[Show abstract][Hide abstract] ABSTRACT: In response to terminal differentiation signals that enable B cells to produce vast quantities of antibodies, a dramatic expansion of the secretory pathway and a corresponding increase in the molecular chaperones and folding enzymes that aid and monitor immunoglobulin synthesis occurs. Recent studies reveal that the unfolded protein response (UPR), which is normally activated by endoplasmic reticulum (ER) stress, plays a critical role in this process. Although B cells activate all three branches of the UPR in response to pharmacological inducers of the pathway, plasma cell differentiation elicits only a partial UPR in which components of the PKR-like ER kinase (PERK) branch are not expressed. This prompted us to further characterize UPR activation during plasma cell differentiation. We found that in response to lipopolysaccharides (LPS)-induced differentiation of the I.29 micro(+) B cell line, Ire1 was activated early, which led to splicing of XBP-1. PERK was partially phosphorylated with similar kinetics, but this was not sufficient to activate its downstream target eIF-2alpha, which initiates translation arrest, or to induce other targets like CHOP or GADD34. Both of these events preceded increased Ig synthesis, arguing this is not the signal for activating these two transducers. Targets of activating transcription factor 6 (ATF6) were up-regulated considerably later, arguing that the ATF6 branch is activated by a distinct signal. Pretreatment with LPS inhibited activation of the PERK branch by pharmacological inducers of the UPR, suggesting that differentiation-induced signals specifically silence this branch. This unique ability to differentially regulate various branches of the UPR allows B cells to accomplish distinct outcomes via the same UPR machinery.
Full-text · Article · Nov 2009 · Cell Stress and Chaperones
[Show abstract][Hide abstract] ABSTRACT: Heat shock proteins of 70 kDa (Hsp70s) and their J domain-containing Hsp40 cofactors are highly conserved chaperone pairs
that facilitate a large number of cellular processes. The observation that each Hsp70 partners with many J domain-containing
proteins (JDPs) has led to the hypothesis that Hsp70 function is dictated by cognate JDPs. If this is true, one might expect
highly divergent Hsp70-JDP pairs to be unable to function in vivo. However, we discovered that, when a yeast cytosolic JDP, Ydj1, was targeted to the mammalian endoplasmic reticulum (ER),
it interacted with the ER-lumenal Hsp70, BiP, and bound to BiP substrates. Conversely, when a mammalian ER-lumenal JDP, ERdj3,
was directed to the yeast cytosol, it rescued the temperature-sensitive growth phenotype of yeast-containing mutant alleles
in two cytosolic JDPs, HLJ1 and YDJ1, and activated the ATP hydrolysis rate of Ssa1, the yeast cytosolic Hsp70 that partners with Hlj1 and Ydj1. Surprisingly,
ERdj3 mutants that were compromised for substrate binding were unable to rescue the hlj1ydj1 growth defect even though they stimulated the ATPase activity of Ssa1. Yet, J domain mutants of ERdj3 that were defective
for interaction with Ssa1 restored the growth of hlj1ydj1 yeast. Taken together, these data suggest that the substrate binding properties of certain JDPs, not simply the formation
of unique Hsp70-JDP pairs, are critical to specify in vivo function.
[Show abstract][Hide abstract] ABSTRACT: ERdj3 was identified as a soluble, lumenal DnaJ family member that binds to unassembled immunoglobulin heavy chains along with the BiP chaperone complex in the endoplasmic reticulum of mammalian cells. Here we demonstrated that ERdj3 binds directly to unfolded substrates. Secondary structure predictions suggested that the substrate binding domain of ERdj3 was likely to closely resemble Ydj1, a yeast cytosolic DnaJ family member, which was previously crystallized with a peptide bound to the C-terminal fragment composed of domains I, II, and III. Mutation of conserved residues in domain I, which formed the peptide binding site in Ydj1, affected ERdj3's substrate binding ability in mammalian cells and in vitro binding studies. Somewhat unexpectedly, we found that domain II, which is highly conserved among ERdj3 homologues, but very different from domain II of Ydj1, was also critical for substrate binding. In addition, we demonstrated that ERdj3 forms multimers in cells and found that the conserved carboxy-terminal residue phenylalanine 326 played a critical role in self-assembly. In vitro binding assays revealed that mutation of this residue to alanine diminished ERdj3's substrate binding ability, arguing that multimerization is important for substrate binding. Together, these studies demonstrate that the Ydj1 structure is conserved in another family member and reveal that among this group of DnaJ proteins domain II, which is not present in the closely related type II family members, also plays an essential role in substrate binding.
[Show abstract][Hide abstract] ABSTRACT: DnaJ proteins often bind to unfolded substrates and recruit their Hsp70 partners. This induces a conformational change in the Hsp70 that stabilizes its binding to substrate. By some unknown mechanism, the DnaJ protein is released. We examined the requirements for the release of ERdj3, a mammalian ER DnaJ, 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 were defective in any step of the ATPase cycle 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 and support a model where ERdj3 must recruit BiP and stimulate its high-affinity association with the substrate through activation of ATP hydrolysis to trigger its own release from substrates. On the basis of similarities among DnaJs and Hsp70s, this is likely to be applicable to other Hsp70-DnaJ pairs.