The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy

Abstract

Autophagy-dependent clearance of ubiquitinated and aggregated proteins is critical to protein quality control, but the underlying mechanisms are not well understood. Here, we report the essential role of the BEACH (beige and Chediak-Higashi) and WD40 repeat-containing protein WDR81 in eliminating ubiquitinated proteins through autophagy. WDR81 associates with ubiquitin (Ub)-positive protein foci, and its loss causes accumulation of Ub proteins and the autophagy cargo receptor p62. WDR81 interacts with p62, facilitating recognition of Ub proteins by p62. Furthermore, WDR81 interacts with LC3C through canonical LC3-interacting regions in the BEACH domain, promoting LC3C recruitment to ubiquitinated proteins. Inactivation of LC3C or defective autophagy results in accumulation of Ub protein aggregates enriched for WDR81. In mice, WDR81 inactivation causes accumulation of p62 bodies in cortical and striatal neurons in the brain. These data suggest that WDR81 coordinates p62 and LC3C to facilitate autophagic removal of Ub proteins, and provide important insights into CAMRQ2 syndrome, a WDR81-related developmental disorder.

Figure 1.

WDR81 inactivation causes accumulation of Ub proteins. (A) Colocalization of endogenous WDR81 and Ub proteins in HeLa and SH-SY5Y cells. Insets show magnified (2×) views of the box in the merged images. (B) Colocalization of endogenous WDR81 and Ub proteins (arrows) to large focal structures in HeLa cells treated with 1 µg/ml puromycin or 10 µM MG132 for 12 h. Arrows indicate the enlarged foci costained with WDR81 and Ub. (C) WDR81 siRNA effectively knocks down WDR81. Left: HeLa cells stably expressing GFP-WDR81 were transfected with siRNA (siCtrl) or WDR81 siRNA (si81) oligos. Cells were subjected to Western blot analysis using GFP antibody 48 h after transfection. Right: HeLa cells were treated with siCtrl or siWDR81 and stained with WDR81 antibody 48 h after transfection. (D) Immunostaining of endogenous WDR81 in control and KO-81 HeLa cells. (E and F) Immunostaining (E) and quantification (F) of Ub protein and p62 foci in HeLa and SH-SY5Y cells treated with siCtrl or si81. Arrows indicate the enlarged foci costained with Ub and p62. (G) Immunostaining of Ub proteins and p62 in control and KO-81 HeLa cells. Arrows indicate the enlarged foci costained with Ub and p62. (H) Immunoblotting of Ub proteins in HeLa cells transfected with siCtrl or siWDR81. (I) Immunoblotting of Ub proteins in control or KO-81 HeLa cells. (J) Immunoblotting of p62 in siCtrl and si81 (top) or Ctrl and KO-81 cells (bottom). (K) Immunoblotting of K63-Ub or K48-Ub in control and KO-81 cells. (L) Colocalization of endogenous WDR81 with K63- or K48-linked Ub proteins (K63-Ub or K48-Ub) in HeLa cells treated with 1 µg/ml puromycin or 10 µM MG132 for 12 h. Insets show images of WDR81 foci (green) and K63-Ub or K48-Ub foci (red) indicated by arrows in the main panels. Quantification of Ub proteins and WDR81 is shown in the right panel. ≥40 WDR81 foci were examined for each Ub antibody. Bars: (all images) 10 µm; (insets) 5 µm. For all quantifications, data (mean ± SEM) were from three independent experiments and analyzed using ANOVA. **, P < 0.01; ***, P < 0.001. Fold changes of Ub proteins or p62 (H–K) were normalized to control and are indicated at the bottom.

Figure 2.

