Quantitative proteomic analysis reveals unique Hsp90 cycle-dependent client interactions.

Hsp90 is an abundant and essential molecular chaperone that mediates the folding and activation of client proteins in a nucleotide-dependent cycle. Hsp90 inhibition directly or indirectly impacts the function of 10-15% of all proteins due to degradation of client proteins or indirect downstream effects. Due to its role in chaperoning oncogenic proteins, Hsp90 is an important drug target. However, compounds that occupy the ATP-binding pocket and broadly inhibit function have not achieved widespread use due to negative effects. More selective inhibitors are needed; however, it is unclear how to achieve selective inhibition. We conducted a quantitative proteomic analysis of soluble proteins in yeast strains expressing wild-type Hsp90 or mutants that disrupt different steps in the client folding pathway. Out of 2,482 proteins in our sample set (approximately 38% of yeast proteins), we observed statistically significant changes in abundance of 350 (14%) of those proteins (log2 fold change ≥ 1.5). Of these, 257/350 (∼73%) with the strongest differences in abundance were previously connected to Hsp90 function. Principal component analysis of the entire dataset revealed that the effects of the mutants could be separated into 3 primary clusters. As evidence that Hsp90 mutants affect different pools of clients, simultaneous co-expression of 2 mutants in different clusters restored wild-type growth. Our data suggest that the ability of Hsp90 to sample a wide range of conformations allows the chaperone to mediate folding of a broad array of clients and that disruption of conformational flexibility results in client defects dependent on those states.

Insights into Hsp90 mechanism and in vivo functions learned from studies in the yeast, Saccharomyces cerevisiae.

The molecular chaperone Hsp90 (Heat shock protein, 90 kDa) is an abundant and essential cytosolic protein required for the stability and/or folding of hundreds of client proteins. Hsp90, along with helper cochaperone proteins, assists client protein folding in an ATP-dependent pathway. The laboratory of Susan Lindquist, in collaboration with other researchers, was the first to establish the yeast Saccharomyces cerevisiae as a model organism to study the functional interaction between Hsp90 and clients. Important insights from studies in her lab were that Hsp90 is essential, and that Hsp90 functions and cochaperone interactions are highly conserved between yeast and mammalian cells. Here, we describe key mechanistic insights into the Hsp90 folding cycle that were obtained using the yeast system. We highlight the early contributions of the laboratory of Susan Lindquist and extend our analysis into the broader use of the yeast system to analyze the understanding of the conformational cycle of Hsp90 and the impact of altered Hsp90 function on the proteome.