellular proteolytic system which degrades ubiquitinated proteins [6]. Genome-wide studies have suggested that the deletion of SCH9 enhances the expression of autophagy related genes [36]. We also found that the transcription of Atg8 gene, the homolog of mammalian LC3 which is a key factor of autophagy [44], was upregulated to 3 folds upon SCH9 deletion (Fig. 3A). It has been demonstrated that PKA and Sch9 cooperatively regulate the induction of autophagy [45]. By using the Atg8-GFP protein as an autophagy activity reporter [46], we found that sch9Δ cells generated more degradation product which diminished when autophagy was inhibited by the deletion of Atg1, confirming that sch9Δ cells have higher autophagy activity than WT cells (Fig. 3B, compare lane 3 to lane 1). If the enhanced autophagy contributes to the decreased ubiquitination in sch9Δ cell, shutting down autophagy should be able to restore the ubiquitination level. However, the depletion of ATG1 did not elevate the level of ubiquitinated proteins in sch9Δ cells despite its autophagy blocking effect (Fig. 3C and D), suggesting that the enhanced autophagy in sch9Δ cells was not the direct mechanism by which the ubiquitinated proteins are decreased.

Bottom Line: In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase.Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function.Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu, Sichuan 610064, China.

No MeSH data available.


Related in: MedlinePlus