(A) IB analysis measured ATF6α and WFS1 levels in MIN6 cells expressing shGFP (control) or shWFS1, as well as in MIN6 cells expressing shWFS1 or expressing shWFS1 and rescued with WFS1 (n = 3). (B) IB analysis measuring ATF6α, WFS1, IRE1α, and PERK levels in INS1 832/13 cells (treated with 2 mM DTT for 3 hours) overexpressing GFP (control) or WFS1 (n = 3). (C) Quantitative real-time PCR analysis of BiP, total Xbp-1, Chop, Ero1-α, Glut2, and Ins2 mRNA levels in INS1 832/13 cells overexpressing GFP (control) or WFS1 (n = 3). (D) IB analysis of ATF6α and WFS1 in COS7 cells transfected with ATF6α-HA or ATF6α-HA and WFS1-FLAG at 2 different ratios, and in INS1 832/13 cells expressing inducible WFS1 and treated with or without MG132. (E) IB analysis of ATF6α and WFS1 in MIN6 cells expressing shWFS1 and transfected with WT WFS1-FLAG or mutant P724L WFS1-FLAG and G695V WFS1-FLAG (n = 3). (F) IB analysis measuring ATF6α and WFS1 levels in INS1 832/13 cells expressing WT WFS1 or P724L WFS1 (n = 3). (G) WFS1 was subjected to IP from COS7 cells expressing ATF6α-HA or ATF6α-HA with WT, P724L, or G695V WFS1-Flag using an anti-Flag antibody. IPs and input proteins were analyzed using anti-HA and anti-Flag antibodies. **P < 0.01.

(A) WFS1 was subjected to IP from INS1 832/13 cells using an anti-WFS1 specific antibody. IPs were subjected to IB with anti–alpha 5 20S proteasome and anti-WFS1 antibodies. (B) IB analysis measuring CREB, actin, and PDI levels using whole cell lysates or ER-isolated lysates of INS1 832/13 cells. ER-isolated lysates of INS1 832/13 cells were also subjected to fractionation using a 10%–40% glycerol gradient. Fractions were analyzed by IB using anti–alpha 5 20s proteosome, anti-ATF6α, and anti-WFS1 antibodies. Lanes were run on separate gels and were not contiguous. (C) WFS1 was subjected to IP from a mixture of fractions 10 and 11 using an anti-WFS1 antibody, and IP products were subjected to IB analysis using anti-alpha 5 20s proteosome, anti-ATF6α, and anti-WFS1 antibodies. ATF6 was subjected to IP from a mixture of fractions 9 and 12, and IP products were analyzed by IB with anti–alpha 5 20s proteosome and anti-ATF6α (n = 3).

(A) HRD1 was subjected to IP from INS1 832/13 cells treated for 3 hours with 30 μM MG132. IPs and input proteins were analyzed by IB using anti-ATF6α and anti-HRD1 antibodies. Lanes were run on the same gel but were noncontiguous (white line). (B) An anti-HRD1 antibody was used to IP HRD1 protein from INS1 832/13 cells untreated or treated with DTT (1 mM) and thapsigargin (Tg; 1 μM) for 3 hours. IPs were then subjected to IB analysis using anti-ATF6α, anti-HRD1, and anti-actin antibodies (n = 3). (C) IB analysis measuring ATF6α levels in MIN6 cells stably expressing shGFP (control) or shRNA shHRD1 treated with 40 μM cycloheximide for 0, 4, and 6 hours (n = 3). (D) COS7 cells transfected with ATF6α-HA expression plasmid (control) or ATF6α-HA together with Hrd1-myc expression plasmids (Hrd1) were treated with 40 μM cycloheximide for 0, 4, and 6 hours. Whole cell lysates were subjected to IB with an anti-HA antibody (n = 3). (E) ATF6α was subjected to IP using an anti-ATF6α antibody from INS1 832/13 cells either mock transfected (control) or transfected with a Hrd1-Myc expression plasmid and treated with MG132 (20 μM) for 3 hours. IPs were then subjected to IB with anti-ubiquitin and anti-ATF6α antibodies, and input lysates were blotted with anti-ATF6α, anti–c-Myc, and anti-actin antibodies (n = 3). (F) ATF6 was subjected to IP using an anti-ATF6α antibody from MIN6 cells stably expressing shGFP (control) or shHRD1 and treated with MG132 (20 μM) for 3 hours. IPs were then subjected to IB with anti-ubiquitin and anti-ATF6α antibodies, and input lysates were blotted with anti-HRD1, anti-ATF6α, and anti-actin antibodies (n = 3).

