Global conformational changes and their allosteric control by ubiquitin regulate protein degradation of the 26S proteasome molecular machine

The 26S proteasome is the main ATP-dependent protease in eukaryotic cells, responsible for protein homeostasis and quality control. It degrades ubiquitin-tagged proteins in several well-coordinated steps that include ubiquitin binding, substrate engagement, de-ubiquitination, mechanical unfolding, translocation into an internal chamber, and proteolytic cleavage. Our recently developed single-molecule FRET measurements, relying on the fluorescent labeling of incorporated unnatural amino acids, provide unprecedented insights into the conformational changes of the proteasome and the progression of individual substrates through the ATPase motor. We found that ubiquitin binding to an allosteric trigger affects the proteasome switching between engagement-competent and processing-competent states, and accelerates motor engagement of the substrate for faster, more efficient degradation. Furthermore, mutational studies revealed important details about the mechanisms of degradation initiation and the role of individual ATPase subunits in substrate sensing and inducing the conformational switch for multi-step processing. These studies indicate how the proteasome may utilize the "ubiquitin code" to prioritize substrates in a complex and crowded cellular environment.