Proteins must fold correctly into a defined three-dimensional structure in order to properly function. Several human diseases, includingCreutzfeldt-Jacob disease and cystic fibrosis, can be considered protein folding diseases because proteins fail to achieve active conformations in their proper cellular compartment. Within the crowded cellular milieu, newly translated proteins are prone to aggregation and nonproductive premature folding because of the exposure to the aqueous environment of hydrophobic residues which are normally sequestered within the folded protein. Molecular chaperones are excellent candidates for proteins which bind to and protect nascent chains from problematic interactions because of the nature of theirtransient interactions with hydrophobic polypeptide segments. Data collected in a variety of systems, in vivo and in vitro, indicate a pathway(s) of protein folding from the time proteins are nascent chains on ribosomes until they are either properly folded or degraded. Molecular chaperones play important roles in these processes. The focus of experiments described in this proposal is to determine the function of Hsp70 (heat shock protein 70K) and Hsp40 as molecular chaperones chaperone using the budding yeast Saccharomyces cerevisiae as a model system. More specifically, the focus is on the role of molecular chaperones during the early events of protein folding on the ribosome. The cytosol of S. cerevisiae contains two classes of abundant, functionally distinct Hsp70s (SSA and SSB proteins) and three Hsp40s (Sisl, Zuol and Ydjl). Of these chaperones, Ssb, Sisl and Zuol associate with translating ribosomes, suggesting a role early in the life of a protein. Using a combination of genetic and biochemical approaches we will analyze the function of Ssb, Zuol and Sisl in translation and early steps of protein folding. Hsp70s and Hsp40s are known to function cooperatively as chaperones. However, the functional relationship between them is not well understood. Our goal is to understand the functional relationship between the two Hsp40s (Sisl and Zuol) and one Hsp70 (Ssb) which are associated with the ribosome. More specifically, we will ascertain through genetic and biochemical experiments whether these two DnaJs functionally and physically interact with Ssb. In addition, we will determine the sequence of interactions of a nascent chain with the ribosome-associated chaperones.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM031107-27
Application #
7459840
Study Section
Special Emphasis Panel (NSS)
Program Officer
Anderson, Richard A
Project Start
1982-07-01
Project End
2009-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
27
Fiscal Year
2008
Total Cost
$409,666
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Yu, Hyun Young; Ziegelhoffer, Thomas; Osipiuk, Jerzy et al. (2015) Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone Sis1. J Mol Biol 427:1632-43
Ciesielski, Grzegorz L; Plotka, Magdalena; Manicki, Mateusz et al. (2013) Nucleoid localization of Hsp40 Mdj1 is important for its function in maintenance of mitochondrial DNA. Biochim Biophys Acta 1833:2233-43
Sahi, Chandan; Kominek, Jacek; Ziegelhoffer, Thomas et al. (2013) Sequential duplications of an ancient member of the DnaJ-family expanded the functional chaperone network in the eukaryotic cytosol. Mol Biol Evol 30:985-98
Kominek, Jacek; Marszalek, Jaroslaw; Neuvéglise, Cécile et al. (2013) The complex evolutionary dynamics of Hsp70s: a genomic and functional perspective. Genome Biol Evol 5:2460-77
Ducett, Jeanette K; Peterson, Francis C; Hoover, Lindsey A et al. (2013) Unfolding of the C-terminal domain of the J-protein Zuo1 releases autoinhibition and activates Pdr1-dependent transcription. J Mol Biol 425:19-31
Prunuske, Amy J; Waltner, Jeanette K; Kuhn, Peter et al. (2012) Role for the molecular chaperones Zuo1 and Ssz1 in quorum sensing via activation of the transcription factor Pdr1. Proc Natl Acad Sci U S A 109:472-7

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