Functional Analysis Of Cytosolic Hsp70 Nucleotide Exchange Factor Networks In Yeast

Author

Jennifer Lynn Abrams, The University of Texas Graduate School of Biomedical Sciences at HoustonFollow

Date of Graduation

5-2014

Document Type

Dissertation (PhD)

Program Affiliation

Microbiology and Molecular Genetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Kevin A. Morano, Ph. D.

Committee Member

Heidi B. Kaplan, Ph. D.

Committee Member

Michael C. Lorenz, Ph. D.

Committee Member

Eric J. Wagner, Ph. D.

Committee Member

Ambro van Hoof, Ph. D.

Abstract

The Hsp70 class of molecular chaperones play critical roles in protein homeostasis via an ATP-dependent folding cycle. Cytosolic Hsp70s in the budding yeast Saccharomyces cerevisiae, Ssa and Ssb, interact with up to three distinct nucleotide exchange factors (NEFs) homologous to human counterparts; Sse1/Sse2/HSP110, Fes1/HspBP1, and Snl1/Bag1. In an effort to understand the differential functional contributions of the cytosolic NEFs to protein homeostasis (“proteostasis”), I carried out comparative genetic, biochemical and cell biological analyses. For these studies, I developed protocols to monitor protein disaggregation and reactivation in a near real-time coupled assay that revealed the importance of aggregate dynamics in the solubilization of proteins for their refolding. This coupled experimental approach, represents an important step toward developing tools necessary to monitor in vivo mechanisms of proteostasis.

This work determined that the Hsp110, Sse1, is the primary NEF contributing to most Hsp70 functions and also uncovered a unique role for Fes1 in Ssa-mediated regulation of the cytosolic heat shock response, while revealing no significant contributions from Snl1 and Sse2. These findings suggest that NEFs do have overlapping functions, but their distinct associations with the Hsp70s as well as unique structural components could contribute to differential roles in proteostasis.

Additionally, this study uncovered that relative levels of Snl1 and Sse1 are important for optimal growth. To probe this relationship, I exploited the Snl1 overexpression toxicity phenotype exhibited in the absence of Sse1 to examine which unique characteristics of Snl1 are important for its function. I discovered that Snl1 localization to the ER membrane is required for toxicity and that Sse1-mediated alleviation of this phenotype is Ssb-dependent. These results demonstrate a network of interactions that supports a hypothesis where Snl1 plays a role in translation regulation.

This investigation was conducted to gain a better understanding of NEF roles within the Hsp70 chaperone network. Understanding these dynamics is critical to obtain successful treatments that can reverse the debilitating effects of neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Pharmacological targeting of molecular chaperones and their co-factors, such as the NEFs, is an attractive therapeutic goal that may contribute to improving human health, most notably in the aging population.

Keywords

chaperones, proteostasis, Hsp70, nucleotide exchange factors, protein networks