The HSP70 co-chaperone DNAJC14 targets misfolded pendrin for unconventional protein secretion

Abstract

Mutations in SLC26A4, which encodes pendrin, are responsible for hearing loss with an enlarged vestibular aqueduct and Pendred syndrome. The most prevalent mutation in East Asia is p.H723R (His723Arg), which leads to defects in protein folding and cell-surface expression. Here we show that H723R-pendrin can be rescued to the cell surface by an HSP70 co-chaperone DNAJC14-dependent unconventional trafficking pathway. Blockade of ER-to-Golgi transport or activation of ER stress signals induced Golgi-independent cell-surface expression of H723R-pendrin and restored its cell-surface Cl(-)/HCO3(-) exchange activity. Proteomic and short interfering RNA screenings with subsequent molecular analyses showed that Hsc70 and DNAJC14 are required for the unconventional trafficking of H723R-pendrin. Moreover, DNAJC14 upregulation was able to induce the unconventional cell-surface expression of H723R-pendrin. These results indicate that Hsc70 and DNAJC14 play central roles in ER stress-associated unconventional protein secretion and are potential therapeutic targets for diseases such as Pendred syndrome, which arise from transport defects of misfolded proteins.

Figure 1. Cell-surface expression of H723R-pendrin by unconventional protein secretion.

(a) PANC-1 cells were transfected with plasmids encoding wild-type (WT)-pendrin. Protein samples were treated with N-glycosidase F (PNGase F), which deglycosylates all N-glycan chains and immunoblotted with anti-pendrin antibodies. Band A: deglycosylated pendrin, band B: immature core-glycosylated pendrin, band C: mature complex-glycosylated pendrin. (b,c) Surface biotinylation assays were performed in PANC-1 cells expressing WT- (b) or H723R- (c) pendrin. In control cells (first lanes in b and c), the band C form of WT-pendrin and no form of H723-pendrin were expressed on the cell surface. Blockade of ER-to-Golgi traffic by overexpression of Arf1-Q71L, Sar1-T39N or syntaxin 5 induced the cell-surface expression of core-glycosylated (band B) WT- and H723R-pendrins. The absence of the cytosolic protein aldolase A in the biotinylated fraction confirmed cell surface protein-specific labelling in each experiment (input: 40 μg of cell lysate, surface biotinylation: 400 μg protein). (d) Treatments with brefeldin A (BFA) or thapsigargin (TG) also induced cell-surface expression of H723R-pendrin in PANC-1 cells. (e,f) Whereas GRASP55 overexpression induced cell-surface expression of ΔF508-CFTR in PANC-1 cells (f), GRASP55 was not effective in inducing cell-surface expression of H723R-pendrin (e). Quantitation of multiple experiments is presented under each immunoblot. ^**P<0.01 by one-way analysis of variance, compared with lane 1, the number of replicates (n) is presented in each panel. Unprocessed original scans of western blots are shown in Supplementary Fig. 10.

Figure 2. Rescue of anion exchange activity of H723R-pendrin by Arf1-Q71L.

The Cl⁻/HCO₃⁻ exchange activity was measured by recording pH_i as detailed in Methods. A clone of HEK 293 cells that exhibited minimal intrinsic anion (Cl⁻/HCO₃⁻) exchange activity (a) was transfected with the indicated plasmids (b–d) and then anion exchange activity was analysed. The quantitation of multiple experiments is depicted in e. Blockade of ER-to-Golgi trafficking by Arf1-Q71L significantly increased Cl⁻/HCO₃⁻ exchange activity in cells expressing H723R-pendrin. ^**P<0.01 by unpaired Student's t-test, the number of replicates (n) is presented in each lane.

Figure 3. Hsc70 is necessary for Arf1-Q71L–mediated unconventional cell-surface expression of H723R-pendrin.

