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Updated: Jul 6, 2026
Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae
Published on: April 18, 2016
Mitochondrial protein quality control by the proteasome involves ubiquitination and the protease Omi
Susanne Radke1, Harish Chander, Patrick Schäfer
1Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA.
Abstract:
We report here that blocking the activity of the 26 S proteasome results in drastic changes in the morphology of the mitochondria and accumulation of intermembrane space (IMS) proteins. Using endonuclease G (endoG) as a model IMS protein, we found that accumulation of wild-type but to a greater extent mutant endoG leads to changes in the morphology of the mitochondria similar to those observed following proteasomal inhibition. Further, we show that wild-type but to a greater extent mutant endoG is a substrate for ubiquitination, suggesting the presence of a protein quality control. Conversely, we also report that wild-type but not mutant endoG is a substrate for the mitochondrial protease Omi but only upon inhibition of the proteasome. These findings suggest that although elimination of mutant IMS proteins is strictly dependent on ubiquitination, elimination of excess or spontaneously misfolded wild-type IMS proteins is monitored by ubiquitination and as a second checkpoint by Omi cleavage when the proteasome function is deficient. One implication of our finding is that in the context of attenuated proteasomal function, accumulation of IMS proteins would contribute to the collapse of the mitochondrial network such as that observed in neurodegenerative diseases. Another implication is that such collapse could be accelerated either by mutations in IMS proteins or by mutations in Omi itself.
Insights
Inhibiting the 26S proteasome causes mitochondrial changes and protein buildup. Mutant proteins are ubiquitinated, while wild-type proteins are cleared by Omi when the proteasome is blocked, impacting mitochondrial health.
Area of Science:
- Cell Biology
- Mitochondrial Biology
- Neuroscience
Background:
- The 26S proteasome is crucial for protein degradation.
- Mitochondrial morphology and function are sensitive to proteasomal activity.
- Intermembrane space (IMS) proteins play roles in mitochondrial homeostasis.
Purpose of the Study:
- To investigate the impact of proteasome inhibition on mitochondrial morphology and IMS protein accumulation.
- To elucidate the mechanisms of IMS protein degradation, particularly endonuclease G (endoG).
- To explore the interplay between the proteasome, ubiquitination, and the mitochondrial protease Omi in protein quality control.
Main Methods:
- Proteasome inhibition using specific inhibitors.
- Morphological analysis of mitochondria.
- Western blotting to detect protein ubiquitination and degradation.
- Studying the role of endonuclease G (endoG) as a model IMS protein.
- Investigating the activity of the mitochondrial protease Omi.
Main Results:
- Proteasome inhibition leads to significant mitochondrial morphological changes and accumulation of IMS proteins.
- Both wild-type and mutant endoG accumulate upon proteasome inhibition, with mutant endoG accumulating more extensively.
- Wild-type and mutant endoG are substrates for ubiquitination, indicating a protein quality control pathway.
- Wild-type endoG, but not mutant endoG, is degraded by Omi when the proteasome is inhibited.
- These findings suggest a dual quality control mechanism for IMS proteins involving ubiquitination and Omi cleavage.
Conclusions:
- Elimination of mutant IMS proteins relies on ubiquitination.
- Excess or misfolded wild-type IMS proteins are managed by ubiquitination and Omi cleavage when proteasomal function is impaired.
- Accumulation of IMS proteins due to impaired proteasomal function contributes to mitochondrial network collapse, relevant to neurodegenerative diseases.
- Mutations in IMS proteins or Omi can accelerate mitochondrial collapse in conditions of compromised proteasome function.
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