UC Berkeley

The 26S proteasome is the major ATP-dependent protease in eukaryotic cells. The 19S proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes poly-ubiquitinated substrates. In this thesis I report the development of heterologous expression systems for the base and lid subcomplexes, and I use these new systems, along with biochemistry and electron microscopy, to map the architecture of the massive 19S regulatory particle, reveal the ordered self-assembly of the lid, and demonstrate structural and functional asymmetries in the AAA+ base unfoldase. I describe the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes, and the unfolding machinery at subnanometer resolution, thereby outlining a substrate's path to degradation, providing the structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes. Furthermore, I show that a helical bundle serves as a hub through which the last-added subunit, Rpn12, monitors proper lid assembly prior to incorporation into the proteasome. Finally, data presented here demonstrate that the six ATPases play distinct roles in degradation, highlighting how the 26S proteasome deviates from simpler, homomeric AAA+ proteases.