Hsp104 and proteostasis yeast

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In this project we will pursue an ongoing effort to elucidate how proteins trapped in irreversible aggregates are solubilized and refolded by the AAA+ ATPase Hsp104 in cooperation with other molecular chaperones. The findings will have implications for the basic understanding the stress ecology of plants and microbes at the molecular level, as well as providing fundamental knowledge that could lead to the engineering of protein homeostasis networks and thereby create rugged organisms for enhanced crop yield, biofuel synthesis, and biopharmaceutical protein production. The hallmark property of molecular chaperones is their ability to prevent the aggregation of unfolded proteins that result from protein folding stresses like heat. Molecular chaperones generally recognize hydrophobic amino acid residues exposed in misfolded proteins, shielding them from non-specific interactions that cause aggregation. The focus of this proposal is Hsp104, a truly unconventional molecular chaperone. Rather than preventing aggregation, Hsp104 is able to disperse aggregates and restore function to proteins that are otherwise irretrievably trapped. Through the proposed experiments we will establish how Hsp104 interacts, both physically and functionally, with other members of a proteostasis network. To understand the biology of Hsp104 we will conduct a screen to identify proteins and pathways that are dependent on remodelling by Hsp104. The specific aims are: 1. We will determine the role of the Hsp104/Hsp70 interaction in protein disaggregation by using a combination of site-directed mutagenesis, biochemical assays for substrate exchange between bacterial Hsp70 and ClpB the bacterial orthologue of Hsp104, and analysis of Hsp70 binding to Hsp104 and its influence on nucleotide exchange and substrate release. 2. We will identify sites important for the binding of distinct substrates by Hsp104. We have used peptides to determine the existence of at least two binding sites in Hsp104 for peptides containing aromatic residues and to identify a region on the yeast prion protein Sup35 that is required for the curing of the [PSI+] prion by Hsp04 overexpression. We will map the amino acid binding site with high resolution using peptides with a photoactivatable crosslinking amino acid derivative that substitutes for aromatic residues in peptide binding to Hsp104. Evidence suggests that Sup35 binds directly to the N-terminal domain of Hsp104, which is also required for prion curing. We will conduct experiments to test this idea and map the binding surface using NMR spectroscopy or crystallography. 3. We will identify targets of Hsp104 under non-stress conditions. When we express an engineered an Hsp104 that redirects substrates to a bacterial protease for degradation, yeast grow significantly more slowly and have a mildly elongated phenotype. This observation suggests that Hsp104 has cellular targets that, when degraded, result in defects in polrarized growth and/or cell cycle. We propose to conduct a screen using the synthetic gene array to identify pathways that may be affected by Hsp104-dependent degradation. Furthermore, we will trap Hsp104 targets inside a protease deficient ClpP oligomer. We will determine if deletion of Hsp104 affects the function or localization of select candidates in yeast (they may, for example, tend to aggregate when Hsp104 is absent) or the components of pathways in which they participate. A list of substrates will be used to gain insight into what types of molecules are subject to Hsp104-dependent remodeling and commonalities among these proteins will help us learn more about the structural determinants of substrate selection.