(a) After tandem affinity purification, binding partners of H723R-pendrin in PANC-1 cells with or without Arf1-Q71L co-expression were separated by electrophoresis. Subsequent analyses by LC-MS/MS identified NckAP1, AP3B1, KIAA0980 and Hsc70 as binding partners of H723R-pendrin (Supplementary Table 1). (b–f) Surface biotinylation assays were performed after siRNA-mediated knockdown of indicated candidate proteins. Representative blots are shown in b–e and a summary of multiple experiments is presented in f. Only treatment with siRNA targeting Hsc70 showed a significant effect. ^**P<0.01 by one-way analysis of variance, difference from control, the number of replicates (n) is presented in each lane. (g) Surface biotinylation assays were performed after overexpression of NckAP1, AP3B1, KIAA0980 and Hsc70. None of the candidate proteins induced cell-surface expression of H723R-pendrin. The data are representative of three independent experiments. Unprocessed original scans of western blots are shown in Supplementary Fig. 10.

Figure 4. Physical interaction with Hsc70 and Hsc70 ATPase activity are necessary for unconventional surface trafficking of H723R-pendrin.

(a,b) Lysates of PANC-1 cells transfected with indicated plasmids were immunoprecipitated with anti-Myc (Hsc70) and immunoblotted. Both and WT- (a) and H723R- (b) pendrins strongly interacted with Hsc70 when ER-to-Golgi traffic was blocked by Arf1-Q71L or syntaxin 5 overexpression. (c) Inhibition of Hsc70 ATPase activity by overexpression of Hsc70-K71M (ATPase activity-ablated dominant-negative mutant) inhibited the surface expression of unconventionally transported H723R-pendrin. (d) Hsc70 ATPase activity inhibitor apoptozole dose-dependently reduced Arf1-Q71L-induced surface expression of H723R-pendrin. *P<0.05, ^**P<0.01 by one-way analysis of variance, the number of replicates (n) is presented in each panel. Unprocessed original scans of western blots are shown in Supplementary Fig. 10.

Figure 5. DNAJC14 is necessary for unconventional surface trafficking of H723R-pendrin.

(a) An siRNA screen was performed in PANC-1 cells transfected with plasmids encoding H723R-pendrin and Arf1-Q71L to identify J proteins or nucleotide exchange factors involved in the unconventional transport of H723-pendrin. Cells were treated with siRNA 24 h before transfection with plasmids. Surface biotinylation assays were performed 24 h after plasmid transfection, and the surface-to-lysate ratio was calculated by densitometry. A summary of multiple experiments is shown, and representative immunoblots are presented in Supplementary Fig. 4. ^**P<0.01 by one-way analysis of variance (ANOVA), n=4–7. (b) Treatment with siRNAs targeting DNAJC14 and Hsc70 cooperatively reduced the surface expression of unconventionally transported H723R-pendrin. ^**P<0.01 by one-way ANOVA, difference from Arf1-Q71L alone (lane 2), n=3. (c) Quantitative PCR analyses of DNAJC14 in PANC-1 cells. Arf1-Q71L or thapsigargin did not significantly alter the mRNA levels of DNAJC14. (d) Proteins samples from PANC-1 cells transfected with indicated plasmids were immunoprecipitated with anti-Flag (DNAJC14) and immunoblotted. Arf1-Q71L-mediated ER-to-Golgi blockade induced the association of DNAJC14 with H723R-pendrin. The data are representative of three independent experiments. Unprocessed original scans of western blots are shown in Supplementary Fig. 10.

Figure 6. Knockdown of Hsc70 and DNAJ14 inhibits the cell-surface expression H723R-pendrin induced by Arf1-Q71L.

