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Genetic and biochemical analyses of Hsp70-Hsp40 interactions in Saccharomyces cerevisiae provide insights into specificity and mechanisms of regulation

Vembar, Shruthi Sridhar (2009) Genetic and biochemical analyses of Hsp70-Hsp40 interactions in Saccharomyces cerevisiae provide insights into specificity and mechanisms of regulation. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Heat shock proteins of 70kDa (Hsp70s) and their J domain-containing Hsp40 cofactors are conserved chaperone pairs that facilitate diverse cellular processes. One essential Hsp70 in the endoplasmic reticulum (ER) lumen, BiP (Kar2p in yeast), participates in polypeptide translocation into the ER, protein folding, and ER-associated degradation (ERAD). Like other Hsp70s, BiP contains an N-terminal ATPase domain, followed by a substrate binding domain and a C-terminal lid domain. To better define how substrate affinity and Hsp40 interaction affect BiP function, I constructed and characterized a mutation, R217A, in the putative J domain-interacting surface of yeast BiP. The mutation compromises ATPase stimulation by Sec63p, an Hsp40 required for translocation, but stimulation by Jem1p, an Hsp40 required for ERAD, is robust. In accordance with these data, yeast expressing R217A BiP exhibit translocation defects, but no ERAD defects, and a genetic interaction study using this mutant yielded data consistent with defects in translocation. In contrast, mutations in the substrate binding domain that either disrupt an ionic contact with the lid or remove this domain are deficient for peptide-stimulated ATPase activity. Expression of these mutants in yeast results in varying translocation and ERAD defects. Taken together, these data indicate that BiP can distinguish between its ER-resident cochaperones, and that optimal substrate binding is a key determinant of BiP function.Next, I tested the hypothesis that the functional specificity of Hsp70s is regulated by cognate Hsp40s. If this is true, one might expect divergent Hsp70-Hsp40 pairs to be unable to function in vivo. However, I discovered that a mammalian ER-lumenal Hsp40, ERdj3, when directed to the yeast cytosol, was able to rescue the temperature-sensitive growth phenotype of yeast containing mutant alleles in two cytosolic Hsp40s, HLJ1 and YDJ1. Moreover, ERdj3 activated the ATPase activity of Ssa1p, the yeast cytosolic Hsp70 that partners with Hlj1p and Ydj1p. Intriguingly, ERdj3 mutants that were compromised for substrate binding were unable to rescue the hlj1ydj1 growth defect, even though they stimulated Ssa1p ATPase activity. These data suggest that the substrate binding properties of certain Hsp40s—not simply the formation of unique Hsp70-Hsp40 pairs—is critical to specify in vivo function.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Vembar, Shruthi Sridharssv5@pitt.eduSSV5
Date: 1 October 2009
Date Type: Completion
Defense Date: 7 May 2009
Approval Date: 1 October 2009
Submission Date: 12 August 2009
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: BiP/Kar2p; ER protein translocation; ER-associated degradation; ERdj3; J-domain containing cochaperones; protein folding
Other ID:, etd-08122009-104313
Date Deposited: 10 Nov 2011 19:59
Last Modified: 15 Nov 2016 13:48


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