The 26S proteasome is responsible for regulated proteolysis in eukaryotic cells. Its substrates are diverse in structure, function, sequence length, and amino acid composition, and are targeted to the proteasome by post-translational modification with ubiquitin. Ubiquitination occurs through a complex enzymatic cascade and can also signal for other cellular events, unrelated to proteasome-catalyzed degradation. Like other post-translational protein modifications, ubiquitination is reversible, with ubiquitin chain hydrolysis catalyzed by the action of deubiquitinating enzymes (DUBs), ~90 of which exist in humans and allow for temporal events as well as dynamic ubiquitin-chain remodeling. DUBs have been known for decades to be an integral part of the proteasome, as deubiquitination is coupled to substrate unfolding and translocation into the internal degradation chamber. Moreover, the proteasome also binds several ubiquitinating enzymes as well as shuttle factors that recruit ubiquitinated substrates. The role of this intricate machinery and how ubiquitinated substrates interact with proteasomes remains an area of active investigation. Here, we review what has been learned about the mechanisms used by the proteasome to bind ubiquitinated substrates, substrate shuttle factors, ubiquitination machinery, and DUBs. We also discuss many open questions that require further study or the development of innovative approaches to be answered. Finally, we address the promise of expanded therapeutic targeting that could benefit from such new discoveries.

The 26S proteasome is an ATP‐dependent molecular machine that degrades ubiquitin‐tagged proteins in several well‐coordinated steps, including ubiquitin binding, substrate engagement, mechanical unfolding, de‐ubiquitination, translocation, and proteolytic cleavage. Recently developed single‐molecule FRET measurements, relying on the labeling of proteasome‐incorporated unnatural amino acids, allow us to follow individual substrate proteins through the central channel and dissect the kinetics and coordination of processing steps. Mutational studies thereby reveal new details about the mechanisms of substrate engagement by the AAA+ ATPase motor and the contributions of individual ATPase subunits depending on their position in the hexameric spiral‐staircase arrangement of the motor. Our FRET‐based measurements with double‐labeled proteasomes provide exciting insights into the conformational dynamics of the proteasomal regulatory particle and how ubiquitin‐chain binding to certain receptor subunits influences the switching between substrate‐free and substrate‐processing states. Through this regulation of the proteasome conformational dynamics, ubiquitin chains accelerate substrate engagement by the AAA+ motor and potentiate mechanical unfolding, leading to faster and more efficient degradation. These effects depend on the length and linkage type of ubiquitin chains, indicating how the “ubiquitin code” may be utilized by the proteasome to prioritize substrates and fine‐tune degradation in a complex cellular environment.

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The degradation of ubiquitin‐tagged proteins by the 26S proteasome is a highly complex process with several well‐coordinated sub‐steps, including ubiquitin recognition, deubiquitination, substrate engagement by the AAA+ motor, mechanical unfolding, translocation, and proteolytic cleavage. Our recently developed FRET‐ and fluorescence‐based assays, relying on the labeling of proteasome‐incorporated unnatural amino acids, allow us to dissect the kinetics and coordination of individual processing steps, and study the coupled conformational changes of the proteasome. Single‐molecule measurements provide exciting details about the conformational dynamics of the proteasomal regulatory particle and how ubiquitin binding, substrate engagement by the AAA+ motor, or the presence of certain receptor subunits affect the switching between substrate‐free and substrate‐processing states, which are furthermore controlled through specific interactions between the ATPase hexamer and the proteasomal lid subcomplex. FRET‐based assays reveal how substrates are propelled through the central channel of the proteasome and progress through the various stages of degradation, including a translocation stall during deubiquitination and the backtracking of substrate upon failure to remove ubiquitin chains prior to substrate entry into the AAA+ motor. Our mutational studies thereby provide important new insight into the mechanisms of substrate engagement and the contributions of individual ATPase subunits depending on their position in the vertical spiral‐staircase arrangement of the AAA+ hexamer. By using substrates with various architectures, we also gained a better understanding of how a substrate’s geometry and ubiquitin modifications affect the degradation rate, and how the proteasome may prioritize its substrates in the cell. Support or Funding Information Howard Hughes Medical Institute; NIH‐NIGMS (R01‐GM094497)