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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.


Sch9 does not regulate the level of ubiquitinated proteins by affecting ubiquitin expression or proteasomal activity. (A) Lysates of log phase yeasts (OD600 nm=0.5) were resolved on 15% SDS gels and proteins of interest were detected by Western blotting. (B) Chymotrypsin-like activity of the proteasome was monitored by Suc-Leu-Leu-Val-Tyr-AMC digestion using lysates with equal amounts of total protein from log phase yeasts at OD600nm of 0.5. The proteasome inhibitor MG132 (75 µM) was added to the isopyknic lysates as the blank control. (C) The log phase yeasts (OD600 nm=0.5) were treated with 75 µM MG132 for 1 h 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. (*represents P<0.05 and “ns” denotes no significance p>0.05 between the indicated comparisons.).
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f0010: Sch9 does not regulate the level of ubiquitinated proteins by affecting ubiquitin expression or proteasomal activity. (A) Lysates of log phase yeasts (OD600 nm=0.5) were resolved on 15% SDS gels and proteins of interest were detected by Western blotting. (B) Chymotrypsin-like activity of the proteasome was monitored by Suc-Leu-Leu-Val-Tyr-AMC digestion using lysates with equal amounts of total protein from log phase yeasts at OD600nm of 0.5. The proteasome inhibitor MG132 (75 µM) was added to the isopyknic lysates as the blank control. (C) The log phase yeasts (OD600 nm=0.5) were treated with 75 µM MG132 for 1 h 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. (*represents P<0.05 and “ns” denotes no significance p>0.05 between the indicated comparisons.).

Mentions: Although overall protein ubiquitination is decreased in sch9Δ cells (Fig. 1), the level of free ubiquitin in these cells was indistinguishable to that in cells expressing Sch9 (Fig. 2A), suggesting that the decrease of ubiquitinated proteins does not result from a shortage of free ubiquitin.The proteasome pathway is one of the major pathways which regulate the dynamics of intracellular polyubiquitinated proteins [1,43]. The decline of ubiquitinated proteins in sch9Δ cells may be caused by the enhanced proteasomal degradation. However, the proteasome activities in WT, sch9Δ, and sch9Δ(Sch9) cells are indistinguishable (Fig. 2B), indicating that the decreased protein ubiquitination in sch9Δ cells is not due to enhanced proteasome activity. In agreement with this result, we found that inhibiting proteasome activity by MG132 on sch9Δ cells could not restore the ubiquitinated proteins to the same level as that in WT and sch9Δ(Sch9) cells (Fig. 2C and D).


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)

Sch9 does not regulate the level of ubiquitinated proteins by affecting ubiquitin expression or proteasomal activity. (A) Lysates of log phase yeasts (OD600 nm=0.5) were resolved on 15% SDS gels and proteins of interest were detected by Western blotting. (B) Chymotrypsin-like activity of the proteasome was monitored by Suc-Leu-Leu-Val-Tyr-AMC digestion using lysates with equal amounts of total protein from log phase yeasts at OD600nm of 0.5. The proteasome inhibitor MG132 (75 µM) was added to the isopyknic lysates as the blank control. (C) The log phase yeasts (OD600 nm=0.5) were treated with 75 µM MG132 for 1 h 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. (*represents P<0.05 and “ns” denotes no significance p>0.05 between the indicated comparisons.).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
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f0010: Sch9 does not regulate the level of ubiquitinated proteins by affecting ubiquitin expression or proteasomal activity. (A) Lysates of log phase yeasts (OD600 nm=0.5) were resolved on 15% SDS gels and proteins of interest were detected by Western blotting. (B) Chymotrypsin-like activity of the proteasome was monitored by Suc-Leu-Leu-Val-Tyr-AMC digestion using lysates with equal amounts of total protein from log phase yeasts at OD600nm of 0.5. The proteasome inhibitor MG132 (75 µM) was added to the isopyknic lysates as the blank control. (C) The log phase yeasts (OD600 nm=0.5) were treated with 75 µM MG132 for 1 h 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. (*represents P<0.05 and “ns” denotes no significance p>0.05 between the indicated comparisons.).
Mentions: Although overall protein ubiquitination is decreased in sch9Δ cells (Fig. 1), the level of free ubiquitin in these cells was indistinguishable to that in cells expressing Sch9 (Fig. 2A), suggesting that the decrease of ubiquitinated proteins does not result from a shortage of free ubiquitin.The proteasome pathway is one of the major pathways which regulate the dynamics of intracellular polyubiquitinated proteins [1,43]. The decline of ubiquitinated proteins in sch9Δ cells may be caused by the enhanced proteasomal degradation. However, the proteasome activities in WT, sch9Δ, and sch9Δ(Sch9) cells are indistinguishable (Fig. 2B), indicating that the decreased protein ubiquitination in sch9Δ cells is not due to enhanced proteasome activity. In agreement with this result, we found that inhibiting proteasome activity by MG132 on sch9Δ cells could not restore the ubiquitinated proteins to the same level as that in WT and sch9Δ(Sch9) cells (Fig. 2C and D).

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.