Genetic and biochemical analysis of the Hsp90 system in Saccharomyces cerevisiae

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The 90 kiloDalton heat shock protein, Hsp90, plays a fundamental and essential role in maintaining proteostasis in all eukaryotes. My research program is focused on understanding the mechanism of action Hsp90, and the basis for its regulation by co-chaperone proteins and post-translational modifications (PTMs). Hsp90 chaperones conformational changes in hundreds of 'client' proteins that are required for things like ligand binding, catalytic activity, or assembly into macromolecular complexes. Hsp90 is a homodimeric ATPase and the ATP-driven client activation cycle is tightly regulated by a cohort of co-chaperone proteins and post-translational modifications. How the global conformational changes associated with the Hsp90 ATPase cycle ultimately drive client protein activation remains an open question in the field. My research program is focused on elucidating the mechanics of the Hsp90 client activation cycle with particular emphasis on two co-chaperones that stimulate ATP hydrolysis, Aha1p and Hch1p, and PTMs that modulate their action. My program is comprised of three themes that provide diverse training opportunities using different models and techniques. 1) Characterize Aha1p/Hch1p regulation of Hsp90 using yeast genetics The Hsp90 system is highly conserved in all eukaryotes and Saccharomyces cerevisiae is the most widely used model for Hsp90 studies. We have a collection of yeast strains expressing different Hsp90 and Hsp90 co-chaperone mutants. We will focus on a set of these strains to characterize the in vivo action of Aha1p and Hch1p. We will test these strains in growth assays as well as in client-specific functional assays. The impact of alteration different structural elements of the Hsp90 machine system or regulatory components (co-chaperones and post-translational modifications) can be dissected in the yeast model system. 2) Characterize regulation of the Hsp90 ATPase activity by Aha1p and Hch1p We use purified Hsp90, Hsp90 co-chaperones, and model client proteins to analyze the kinetics of ATP hydrolysis and client activation in vitro. Hsp90 or Hsp90 co-chaperones harbouring mutations that confer growth defects or client-specific activation defects in yeast are purified and studied in a variety of in vitro assays. We measure kinetic parameters of ATP hydrolysis by Hsp90 as well as co-chaperone-mediated activation of client activation. These in vitro strategies allow us to determine the molecular underpinnings of the phenotypes we observe in yeast. 3) Physically characterize Hsp90 complexes We collaborate with other research groups specializing in NMR, crystallography, small-angle light scattering, and fluorescence techniques to interrogate the structural basis for the enzymatic and biological activity of Hsp90. This aspect of my program not only yields important scientific insights into Hsp90 biology but also provides valuable training opportunities for my trainees because of the extensive collaborations it involves.