Project Summary Abstract The 26S proteasome conducts most regulated protein degradation and eliminates toxic proteins from cells. The proteasome is a validated anti-cancer target, and holds substantial promise as a target for treatment of neurodegenerative disorders and some infectious diseases. Our long-term goal is to understand how the three major complexes of the proteasome—the lid, base, and core particle—engage and communicate within and between one another. We have thus far made significant progress toward this goal and have developed a number of novel tools and reagents that have furthered our understanding of intra- and inter-complex communication. Conceptual advances have included: i) discovery of additional conformational states of the yeast proteasome relevant to substrate catalysis; ii) demonstration that six highly similar ATP-hydrolyzing subunits differentially influence the activation state of the proteasome; iii) discovery of a link between the conformational state of the proteasome and release of a dedicated proteasome assembly chaperone; and iv) and the finding that proteasomal subcomplexes disengage one another prior to their destruction by autophagy. A paradigm emerging from this initial budget period is that rather small binding events or molecular movements are transmitted, often over long distances, to enact largescale conformational changes. Understanding how such local events are amplified and transmitted to distant areas of the proteasome to coordinate assembly and catalysis is thus a critical knowledge gap. In this first renewal, we propose three Aims that explore examples of this paradigm newly discovered by us during the initial budget period. Together, they will push our knowledge of proteasome dynamics and inter-complex communication into new arenas. In the first, we will use newly developed FRET-based kinetic assays to decipher how local changes to the lid-base interface regulate the timely binding and release of dedicated assembly chaperones from nascent proteasomes. In the second, we will explore a surprising allosteric conduit originating from the substrate unfolding center of the proteasome that regulates the stability between two key subcomplexes. In the third Aim, we will investigate an unusual eukaryotic proteasome from a poorly studied human parasite from the phylum of Microsporidia. Microsporidia lack several proteasome subunits that normally span a key inter-complex interface. The missing subunits contain several small sequence elements with essential roles in assembly and catalysis in other eukaryotes, so exploring these unusual proteasomes will thus reveal both conserved and unique elements of inter-complex communication. These studies are anticipated to produce important insights into the engagement and communication between the proteasomal subcomplexes, significantly advancing several aspects of proteasome biology and drug discovery. Further, microsporidia are NIH priority pathogens of interest for w...