Most of the fundamental processes of cells are mediated by proteins. However, the biologically-relevant mechanism of most proteins are poorly understood. Dominant negative mutations have provided a valuable tool for investigating mechanism, but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach to identify dominant negative mutations in yeast Hsp90. Hsp90 is a chaperone that forms dynamic complexes with many co-chaperones and client proteins. In vitro analyses have elucidated some key biochemical states and structures of Hsp90, co-chaperones, and clients; however, the biological mechanism of Hsp90 remains unclear. For example, high throughput studies have found that many E3 ubiquitin ligases bind to Hsp90, but it is unclear if these are primarily clients or acting to tag other clients for degradation. Our analysis of all point mutations in Hsp90 identified 205 that dramatically slowed the growth of yeast harboring a second WT copy of Hsp90. 75% of the dominant negative mutations that we identified were located in a loop that closes over bound ATP. We analyzed a small panel of individual dominant mutations in this loop in detail. In this panel, addition of the E33A mutation that prevents ATP hydrolysis by Hsp90 abrogated the dominant negative phenotype. Pull-down experiments did not reveal any stable binding partners, indicating that the dominant effects were mediated by dynamic complexes. We examined the stability to proteolysis of glucocorticoid receptor (GR) as a model Hsp90 substrate. Upon expression of dominant negative Hsp90 variants, GR was rapidly destabilized in a proteasome-dependent fashion. These findings provide evidence that the binding of E3 ligases to Hsp90 may serve a quality control function fundamental to eukaryotes.