As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.

Graphical AbstractHighlights d The rate-limiting step of protein degradation by the proteasome is unfolding d Substrate contact with the motor triggers a conformational switch of the proteasome d Substrates with poor initiation regions are quickly rejected d Supernumerary ubiquitin chains promote degradation of otherwise poor substrates A complete kinetic picture of proteasomal degradation reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. SUMMARYThe 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states.Here, we used reconstituted 26S proteasomes with unnatural amino-acid-attached fluorophores in a series of FRET-and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.(A) Schematic for the substrate processing pathway. Ubiquitin chains (pink) target a substrate (gold) to the 26S proteasome in the s1 state. The substrate's unstructured tail is inserted into the pore of the AAA + motor (blue), where it interacts with pore loops (red) that drive translocation. After substrate engagement, the regulatory particle changes its conformation to an s3-like state and Rpn11 (green) shifts to a central position that allows translocation-coupled deubiquitination. Ubiquitin-chain removal is followed by mechanical substrate unfolding and threading of the polypeptide into the 20S core for proteolytic cleavage.

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