(A) COS7 cells were transfected with a full-length ATF6α expression plasmid or ΔATF6α with a WFS1 plasmid together with the following luciferase reporter genes: ATF6α binding site reporter gene ATF6GL3, ATF6α mutant site reporter ATF6m1GL3, and GRP78 promoter reporter gene ERSE. Relative intensity of luciferase was then measured (n = 3). (B) Protein lysates from the luciferase assay were analyzed by IB using anti-HA (ATF6α), anti-Flag (WFS1), and anti-actin antibodies. ATF6α and ΔATF6α are denoted by single and double asterisks, respectively. (C) COS7 cells were transfected with a full-length ATF6α expression plasmid with a BiP expression plasmid, WFS1 expression plasmid, or WFS1 and BiP expression plasmid together with the GRP78 reporter gene (n = 3). (D) An anti-WFS1 antibody was used to IP WFS1 protein from INS1 832/13 cells untreated (UT) or treated with the ER stress inducer DTT (1 mM) for 0.5, 1.5, or 3 hours. IPs were then subject to IB analysis using anti-ATF6α, anti-WFS1, and anti-actin antibodies (n = 3). (E) INS1 832/13 cells were treated with DTT (1 mM) for 2 hours and then chased in normal media for 0, 1, or 2 hours. WFS1 was subjected to IP from cell lysates, and IPs were analyzed by IB using anti-ATF6α, anti-WFS1, and anti-actin antibodies (n = 3).

(A) IB analysis measuring ATF6α, WFS1, and actin levels in MIN6 cells stably expressing shGFP (control) or shWFS1 treated with 40 μM cycloheximide (CX) for 0, 2, and 4 hours (n = 3). (B) IB analysis measuring ATF6α, WFS1, and actin levels in INS1 832/13 cells expressing GFP (control) or WFS1 treated with 40 μM cycloheximide for 0, 2, and 6 hours (n = 3). (C) ATF6α was subjected to IP using an anti-ATF6α antibody from an INS1 832/13 cells inducibly expressing shWFS1 (treated for 48 hours with 2 μM doxycycline) and treated with MG132 (20 μM) for 3 hours. IPs were then subjected to IB with anti-ubiquitin and anti-ATF6α antibodies, and input lysates were blotted with anti-ATF6α, anti-WFS1, and anti-actin antibodies (n = 3). (D) ATF6α was subjected to IP using an anti-ATF6α antibody, from INS1 832/13 cells overexpressing GFP (control) or WFS1, then treated with MG132 (0.1 μM) overnight. IPs were subjected to IB with anti-ubiquitin and anti-ATF6α antibodies. Input lysates were subjected to IB with anti-ATF6α, anti-WFS1, and anti-actin antibodies (n = 3). (E) Wfs1^–/– and WT littermate mouse pancreata were analyzed by immunohistochemistry using anti-ATF6α and anti-insulin antibodies. Scale bars: 50 μm.

(A) Hrd1 was subjected to IP from INS1 832/13 cells, and IPs were subjected to IB analysis using anti-WFS1 and anti-Hrd1 antibodies (n = 3). (B) Total lysates from INS1 832/13 cells inducibly expressing shWFS1 (treated with 2 μM doxycycline for 48 hours) were analyzed by IB using anti-WFS1, anti-Hrd1, and anti-actin antibodies (n = 3). (C) IB analysis measuring HRD1 levels in MIN6 cells stably expressing shGFP (control) or shWFS1 treated with 40 μM cycloheximide for 0, 0.5, 1, and 2 hours (n = 3). (D) Wfs1^–/– and WT littermate mouse pancreata were analyzed by immunohistochemistry using anti-HRD1 and anti-insulin antibodies (n = 3). Scale bars: 100 μm. (E) COS7 cells were transfected with pcDNA3, HRD1–c-Myc, HRD1–c-Myc and WT WFS1, or HRD1–c-Myc and WFS1 mutants (P724L, G695V, and ins483fs/ter544) expression plasmids. Expression levels of HRD1–c-Myc, WFS1, and actin were measured by IB using anti–c-Myc, anti-WFS1, and anti-actin antibodies, respectively. WT and mutant WFS1 are denoted by single and double asterisks, respectively. (F) COS7 cells were transfected with pcDNA3, HRD1–c-Myc, HRD1–c-Myc and WT WFS1-Flag, HRD1–c-Myc and WFS P724L-Flag, and HRD1–c-Myc and WFS1 G695V-Flag expression plasmids. The lysates were subjected to IP with anti-Flag antibody and IB with anti–c-Myc antibody to study the interaction between HRD1 and WFS1.

(A) In normal cells, WFS1 recruits the ER transcription factor ATF6α to the E3 ligase Hrd1 under non–ER stress conditions. Hrd1 marks ATF6α with ubiquitin for proteasomal degradation. Under ER stress, ATF6α dissociates from WFS1 and undergoes proteolysis, and its soluble aminoportion, p60ATF6α, translocates to the nucleus, where it upregulates ER stress target genes, such as BiP, CHOP, and XBP-1. At later time points, WFS1 is induced by ER stress, which causes the eventual degradation of ATF6α. (B) In patients with Wolfram syndrome or Wfs1^–/– mice, ATF6α escapes from the proteasome-dependent degradation, leading to chronic hyperactivation of ATF6α signaling. This ATF6α hyperactivation is involved in apoptosis through apoptotic effectors of the UPR, such as CHOP.