UC Berkeley

The 26S proteasome is essential for proteostasis and the regulation of vital processes through ATP-dependent degradation of ubiquitinated substrates. Proteasome substrate selection is paramount to maintaining the proteome. My graduate work has focused on the interplay between conformational properties of the proteasome and its substrates to coordinate commitment to the degradative process. To accomplish the multi-step degradation process, the proteasome’s regulatory particle, consisting of lid and base subcomplexes, undergoes major conformational changes whose molecular determinants and regulation is unknown. Investigating the S. cerevisiae proteasome through in vitro biochemical and structural studies, I found that peripheral interactions between the lid subunit Rpn5 and the base AAA+-ATPase ring are important for stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states upon substrate engagement. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later processing steps remain unaffected. Similar defects in early degradation steps are observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control proteasome conformational transitions. We found that perturbed proteasome conformational distributions can make substrate engagement the rate limiting step in a substrate dependent manner. These results provide important insight into interaction networks that coordinate conformational changes with various stages of degradation, and how modulators of conformational equilibria may influence substrate turnover.

In addition to ubiquitin modification for substrate binding to the 26S proteasome, proteasome substrates require an initiation region of greater than 10 amino acids for engagement by the 19S AAA+ motor. While many substrates in the cell contain disordered regions, many substrates lack this requirement. The Marqusee lab found that ubiquitination of lysine residues within structured domains can confer a dramatic destabilization of the substrate proteins that is both substrate specific and dependent on the site of ubiquitination. In collaboration, we provided the first in vitro experimental evidence that ubiquitin mediated destabilization of substrates lacking disordered regions was sufficient to induce an unstructured region for proteasomal engagement. Degradation rates of these substrates were also sensitive to our previously characterized mutations to Rpn5 and Rpt6 in the proteasome, thus providing evidence that engagement, rather than mechanical unfolding, is the rate limiting step for degradation of these substrates that rely on spontaneous partial unfolding to reveal unstructured initiation regions. All ubiquitin-mediated effects on substrate stability could further be modulated by deubiquitination, binding to ligand, or stabilizing mutations, leading us to conclude that substrate energetics and their modulation by ubiquitination is paramount to both substrate selection, and the rate of degradation by the proteasome.

Substrate thermodynamics play an important role in influencing degradation rate and efficiency, yet there is a dearth of evidence regarding which thermodynamic, topological, or kinetic factors contribute most to this defining role. I thus present an experimental approach based on single-molecule FRET and the determination of proteasome degradation rates that are comparable across various substrates to assess the detailed biophysical principles underlying proteasomal substrate processing. In principle, this approach is generalizable to multitudes of protein domains. Additionally, evidence of potential intermediary substrate protein structures is presented. Taken together, the studies described herein provide evidence for multiple different means of regulating the ubiquitin-proteasome system.