Date of Award

12-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Molecular Sciences

Research Advisor

Linda M. Hendershot, Ph.D.

Committee

John Cox, Ph.D. Susan E. Senogles, Ph.D. Dario A. Vignali, Ph.D. Michael A. Whitt, Ph.D.

Keywords

BiP, DnaJ-like proteins, Endoplasmic reticulum, Protein folding

Abstract

Heat shock protein 70s (Hsp70s) and their DnaJ co-factors exist in all organisms and in all eukaryotic organelles. These highly conserved chaperone pairs facilitate a large number of cellular processes. ERdj3 was identified as a soluble, lumenal DnaJ family member that binds to unassembled immunoglobulin heavy chains (HC) along with the BiP chaperone complex in the endoplasmic reticulum of mammalian cells. Here we demonstrate that ERdj3 binds directly to two unfolded substrates: immunoglobulin γHC and denatured firefly luciferase. Using mutagenesis studies on ERdj3 in both in vivo and in vitro binding assays, we defined ERdj3’s critical amino acids in domain I that contribute to substrate binding and demonstrated that ERdj3 forms dimers, which are important for substrate binding. We suggested that these features are conserved among all type I and type II DnaJ proteins. Somewhat unexpectedly, we found that domain II, which is highly conserved among ERdj3 homologues but very different from domain II of Ydj1, was also essential for substrate binding. Recent studies have demonstrated that Hsp70s can interact in vitro with some but not all DnaJ proteins from different organelles or even different organisms. To better understand restrictions on Hsp70/DnaJ interactions, we expressed ERdj3 in both the yeast ER and cytosol. Our data revealed that the ability to complement loss of the resident DnaJ proteins in either of these organelles was most dependent on its ability to interact with the resident Hsp70. Mutations in ERdj3 that affected substrate binding were unable to complement loss of Ydj1, arguing that substrate binding ability is also important. Finally, previous studies suggested that DnaJ proteins often bind to unfolded substrates initially and recruit their Hsp70 partners. DnaJ proteins stimulate the ATPase activity of its Hsp70 partner and induce a conformational change in the Hsp70 to stabilize its binding to substrate. By some unknown mechanism the DnaJ protein is then released. We established an in vitro model to examine the requirements for the release of ERdj3 from substrates and found that BiP promoted the release of ERdj3 only in the presence of ATP. Mutations in ERdj3 or BiP that disrupted their interaction interrupted the release of ERdj3. BiP mutants that cannot bind to ATP or undergo a nucleotide-induced conformational change were also unable to release ERdj3. These results demonstrate that a functional interaction between ERdj3 and BiP, including both a direct interaction and the ability to stimulate BiP’s ATPase activity are required to release ERdj3 from substrate. Furthermore they suggest that the interaction with BiP may induce a reciprocal change in ERdj3 that triggers its release from substrates. Based on similarities among DnaJs and Hsp70s, this is likely to be applicable to other Hsp70/DnaJ pairs.

DOI

10.21007/etd.cghs.2008.0155

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