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Assaying Proteasomal Degradation in a Cell-free System in Plants
Published on: March 26, 2014
Identification of ubiquitin-proteasome system components affecting the degradation of the transcription factor Pap1
Luis Marte1, Susanna Boronat1, Sarela García-Santamarina1
1Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, C/ Doctor Aiguader 88, 08003, Barcelona, Spain.
Abstract:
Signaling cascades respond to specific inputs, but also require active interventions to be maintained in their basal/inactive levels in the absence of the activating signal(s). In a screen to search for protein quality control components required for wild-type tolerance to oxidative stress in fission yeast, we have isolated eight gene deletions conferring resistance not only to H2O2 but also to caffeine. We show that dual resistance acquisition is totally or partially dependent on the transcription factor Pap1. Some gene products, such as the ribosomal-ubiquitin fusion protein Ubi1, the E2 conjugating enzyme Ubc2 or the E3 ligase Ubr1, participate in basal ubiquitin labeling of Pap1, and others, such as Rpt4, are non-essential constituents of the proteasome. We demonstrate here that basal nucleo-cytoplasmic shuttling of Pap1, occurring even in the absence of stress, is sufficient for the interaction of the transcription factor with nuclear Ubr1, and we identify a 30 amino acids peptide in Pap1 as the degron for this important E3 ligase. The isolated gene deletions increase only moderately the concentration of the transcription factor, but it is sufficient to enhance basal tolerance to stress, probably by disturbing the inactive stage of this signaling cascade.
Insights
Researchers identified gene deletions in fission yeast that enhance stress tolerance by affecting the Pap1 transcription factor. These deletions disrupt Pap1
Area of Science:
- Cellular biology
- Molecular genetics
- Biochemistry
Background:
- Signaling pathways require regulation to maintain basal activity levels.
- Oxidative stress and caffeine tolerance in fission yeast involve complex regulatory mechanisms.
- Protein quality control is essential for cellular homeostasis.
Purpose of the Study:
- To identify protein quality control components affecting oxidative stress and caffeine tolerance in fission yeast.
- To elucidate the role of the transcription factor Pap1 in dual stress resistance.
- To understand the regulation of Pap1 stability and activity.
Main Methods:
- Fission yeast screening for gene deletions conferring resistance to H2O2 and caffeine.
- Analysis of Pap1 (Activating Protein 1) protein levels and localization.
- Ubiquitination and proteasomal degradation assays.
- Identification of Pap1 degron sequence.
Main Results:
- Eight gene deletions were identified conferring dual resistance to H2O2 and caffeine.
- Dual resistance is dependent on the transcription factor Pap1.
- Pap1 interacts with Ubr1 (E3 ubiquitin ligase) via a 30 amino acid degron.
- Disruption of Pap1 basal degradation enhances stress tolerance.
Conclusions:
- Protein quality control mechanisms, particularly ubiquitin-proteasome system components, regulate Pap1 activity.
- Basal nucleo-cytoplasmic shuttling of Pap1 is crucial for its ubiquitination and degradation.
- Targeting Pap1 degradation offers a strategy to enhance basal stress tolerance in yeast.
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