In the ubiquitin (Ub)/proteasome proteolytic pathway, substrate-linked polyUb is the specific targeting signal that leads proteins to be degraded by the multisubunit ATP-dependent protease complex called the 26S proteasome. The mechanisms by which the proteasome recognizes, unfolds, and translocates the substrate into its internal proteolytic chamber are poorly understood. In the proposal, in vitro biochemistry augmented by yeast genetics will be used to address how key events such as substrate unfolding and translocation are orchestrated by the six different ATPase subunits of the 19S regulatory complex of 26S proteasomes. For this purpose, well-defined polyUb-conjugates of wild-type and destabilized variants of two model proteins, dihydrofolate reductase and titin I27 domain, have been developed. By using these substrates to measure degradation and ATPase activities with purified wild-type and ATPase-mutated 26S proteasomes, we will be able to resolve the contributions that ATP hydrolysis and, potentially, individual ATPase subunits make toward the substrate binding, unfolding, and translocation steps of the degradation reaction. Our results will provide a basic understanding of how the 26S proteasome complex uses ATP to promote proteolysis. Overall, the results of the proposed research will significantly enhance our understanding of how the 26S proteasome degrades proteins and how the complex is regulated. The significance of proteasome research to human health has been highlighted by the recent FDA approval of the use of proteasome inhibitor as a treatment for multiple myeloma.