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. 2004 Autumn;9(3):253-64.
doi: 10.1379/csc-52.1.

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

Katsuya Nakanishi  1 , Kenjiro Kamiguchi, Toshihiko Torigoe, Chika Nabeta, Yoshihiko Hirohashi, Hiroko Asanuma, Hirotoshi Tobioka, Norie Koge, Oi Harada, Yasuaki Tamura, Hideki Nagano, Shoki Yano, Susumu Chiba, Hiroyuki Matsumoto, Noriyuki Sato
Affiliations

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

Katsuya Nakanishi et al. Cell Stress Chaperones. 2004 Autumn.

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.

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Figures

Fig 1.
Fig 1.
The structure and expression of ERdj3. (A) The deduced amino acid sequence of ERdj3. ERdj3 is composed of 358 amino acids. Italic letters indicate the predicted signal peptide. J-domain is underlined. The peptide sequence that the antibody was raised against is indicated in bold type. (B, C) Western blotting analysis of ERdj3 protein. ERdj3-myc expression vector or mock vector was transfected into Vero cells (B) and SH-SY5Y cells (C). Twenty-four or 48 hours after transfection, Western blotting was performed using the anti-myc, anti-ERdj3, or anti–β-actin antibody. β-Actin was detected as an internal control. (D) Western blotting analysis of total cellular protein isolated from various cell lines
Fig 2.
Fig 2.
Subcellular localization of endogenous ERdj3. (A) MCF7 cells were immunostained with polyclonal anti-ERdj3 antibody followed by fluorescein isothiocyanate–conjugated anti-rabbit immunoglobulin G (IgG) antibody or monoclonal anti-KDEL antibody followed by rhodamine-conjugated anti-mouse IgG antibody. After mounting, cells were visualized by confocal laser microscopy. (a) Immunostaining of ERdj3, (b) immunostaining of KDEL, and (c) merge. SH-SY5Y cells were transfected with ERdj3-myc and immunostained with polyclonal anti-myc antibody followed by fluorescein isothiocyanate–conjugated anti-rabbit antibody or monoclonal anti-KDEL antibody followed by rhodamine-conjugated anti-mouse IgG antibody. After mounting, cells were visualized by confocal laser microscopy. (d) Immunostaining of myc tag, (e) immunostaining of KDEL, and (f) merge. (B) Proteinase K digestion of ERdj3 and BiP. A rat microsome was incubated in phosphate-buffered saline supplemented with (lanes 3 and 4) or without (lanes 1 and 2) 10 μg/mL proteinase K in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of 1% Triton X-100 at 4°C for 30 minutes, followed by sodium dodecyl sulfate–polyacrylamide sulfate gel electrophoresis and Western blotting with anti-BiP or anti-ERdj3 antibody
Fig 3.
Fig 3.
Cellular response to vero toxin treatment. Cells were transiently transfected with ERdj3 expression vector or mock vector. Twenty-four hours after transfection, cells were treated with the indicated concentrations of vero toxin for 18 hours followed by 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide assay to assess cell viability. (A) Vero cells. (B) SH-SY5Y cells. Vero cells were treated with 0.25 ng/mL of vero toxin, and cell lysates were collected at the indicated time points after treatment. (C) The lysates were subjected to Western blotting using anti-ERdj3, anti-BiP, or anti–β-actin antibody. (D) Signal intensities were quantified, and relative ERdj3 or BiP expression level was calculated as: relative expression ratio = (sample ERdj3 or BiP signal intensity/sample actin signal intensity)
Fig 4.
Fig 4.
Cellular response to tunicamycin treatment. (A) SH-SY5Y cells were treated with 1 μg/mL of tunicamycin, and cell lysates were collected at the indicated time points after the treatment. The lysates were subjected to Western blotting using anti-BiP, anti-ERdj3, or anti–β-actin antibody. An arrowhead indicates the aglycosylated form of ERdj3. (B) An ERdj3-transfected SH-SY5Y clone and a mock-transfected clone were transfected with BiP expression vector or a mock vector. Twenty-four hours after the transfection, cells were treated with the indicated concentrations of tunicamycin for 18 hours, followed by 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay to assess cell viability. (C) ERdj3-transfected SH-SY5Y clone (lanes 2 and 4) or mock-transfected clone (lanes 1 and 3) was transiently transfected with BiP expression vector (lanes 3 and 4) or mock vector (lanes 1 and 2), followed by Western blotting with anti-myc, anti-ERdj3, anti-BiP, or anti–β-actin antibody. (D) SH-SY5Y cells were transfected with pERdj3-IRES-EGFP vector. The rate of propidium iodide–positive cells in the enhanced green fluorescence protein (EGFP)–expressing cells (closed circles) or that in the EGFP-negative cells (open circles) was assessed. (E) Vero cells were transfected with ERdj3 expression vector or mock vector. Twenty-four hours after transfection, cells were treated with the indicated concentrations of tunicamycin for 18 hours, followed by MTT assay to assess cell viability
Fig 5.
Fig 5.
Cellular response to thapsigargin treatment. (A) SH-SY5Y cells were treated with 1 μM of thapsigargin, and cell lysates were collected at the indicated time points after the treatment. The lysates were subjected to Western blotting using anti-BiP, anti-ERdj3, or anti–β-actin antibody. (B) An ERdj3-transfected SH-SY5Y clone and a mock-transfected clone were transfected with BiP expression vector or a mock vector, respectively. Twenty-four hours after the transfection, cells were treated with the indicated concentrations of thapsigargin for 18 hours, followed by 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay to assess cell viability. (C) SH-SY5Y cells were transfected with pERdj3-IRES-EGFP vector. The rate of propidium iodide–positive cells in the enhanced green fluorescence protein (EGFP)–expressing cells (closed circles) or that in the EGFP-negative cells (open circles) was assessed. (D) Vero cells were transfected with ERdj3 expression vector or mock vector. Twenty-four hours after the transfection, cells were treated with the indicated concentrations of tunicamycin for 18 hours, followed by MTT assay to assess cell viability
Fig 6.
Fig 6.
Reduction of ERdj3 level by small interfering ribonucleic acid (siRNA) and cellular response against ER stress treatment. (A) SH-SY5Y cells were transfected with either ERdj3-siRNA or green fluorescence protein (GFP)–siRNA. Seventy-two hours after the transfection, cell lysates were collected and subjected to Western blotting using anti-ERdj3, anti-BiP, anti-Hsp70, or anti–β-actin antibody. Signal intensities were quantified, and relative ERdj3, BiP, or Hsp70 expression level was calculated as: relative expression ratio = (sample ERdj3, BiP, or Hsp70 signal intensity/sample actin signal intensity). (B, C) SH-SY5Y cells were transfected with either ERdj3-siRNA or GFP-siRNA. Seventy-two hours after transfection, cells were cultured in media containing the indicated concentrations of thapsigargin (B) or tunicamycin (C) for 48 hours, followed by 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide assay to assess cell viability
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