ABSTRACT Heat shock protein (Hsp) 104, from budding yeast, belongs to the AAA+ (ATPase associated with various cellular activities) family of Hsp100 protein disaggregases, and plays vital roles in protein homeostasis and cell stress response. Hsp104 collaborates with the Hsp70 molecular chaperone system to target and solubilize amorphous and amyloid aggregates. These aggregates are associated with toxic phenotypes and, in humans, are associated various neurodegenerative diseases. Our lab and others discovered how Hsp104 utilizes an ATP hydrolysis-driven ‘hand over hand’ substrate translocation mechanism to solubilize aggregates. However, the contribution and interaction mechanisms of the Hsp70 molecular chaperone system in initiating and enhancing Hsp104’s activity remains poorly understood. Using cryo-EM, we determined the first substrate-bound structures of Hsp104, which reveal how large conformational changes of the two-nucleotide binding AAA+ domains can facilitate processive translocation while simultaneously maintaining substrates in an unfolded state along the central channel. While previous work has indicated that Hsp70 binds the Hsp104 middle domain to facilitate substrate loading, a recent cryo-EM study proposes that Hsp70 promotes substrate re-folding downstream of Hsp104 activity through these same interactions. Here, my goal is to dissect the exact mechanism of the substrate-loading model by defining the structure and mechanism of the Hsp70 molecular chaperone system’s contribution to assisting Hsp104 in substrate targeting and disaggregation. To achieve this, I will employ a biochemical proteolysis assay to allow substrate disaggregation, but prevent potential refolding by the Hsp70 molecular chaperone system, thereby assessing specifically the substrate loading model. Additionally, I will utilize single-particle cryo-EM to determine the precise interaction between Hsp70 and Hsp104 and how this affects Hsp104’s substrate-bound conformations. Capturing a high-resolution structure of Hsp104 interacting with Hsp70 has proven challenging due to Hsp104 flexible domains and transient interaction with Hsp70. However, through our collaboration with Dr. James Shorter at the University of Pennsylvania, we have access to various Hsp104 mutants that can stabilize interaction with Hsp70. I plan to utilize these mutants to form a stable interaction between Hsp104 and Hsp70, thereby allowing me to capture novel substrate-bound Hsp104 conformations and resolve transient interactions. These planned studies will significantly advance our knowledge of how AAA+ disaggregases interact with other chaperone proteins, thereby permitting targeting and disaggregation of toxic amyloid aggregates, shedding light on essential processes underlying protein homeostasis and neurodegenerative diseases.