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Substrate Polyubiquitination Increases Proteasomal Processivity

The 26S proteasome in eukaryotes is the macromolecular machine responsible for the degradation of misfolded and damaged polypeptides, as well as signaling and regulatory proteins that accumulate intracellularly. Most proteins are marked for degradation and targeted to the proteasome by the covalent attachment of polyubiquitin chains, which are recognized by receptors on the 19S regulatory particle of the proteasome. After initiating degradation, the polyubiquitin tag is removed and the proteasome moves processively along the protein substrate, unfolding domains as they are encountered and translocating the unfolded polypeptide to the protease active sites in the 20S core for degradation into small peptides. However, the proteasome can stall during this process and rather than successfully unfolding the encountered domain, the proteasome will release the substrate as a partially degraded protein, which can assume a new biological function or form a potentially toxic byproduct. While our understanding of proteasomal processivity remains limited, it has been shown that ubiquitinated substrates can activate the proteasome's ATPase and peptidase activities. We set out to determine if substrate ubiquitination affected the proteasome's ability to unfold its substrates, since the ubiquitin chain is removed early on in the degradation process. To accomplish this, a previously established processivity assay was used, in which the unfolding and subsequent degradation versus the release of a stabilized dihydrofolate reductase (DHFR) domain are quantified. The ratio of degradation to release rates is defined as the unfolding ability U, a measure of the proteasome's processivity. We compared the unfolding abilities of ubiquitin‐independent substrates, as well as substrates ubiquitinated with the E3 ligase Rsp5 (reported to give K63‐linked chains) or the E3 ligase complex Cul3/Rbx1/Keap1 (reported to give primarily K48‐linked chains). We found that significantly less folded DHFR fragment was produced during degradation of the Cul3/Rbx1/Keap1‐ubiquitinated substrate, resulting in a higher unfolding ability, compared to the Rsp5‐ubiquitinated substrate. The unfolding ability for ubiquitin‐independent substrates was much lower than that of either Rsp5‐ or Keap1 ubiquitinated substrates. These results suggest that the presence and chain architecture of polyubiquitin on a substrate can shift the proteasome towards a more active state during engagement, which results in a greater unfolding ability.