The ubiquitin proteasome system (UPS) controls essential cellular processes such as cell cycle progression, cellular signaling, stress responses and maintenance of protein folding homeostasis via regulated proteolysis of substrates by the 26S proteasome. Proteins destined for degradation need to meet two criteria: i) carry a polyubiquitin degron signal and ii) have an unstructured initiation region that allows for the engagement by the proteasome and subsequent unfolding, translocation and degradation. Yeast Cdc48, also known as p97 or VCP in higher eukaryotes, is an essential, abundant and highly conserved AAA+ ATPase that uses its unfoldase activity to extract ubiquitinated protein substrates from complexes and membranes. It has been hypothesized that Cdc48 also plays an important role in protein degradation pathways, working upstream of the 26S proteasome. For my dissertation, I established an in vitro reconstituted system that allowed me to study the interactions between Cdc48 and its cofactors, determine the kinetics of substrate unfolding by Cdc48 and demonstrate the functional role of Cdc48 in the proteasomal degradation.
First, I established that recombinant Cdc48 can bind to the Ufd1/Npl4 substrate recognition heterodimer, as well as to the Otu1 deubiquitinase. Those interactions are mutually exclusive, meaning that Ufd1/Npl4 and Otu1 cannot both form a complex with Cdc48 in the absence of substrate. I also observed interaction between the Cdc48 unfoldase and the Ufd2 ubiquitin ligase. Additionally, I tested the activity of Ufd2 and developed conditions under which it can ubiquitinate model substrates containing linear ubiquitin fusion. That substrate can be used to study the activity of Otu1 and its modulation by Cdc48, as well as to measure unfolding by Cdc48.
Furthermore, by utilizing the fluorescent protein mEOS3.2 N-terminally fused to tetra ubiquitin as a model substrate, and extending this ubiquitin using Ufd2, I was able to measure kinetics of Cdc48 unfolding under single and multiple turnover conditions.
Finally, I demonstrated for the first time that compact substrates lacking an initiation segment can still be degraded by the 26S proteasome when first processed by Cdc48. Overall, my data suggest that Cdc48’s primary role in the UPS is to create unstructured initiation regions in compact substrates refractory to proteasomal engagement.