In humans, defects in protein folding can lead to neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's disease and debilitating forms of ataxia. The 70 kD heat-shock proteins (Hsp70) are a ubiquitous family of protein chaperones involved in all aspects of protein homeostasis including protein biogenesis, repair and degradation. Hsp70 activity is governed by two types of cofactors;J domain-containing proteins activate the ATP hydrolysis step leading to high affinity substrate binding, while nucleotide exchange factors (NEFs) promote ADP dissociation and polypeptide release. Eukaryotes from yeast to humans express three recently described families of structurally diverse cytosolic NEFs (Hsp110 (Sse1/2 in yeast), HspBP1 (Fes1), and Bag-domain (Snl1)) that perform essentially the same biochemical function. The distinct contributions these proteins make to Hsp70-dependent activities are unknown in any biological system. The primary goal of this proposal is to determine how these NEFs partner with cytosolic Hsp70 chaperones to mediate proteostasis in eukaryotic cells. We hypothesize that while the NEFs share a common ability to stimulate Hsp70 activity, they differentially interact with the Ssa and Ssb families of cytosolic Hsp70 to promote protein folding and repair. In addition, we hypothesize that the Bag homolog Snl1 simultaneously recruits Hsp70 and the ribosome to promote protein biogenesis at the endoplasmic reticulum (ER). Three lines of investigation are proposed to test these hypotheses. In the first aim, we will determine how the apparently principal NEF Hsp110 is integrated into the cytosolic NEF network to promote protein biogenesis and repair. A major component of this aim will be genetic and cell biological experiments to assess the contribution of the Hsp110 substrate binding domain to Hsp70- dependent protein folding in vivo. In contrast to published results that Fes1 binds Ssa and Ssb in vitro, we have obtained preliminary evidence that Fes1 interacts solely with Ssa in vivo.
In Aim 2 we will resolve these conflicting findings and determine the mechanism of Fes1-Hsp70 specificity. Lastly, we have discovered that the ER membrane-associated Snl1 binds the ribosome in addition to Hsp70.
Aim 3 will be focused on determining the physiological significance of membrane recruitment of the translation machinery by an Hsp70 NEF. These studies represent the first comprehensive analysis of cytosolic Hsp70 NEFs by genetic, biochemical, and genomic approaches. Due to the high degree of conservation of these and other components of the Hsp70 chaperone network in eukaryotes, the results will be directly applicable to understanding the roles of Hsp70 NEFs in protein biogenesis and quality control in human cells.

Public Health Relevance

In humans, defects in protein folding can lead to neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's disease and debilitating forms of ataxia. Dedicated cellular machines called molecular chaperones assist newly made proteins to fold and help repair proteins damaged by stress or age. This proposal seeks to understand how the Hsp70 molecular chaperone performs this task inside cells. More specifically, we will focus on helper proteins called exchange factors that control how fast the Hsp70 machine works. These studies will be carried out using yeast cells, a convenient, tractable a tested model for understanding how human cells function. The outcomes of this project should allow us to more accurately predict the disease consequences of genetic defects in the protein repair machinery, and facilitate therapeutic intervention to ameliorate protein misfolding disorders.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Maas, Stefan
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University of Texas Health Science Center Houston
Schools of Medicine
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Abrams, Jennifer L; Verghese, Jacob; Gibney, Patrick A et al. (2014) Hierarchical functional specificity of cytosolic heat shock protein 70 (Hsp70) nucleotide exchange factors in yeast. J Biol Chem 289:13155-67
Abrams, Jennifer L; Morano, Kevin A (2013) Coupled assays for monitoring protein refolding in Saccharomyces cerevisiae. J Vis Exp :e50432
Naticchia, Matthew R; Brown, Haley A; Garcia, Francisco J et al. (2013) Bifunctional electrophiles cross-link thioredoxins with redox relay partners in cells. Chem Res Toxicol 26:490-7
Tapia, Hugo; Morano, Kevin A (2010) Hsp90 nuclear accumulation in quiescence is linked to chaperone function and spore development in yeast. Mol Biol Cell 21:63-72
Mandal, Atin K; Gibney, Patrick A; Nillegoda, Nadinath B et al. (2010) Hsp110 chaperones control client fate determination in the hsp70-Hsp90 chaperone system. Mol Biol Cell 21:1439-48
Shaner, Lance; Gibney, Patrick A; Morano, Kevin A (2008) The Hsp110 protein chaperone Sse1 is required for yeast cell wall integrity and morphogenesis. Curr Genet 54:1-11
Trott, Amy; West, James D; Klaic, Lada et al. (2008) Activation of heat shock and antioxidant responses by the natural product celastrol: transcriptional signatures of a thiol-targeted molecule. Mol Biol Cell 19:1104-12
Gibney, Patrick A; Fries, Thomas; Bailer, Susanne M et al. (2008) Rtr1 is the Saccharomyces cerevisiae homolog of a novel family of RNA polymerase II-binding proteins. Eukaryot Cell 7:938-48
Shaner, Lance; Morano, Kevin A (2007) All in the family: atypical Hsp70 chaperones are conserved modulators of Hsp70 activity. Cell Stress Chaperones 12:1-8
Fan, Qing; Park, Kyung-Won; Du, Zhiqiang et al. (2007) The role of Sse1 in the de novo formation and variant determination of the [PSI+] prion. Genetics 177:1583-93

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