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. 2008 Jun;19(6):2620-30.
doi: 10.1091/mbc.e07-07-0674. Epub 2008 Apr 9.

ERdj4 and ERdj5 are required for endoplasmic reticulum-associated protein degradation of misfolded surfactant protein C

Mei Dong  1 , James P Bridges, Karen Apsley, Yan Xu, Timothy E Weaver
Affiliations

ERdj4 and ERdj5 are required for endoplasmic reticulum-associated protein degradation of misfolded surfactant protein C

Mei Dong et al. Mol Biol Cell. 2008 Jun.

Abstract

Mutations in the SFTPC gene associated with interstitial lung disease in human patients result in misfolding, endoplasmic reticulum (ER) retention, and degradation of the encoded surfactant protein C (SP-C) proprotein. In this study, genes specifically induced in response to transient expression of two disease-associated mutations were identified by microarray analyses. Immunoglobulin heavy chain binding protein (BiP) and two heat shock protein 40 family members, endoplasmic reticulum-localized DnaJ homologues ERdj4 and ERdj5, were significantly elevated and exhibited prolonged and specific association with the misfolded proprotein; in contrast, ERdj3 interacted with BiP, but it did not associate with either wild-type or mutant SP-C. Misfolded SP-C, ERdj4, and ERdj5 coprecipitated with p97/VCP indicating that the cochaperones remain associated with the misfolded proprotein until it is dislocated to the cytosol. Knockdown of ERdj4 and ERdj5 expression increased ER retention and inhibited degradation of misfolded SP-C, but it had little effect on the wild-type protein. Transient expression of ERdj4 and ERdj5 in X-box binding protein 1(-/-) mouse embryonic fibroblasts substantially restored rapid degradation of mutant SP-C proprotein, whereas transfection of HPD mutants failed to rescue SP-C endoplasmic reticulum-associated protein degradation. ERdj4 and ERdj5 promote turnover of misfolded SP-C and this activity is dependent on their ability to stimulate BiP ATPase activity.

