Project Summary Abstract The 26S proteasome conducts most regulated protein degradation and eliminates toxic proteins in vivo. The proteasome is an unusually large and complex ATP-dependent protease comprising nearly 70 individual polypeptide subunits. Although the conventional thinking has been that the proteasome is assembled from these subunits in a single, rigid stepwise sequence, recent evidence from our group and others unexpectedly suggests a broader “landscape” of assembly routes may exist in vivo. Although this possibility has not yet been tested, such an assembly landscape would ensure that this essential biological process can continue effectively in the face of assembly roadblocks, and would provide a powerful means to adjust the speed or volume of proteasome biogenesis in response to the cellular environment. There is an increasing interest in harnessing proteasome biogenesis to help treat conditions as diverse as cancer and neurodegenerative disorders. Understanding whether such an assembly landscape exists, and if so, how it is harnessed to ensure rapid and faithful proteasome biogenesis, will be critical to guide development of such assembly-targeted therapies. The goal of this multi-PI application is to test the hypothesis that a proteasome assembly landscape exists in vivo, and that the relative flux through possible routes within this landscape is governed largely by kinetic factors that change in response to the intracellular environment. By combining the PIs’ respective expertise in proteasome biology and in enzyme kinetics and single molecular biophysics, we hope to validate this new paradigm for proteasome biogenesis. The proposed studies, described below, will add a critical new dimension— time—to our understanding of proteasome assembly in vivo. Our experimental approach contains two complementary but independent Aims. In Aim 1, we will utilize a newly established collection of cutting-edge single-molecule and ensemble fluorescence assays to characterize the kinetics of specific proteasome assembly steps. Experiments under this aim are designed to test the hypothesis that the relative flux through two possible assembly routes is primarily under kinetic control, but can be tuned by exogenous factors such as ligands or proteasome-interacting accessory proteins. Aim 2 will employ a suite of newly developed chemical-genetic approaches to assess the relative flux through two possible assembly routes in vivo, and to understand how the flux changes in response to environmental stimuli. Experiments under this Aim will also test in living cells the predictions derived from our in vitro kinetic model of assembly established in Aim 1. The outcomes of these studies will lead to a deeper understanding of proteasome biology and of macromolecular assembly in general, and also promise to illuminate new therapeutic avenues for cancer, neurodegeneration, and other diseases.