Abstract
ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates.
Highlights
ERdj3 is a dimeric type I DnaJ co-chaperone for BiP, the endoplasmic reticulum (ER) Hsp70
When each substrate was transiently expressed in COS-1 cells along with either wild-type or mutant ERdj3, we found that significantly more of the QPD mutant bound NS-1 LC (Fig. 1A) and the ␥ HC (Fig. 1B, lanes 2 and 4 in both immunoprecipitation panels) than the wild-type ERdj3, both ERdj3 proteins were expressed at similar levels, which was independent of the presence of the HA tag
When signals from multiple experiments were quantified, we found that under steady state conditions, the QPD mutant bound ϳ4 times more than the wild-type ERdj3 protein to these substrates when each protein was expressed alone
Summary
ERdj is a dimeric type I DnaJ co-chaperone for BiP, the ER Hsp. Results: Mutational analyses revealed the requirements for ERdj substrate binding and release in cells. We found that ERdj homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3 This was due to reduced release from these substrates as opposed to enhanced binding, in both cases dimerization was strictly required for substrate binding. The substratebinding domain of ERdj could be readily modeled [16] using the crystal structures of the substrate-binding domains of yeast cytosolic Ydj1p [15], which is very similar to that of Escherichia coli DnaJ [17, 24], and the less well conserved yeast Sis1p [14] Their structures suggest that these DnaJ proteins form a dimeric, tong-like structure, which is required for them to grasp and chaperone unfolded substrates [25, 26]. In the case of two incompletely folded ER proteins, we found that release of wild-type ERdj was slower with a long-lived substrate when compared with a substrate that turned over more rapidly
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