Organisms encode multiple Hsp70s to increase capacity to regulate abundance of Hsp70 in accordance with need and to provide a range of distinct Hsp70 functions for carrying out many different tasks within cells. We constructed a yeast system to evaluate Hsp70s from any source and have found that all of the functionally redundant essential yeast Hsp70s possess distinct activities. We are continuing to use this system to investigate how Hsp70s within and across species influence propagation of amyloid in vivo and act in cellular protein quality control (PQC) processes. The wide range of responses that prions have to alterations of Hsp70s and their co-chaperones provides a sensitive way to investigate even subtle functional distinctions among highly redundant Hsp70s and an approach to uncover the underlying mechanisms. Hsp70s act by binding and releasing client substrate proteins and co-chaperones interact with Hsp70s to regulate their activity. The large number of co-chaperones that act on different steps of the Hsp70 reaction cycle can cooperate to provide both a broad range of function and fine-tuning of Hsp70 activity to specify its proper functions in defined roles in cells. After Hsp70 binds client proteins, nucleotide exchange factors (NEFs) facilitate release of the substrates and restarting of the binding cycle. One aspect of our work focuses on determining how functions of Hsp70s are mediated by ways they cooperate with NEFs. In pursuing whether different functions of nearly identical Hsp70s Ssa1 and Ssa2 could be due to differential interactions with NEFs, we uncovered unexpected Hsp70-independent roles of an Hsp70 NEF in degradation of gluconeogenic enzymes and in cell wall integrity. These findings raise new awareness about roles of such conserved NEFs in yeast and possibly other eukaryotic cells. Altering abundance or function of Hsp70 can moderate pathology in models of protein folding disorders, while in the same models reducing chaperone activity can exacerbate, or alone even cause pathology. Hsp70 is therefore a promising therapeutic candidate for amyloid and other protein folding disorders and it is being evaluated intensively as a drug target. Altering Hsp70 co-chaperones also moderates pathology in several models of amyloid and other protein folding disorders. Our work toward understanding what underlies specificity in activities of functionally redundant Hsp70s can help guide decisions about which Hsp70-family members would be most useful for such applications, or identify potential problems that could arise due to ways different Hsp70s respond differently to specific compounds. Overall our work provides insight into functions of these chaperones that can help guide strategies for using chaperones as targets for therapy in protein folding disorders.