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Figures

Figure 1.
Figure 1.
ERdj4 and ERdj5 are required for degradation of SP-CΔexon4. (A) HEK293 cell lines stably expressing SP-CWT, SP-CΔexon4 (x4) or SP-CL188Q were transfected with plasmids encoding ERdj4 (middle), ERdj5 (right), or vector (left). Forty-eight hours after transfection, cells were treated with the proteasome inhibitor MG-132 to stabilize misfolded SP-C. After cross-linking with DSP, cells were harvested and equal amounts of cell lysates were subjected to IP with SP-C proprotein antibody followed by reducing SDS-PAGE, and sequential Western blotting (IB) with HA or SP-C antibodies. (B) HEK293 cells stably expressing SP-CΔexon4 were not transfected (top row) or transfected with siRNA for ERdj4, ERdj5, or both ERdj4 and ERdj5. Twenty-four hours later, cells were labeled with [35S]Met/Cys for 15 min, chased for the indicated period, and immunoprecipitated with SP-C antibody (left). The radioactivity of each SP-CΔexon4 band was analyzed by phosphorimaging analyses and normalized to the value at 0 chase time; averages of three independent experiments were plotted with SD values (right). (C) HEK293 cells stably expressing SP-CWT were transfected with siRNA for both ERdj4 and ERdj5 (lanes 5–8), or they were not transfected with siRNA (lanes 1–4). Pulse-chase, immunoprecipitation for SP-CWT proprotein, and phosphorimaging analyses were performed as described in B. (D) HEK293 cells stably expressing SP-CΔexon4 were cotransfected with plasmids encoding ERdj4 and ERdj5 (lanes 5–8), or they were left untransfected (lanes 1–4). Pulse-chase, immunoprecipitation, and phosphorimaging analyses were performed as described in B. The radioactivity of each SP-CΔexon4 band was normalized to the value at 0 chase time (bottom); pulse-chase studies represent the average of three independent experiments ± SD values.
Figure 2.
Figure 2.
BiP is required for binding of ERdj5 but not ERdj4 to misfolded SP-C. (A) Cell lines stably expressing WT or SP-CΔexon4 (x4) were transfected with plasmids encoding BiP and treated with MG-132; no cross-linking agent was added in this experiment. Cell lysates were immunoprecipitated with SP-C proprotein antibody followed by Western blotting with FLAG antibody to detect BiP. EV, HEK293 cells transfected with empty vector (pIRES2-EGFP). (B) a, HEK293 cells were cotransfected with plasmids expressing ERdj4 or ERdj4ΔJ and BiP. After cross-linking, cell lysates were immunoprecipitated with HA antibody and analyzed by Western blotting with FLAG antibody to detect BiP or HA antibody to detect ERdj4. b, HEK293 cells stably expressing SP-CWT (lanes 1) or SP-CΔexon4 (lanes 2) were transfected with plasmids encoding ERdj4ΔJ, treated with MG-132, and cross-linked. Cell lysates were immunoprecipitated with SP-C antibody and analyzed by Western blotting with HA antibody to detect ERdj4ΔJ. (C) a, HEK293 cells were cotransfected with plasmids encoding ERdj5 or ERdj5ΔJ and BiP. After cross-linking with DSP, cell lysates were immunoprecipitated with FLAG antibody (BiP) and analyzed by Western blotting with HA antibody to detect ERdj5 or FLAG antibody to detect BiP. b, HEK293 cells stably expressing SP-CWT (lanes 1) or SP-CΔexon4 (lanes 2) were transfected with plasmids encoding ERdj5ΔJ, treated with MG-132, and cross-linked. Cell lysates were immunoprecipitated with SP-C antibody and analyzed by Western blotting with HA antibody to detect ERdj5ΔJ. (D) HEK293 cells stably expressing SP-CWT or SP-CΔexon4 were transfected with plasmids encoding ERdj4 or ERdj4H54Q. Forty-eight hours after transfection, cells were pulse-labeled with [35S]Met/Cys for 15 min, and then they were chased for the indicated times. After cross-linking with DSP, cell lysates were immunoprecipitated with HA antibody followed by reducing SDS-PAGE, and autoradiography.
Figure 3.
Figure 3.
ERdj4 and ERdj5 restore degradation of SP-CΔexon4 in XBP-1−/− MEFs. (A) XBP-1+/+ and XBP-1−/− MEFs were transiently transfected with plasmid encoding SP-CΔexon4 followed by Western blotting of cell lysates with SP-C and actin antibodies at 0, 24, and 48 h posttransfection. (B) XBP-1−/− MEFs were cotransfected with plasmids encoding SP-CΔexon4 and ERdj4 (left), ERdj4H54Q (middle) or ERdj4 plus ERdj4 siRNA (right). Cell lysates were prepared at 0, 24, and 48 h posttransfection, and then they were analyzed by Western blotting with SP-C, HA (ERdj4), and actin antibodies. (C) XBP-1−/− MEFs were cotransfected with plasmids encoding SP-CΔexon4 and ERdj5 (left), ERdj5H63Q (middle), or ERdj5 plus ERdj5 siRNA (right). Cell lysates were prepared at 0, 24, and 48 h posttransfection, and then they were analyzed by Western blotting with SP-C, HA (ERdj5) and actin antibodies. (D) XBP-1−/− MEFs were cotransfected with plasmids encoding SP-CΔexon4, ERdj4, and ERdj5. Cell lysates were prepared at 0, 24, and 48 h posttransfection, and then they were analyzed by Western blotting with SP-C, HA (ERdj4 and ERdj5), and actin antibodies. Within each set of experiments (A–D), samples were run on one gel to ensure identical exposure times. Actin was used to adjust exposure times across experiments.
Figure 4.
Figure 4.
Association of p97 with ERdj4, ERdj5, and SP-C. (A) HEK293-SP-CWT and HEK293-SP-CΔexon4 (x4) stable cell lines were treated with MG-132 and cross-linked with DSP. Cell lysates were immunoprecipitated with p97 antibody and analyzed by Western blotting with SP-C antibody. (B) HEK293 cells were transfected with plasmids encoding ERdj4 or ERdj5; no cross-linking reagent was added in this experiment. Cell lysates were immunoprecipitated with p97 antibody, and then they were analyzed by Western blotting with HA antibody.
Figure 5.
Figure 5.
ERdj4 and ERdj5 associate with WT and mutant insulin 2. (A) HEK293 cells were transfected with empty vector (EV) or cotransfected with plasmid expressing ERdj4 or ERdj5 and plasmid encoding insulin 2 or mutant insulin 2 (insulin 2C96Y). Cells were treated with MG-132 followed by cross-linking with DSP 48 h after transfection. Cell lysates were immunoprecipitated with insulin 2 antibody followed by Western blotting with HA antibody to detect ERdj4 and ERdj5. Quantitative analyses from three independent experiments are shown in Table 2. (B) HEK293 cells were cotransfected with plasmids encoding ERdj4 or ERdj5 and insulin 2 or insulin 2C96Y. After 48 h, cells were treated with MG-132 followed by cross-linking. Cell lysates were immunoprecipitated with insulin 2 antibody followed by Western blotting with p97 antibody. (C and D) HEK293 cells were cotransfected with plasmids encoding insulin2WT (C) or insulin2C96Y (D). Pulse-chase analyses were performed as described in Figure 1B. Cell lysates were immunoprecipitated with insulin 2 antibody, subjected to SDS-PAGE, and band intensity was quantitated by phosphorimaging analyses.
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