Localization and function in endoplasmic reticulum stress tolerance of ERdj3, a new member of Hsp40 family protein

Abstract

Heat shock protein 40 (Hsp40) family proteins are known to bind to Hsp70 through their J-domain and regulate the function of Hsp70 by stimulating its adenosine triphosphatase activity. In the endoplasmic reticulum (ER), there are 5 Hsp40 family proteins known so far, 3 of which were recently identified. In this report, one of the novel Hsp40 cochaperones, ERdj3, was characterized in terms of its subcellular localization, stress response, and stress tolerance of cells. By using ERdj3-specific polyclonal antibody, endogenous ERdj3 protein was shown to reside in the ER as gene transfer-mediated exogenous ERdj3. Analysis of the expression level of endogenous ERdj3 protein revealed its moderate induction in response to various ER stressors, indicating its possible action as a stress protein in the ER. Subsequently, we analyzed whether this molecule was involved in ER stress tolerance of cells, as was the case with the ER-resident Hsp70 family protein BiP. Although overexpression of ERdj3 by gene transfection could not strengthen ER stress tolerance of neuroblastoma cells, reduction of ERdj3 expression by small interfering ribonucleic acid decreased the tolerance of cells, indicating that ERdj3 might have just a marginal role in the ER stress resistance of neuroblastoma cells. In contrast, overexpression of ERdj3 notably suppressed vero toxin-induced cell death. These data suggest that ERdj3 might have diverse roles in the ER, including that of the molecular cochaperone of BiP and an as yet unknown protective action against vero toxin.

ERdj3 Regulates BiP Occupancy in Living Cells

“…When expressed in cells, our construct correctly colocalizes in a tubular network pattern with ER-RFP, consistent with previous reports of ER localization (Fig. 1B) (Nakanishi et al, 2004;Yu et al, 2000). In an immunoblot, ERdj3-sfGFP migrates more slowly (70 kDa) than endogenous ERdj3 (,40 kDa), consistent with the addition of the 25 kDa sfGFP (Fig.…”

“…Previously, our lab reported that BiP is highly mobile in the ER lumen (Lai et al, 2010) and we postulated that ERdj3 would be similarly mobile. Since ERdj3 appears to assist BiP in folding and QC of nascent peptides Nakanishi et al, 2004;Shen and Hendershot, 2005), ERdj3 must be able to encounter clients and BiP in the ER. A sufficient concentration and mobility of ERdj3 would ensure efficient encounters with clients and BiP.…”

“…Yet, overexpressed ERdj3 did not result in obvious UPR activation. Even though ERdj3 is upregulated during the UPR, overexpression of ERdj3, alone did not confer resistance to the Tm-induced or thapsigargin UPR (Nakanishi et al, 2004). Occupancy of BiP is thought to buffer cells against increasing levels of unfolded proteins (Pincus et al, 2010).…”

“…The ability of co-chaperones to encounter their dependent chaperones is likely to significantly impact the overall functionality of the global chaperone pool. While much is known regarding the biochemistry and functions of chaperones and their co-chaperones (Jin et al, 2008;Jin et al, 2009;Nakanishi et al, 2004;Shen and Hendershot, 2005), the cell biology of co-chaperones and their impact on the general availability of chaperones in cells are less well understood. It remains unclear whether co-chaperones are generally mobile and can diffuse to chaperones as needed or if co-chaperones are restricted in mobility by incorporation into large molecular complexes .…”

A Novel Isoform of TUCAN Is Overexpressed in Human Cancer Tissues and Suppresses Both Caspase-8– and Caspase-9–Mediated Apoptosis

“…To characterize the expression of endogenous TUCAN, we established rabbit antisera against TUCAN as described previously (15). Briefly, we immunized a New Zealand white rabbit with a synthetic peptide (residues 99-115 of TUCAN-54 conjugated with carrier protein keyhole limpet hemocyanin; PIERCE, Rockford, IL).…”