Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 118, Issue 25, Pages -Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2101004118
Keywords
proteasome; protein unfolding; protein degradation; ATPases associated with diverse cellular activities (AAA); ATP-dependent protease
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Funding
- Beckman Scholars Program
- NSF [1935596]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1935596] Funding Source: National Science Foundation
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The 26S proteasome, responsible for protein degradation in eukaryotic cells, transitions between substrate-accepting and substrate-processing conformations, with important intramolecular interactions stabilizing these conformations. A new conformationally sensitive assay revealed that interactions involving Rpn5 and Rpn2 are crucial for stabilizing substrate-processing conformations, impacting the proteasome's ability to successfully unfold and degrade difficult substrates.
The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrateprocessing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome's various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Forster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome's ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome's conformational state and its unfolding ability.
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