Cell survival fundamentally depends on protein degradation, which in eukaryotes is carried out to a large extent by the ubiquitin-proteasome system (UPS) 1,2. Cells not only must maintain the proteome and degrade misfolded or damaged polypeptides, but degradation of regulatory and signaling proteins mediates numerous vital processes, ranging from transcription to cell division3. As the final destination in the ubiquitin-proteasome system, the essential 26S proteasome is a compartmental protease of the AAA+ family that mechanically unfolds and degrades protein substrates in an ATP-dependent manner. Most proteasomal substrates are marked for degradation and targeted to the proteasome by the enzymatic attachment of ubiquitin chains, which need to be removed by intrinsic deubiquitinases (DUBs) at the proteasome to allow efficient turnover.

The 26S proteasome recognizes post-translationally added polyubiquitin chains attached to substrates by a complex network of ubiquitination enzymes. Degradation of a substrate requires binding of a polyubiquitin chain to proteasomal ubiquitin receptors, engagement of an unstructured region followed by translocation and unfolding by an ATPase motor subcomplex, and removal of ubiquitin chains by deubiquitinases Rpn11 and Ubp6 prior to translocation into the degradation chamber. The 26S proteasome utilizes over thirty different subunits and distinct holoenzyme conformations to accomplish these tasks.

Here I investigate two distinct aspects that modulate proteasome function. The deubiquitinase Ubp6 has previously been shown to trim ubiquitin chains and affect substrate processing by the proteasome, but the involved mechanisms and its location in the 26S proteasome complex remained elusive. Here we show that Ubp6 deubiquitination strongly responds to interactions with the base ATPase and the conformational state of the proteasome. Our electron-microscopy studies revealed that ubiquitin-bound Ubp6 contacts the N-ring and AAA+ ring of the ATPase hexamer, placing it in close proximity to the deubiquitinase Rpn11. In complex with ubiquitin, Ubp6 inhibits substrate deubiquitination by Rpn11, stabilizes the substrate-engaged conformation of the proteasome, and allosterically interferes with the engagement of a subsequent substrate. Ubp6 may thus act as an ubiquitin-dependent timer to coordinate individual processing steps at the proteasome and modulate substrate degradation.

Secondly, although the mechanism of substrate proteolysis has been well characterized, little is known about how the processing steps prior to degradation culminate to give an overall degradation rate. The complexity of substrate processing and the requisite of proteasome function for cell viability pose a significant challenge to quantitative analysis. We have developed an in vitro system to prepare defined proteasomal substrates and designed assays to determine the rates of degradation and deubiquitination for a given substrate input. We used these tools to show that deubiquitination by Rpn11depends on translocation of the substrate polypeptide by the ATPase. Moreover we also observe that unlike prokaryotic ATP dependent protease ClpXP, unfolding is not rate limiting in degradation by the proteasome. Overall these studies provide insight on how the complex 26S proteasome functions to degrade such a diversity of substrates and is integral to the cell.