(a) Cellular localization of pendrin was examined in PANC-1 cells permeabilized with ethanol and acetone. Cells stained for WT- and H723R-pendrin were co-stained for the ER marker protein calnexin. Arrows indicate the cell-surface expression of WT- or H723R-pendrin. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 20 μm. (b,c) Cell-surface expression of WT- and H723R-pendrin was examined in non-permeabilized PANC-1 cells (4% formaldehyde fixation) using antibodies against the extracellular N terminus of pendrin (Abcam, ab66702). The plasmid encoding the ER marker protein ER-yellow fluorescent protein (YFP) was co-transfected. Arf1-Q71L induced the cell-surface expression of H723R-pendrin, which was blocked by siRNAs targeting Hsc70 and DNAJC14. Quantification of the ratio of cells expressing pendrin on the cell surface relative to total cells in five independent experiments is shown in c. Scale bar, 20 μm. ^**P<0.01 by one-way analysis of variance, difference from H723R-Pendrin+Arf1-Q71L alone (lane 3), n=5.

Figure 7. DNAJC14 upregulation restores the cell-surface expression of H723R-pendrin.

(a) The surface expression of WT-pendrin is not affected by upregulation of Hsc70 and DNAJC14 or by inhibition of Hsc70 ATPase activity (Hsc70-K71M) in PANC-1 cells. (b) Overexpression of DNAJC14 alone or with Hsc70 induces the cell-surface expression of core-glycosylated H723R-pendrin in PANC-1 cells. Note that K71M abolished the effect of DNAJC14 to rescue H723R-pendrin at the surface. (c) Expression of BiP was analysed as an indicator of ER stress. Cells treated with thapsigargin were used as a positive control. Overexpression of DNAJC14, GRASP55 or GRASP65 did not increase BiP in PANC-1 cells. (d) The DNAJC14-H471Q mutation, which results in the loss of the Hsc70 ATPase accelerating activity, abolished the ability of DNAJC14 to rescue H723R-pendrin at the surface. Quantitation of multiple experiments is presented under each immunoblot. ^**P<0.01 by one-way analysis of variance, difference from lane 1, the number of replicates (n) is presented in each panel. Unprocessed original scans of western blots are shown in Supplementary Fig. 10.

Figure 8. Rescue of anion exchange activity of H723R-pendrin by DNAJC14.

PANC-1 cells were transfected with the indicated plasmids and Cl⁻/HCO₃⁻ exchange activity was examined. Representative traces are shown in a–d, and a summary of multiple experiments is depicted in e. Overexpression of DNAJC14 alone, but not Hsc70 alone, significantly increased the Cl⁻/HCO₃⁻ exchange activity in H723R-pendrin-expressing PANC-1 cells. ^**P<0.01 by one-way analysis of variance, difference from H723R-pendrin alone (lane 1), the number of replicates (n) is presented in each lane.

Figure 9. DNAJC14 restores cell-surface expression of H723R-pendrin in immunostainings.

The cell-surface expression of wild-type (WT) and H723R pendrins were examined in non-permeabilized PANC-1 cells. The ER marker protein ER-YFP was co-expressed. Representative images are shown in a. Quantitation of results from five independent experiments is shown in b. Note that the Hsc70-K71M and DNAJC14-H471Q mutations strongly inhibited the ability of DNAJC14 to rescue H723R-pendrin at the cell surface. Scale bar, 20 μm. **P<0.01 by one-way analysis of variance, n=5.

Figure 10. Localization of DNAJC14 and Hsc70 in PANC-1 cells.

(a,b) Subcellular localization of Flag-DNAJC14 and Myc-Hsc70 was examined in permeabilized PANC-1 cells. Representative images are shown in a. Quantitation of results from multiple experiments is summarized in b. A high degree of colocalization (an average of 52%) was observed in cells stained doubly with Hsc70 and DNAJC14. The Hsc70-K71M and DNAJC14-H471Q mutations significantly reduced colocalization of Hsc70 and DNAJC14. Scale bar, 20 μm. **P<0.01 by one-way analysis of variance, difference from lane 1, n=15. (c–e) Endogenous DNAJC14 and Rab18 in PANC-1 cells were immunostained. ER-to-Golgi blockade by Arf1-Q71L (c) or Sar1-T39N (d) induced the movement of endogenous DNAJC14 into punctate structures and increased colocalization with Rab18 (d,e). Scale bar, 20 μm. **P<0.01 by paired Student's t-test, n=15.