WDR81 acts in the autophagy pathway for clearance of Ub proteins. (A) Immunoblotting of Ub proteins in siCtrl or si81 (left) and control or KO-81 (right) HeLa cells treated without or with MG132. (B) Immunoblotting of p62 in siCtrl or siWDR81 (left) and control or KO-81 (right) HeLa cells treated without or with MG132. (C and D) Immunoblotting of Ub proteins (C) and p62 (D) in siCtrl- or si81-treated wild-type (WT) and ATG5^−/− MEF cells. In A–D, fold changes of Ub proteins or p62 were normalized to control and are indicated at the bottom. (E) Immunostaining of WDR81 and Ub proteins in WT and ATG5^−/− MEF cells. Insets in the bottom left corner show magnified (1.8×) views of the box in the merged images. Bars: (all images) 10 µm; (insets) 5 µm. Quantification of WDR81 intensity is shown in the right panel. Data (mean ± SEM) were from three independent experiments and analyzed using unpaired t tests. ***, P < 0.001. (F) Colocalization of BFP-WDR81, Myc-Ub and GFP-LC3B in HeLa cells treated without (Ctrl) or with MG132. In the Ctrl group, insets show magnified (1.8 x) views of the boxed region in the merged image. In the MG132 group, arrows indicate the large foci containing all three tagged proteins. Bars: (main images) 10 µm; (insets) 5 µm. (G) WDR81 is degraded by the autophagy pathway. HeLa cells stably expressing Flag-WDR81 were treated with siAtg5 or siBeclin1. Knockdown efficiency, determined by immunoblotting, is shown in the left panel. The siRNA-induced change in Flag-WDR81 levels is shown in the right panel. (H) Immunoblotting of Flag-WDR81 cells treated with 0.4 µM BFA1 and 10 µM MG132 for the indicated times. In G and H, fold changes of Flag-WDR81 were normalized to control and are indicated at the bottom. The treatment of cells with puromycin or MG132 was performed as in Fig. 1 (except in H).

Figure 3.

WDR81 acts in aggrephagy independently of the endosomal WDR81-WDR91 complex. (A) Colocalization of GFP-WDR81 with mCh-WDR91 in HeLa cells treated with or without puromycin or MG132 (left). Quantification of WDR91-vesicles positive for WDR81 (right). (B) Colocalization of GFP-WDR81 with mCh-Rab7 in HeLa cells treated with or without puromycin or MG132 (left). Arrows indicate WDR81-positive Rab7 vesicles; arrowheads indicate WDR81 foci that do not localize to Rab7 vesicles. Quantification of Rab7-vesicles positive for WDR81 (right). (C) Colocalization of GFP-Rab7 with mCh-WDR91 in HeLa cells treated without or with puromycin or MG132 (left). Quantification of Rab7-vesicles positive for WDR91 (right). (D) Loss of WDR81 or WDR91 does not affect the activity of the Atg14–Beclin1–Vps34 complex. Beclin1 or Atg14L was immunoprecipitated from control (ctrl), KO-81, and KO-91 HeLa cells. Precipitated proteins were detected with antibodies for the indicated proteins (bottom). Equal amounts of precipitated proteins from each genotype were examined for Vps34 activity by measuring the luminescence (relative light units [RLU]) of ADP converted from ATP. Data (mean ± SEM) were from three independent experiments and are normalized to Vps34 activity in Ctrl cells (top). (E) Representative TEM images of control and KO-81 HeLa cells with MG132 treatment. Red arrows indicate cargo-containing autophagic vesicles (AVs) with intact membranes in control HeLa cells. Pink arrows indicate proteinaceous structures with partial membranes in KO-81 cells. Blue arrows indicate non–membrane-enclosed proteinaceous structures in KO-81 cells. Representative structures indicated by red, pink, and blue arrows are magnified at the bottom. (F–H) Quantification of AV numbers (F), non–membrane-enclosed proteinaceous structures (G), and intact or defective AVs observed by TEM (H). The treatment of cells with puromycin or MG132 was performed as in Fig. 1. For all quantifications, data (mean ± SEM) were from three independent experiments and analyzed using ANOVA. ***, P < 0.001; NS, not significant. Bars: (fluorescence images) 10 µm; (TEM images) 5 µm.

Figure 4.

WDR81 interacts with p62. (A) Immunostaining of p62 and WDR81 in wild-type (WT) and ATG5^−/− MEF cells. Insets show a magnified view (1.8×) of the boxed area in the merged images. (B) Images of BFP-WDR81 and GFP-p62 coexpressed in HeLa cells (top row); and images of overexpressed GFP-p62 and immunostained endogenous WDR81 (bottom). Dashed lines indicate the cell outline. (C) Colocalization of BFP-WDR81 with GFP-p62 (green arrows) or mCh-WDR91 (red arrows) in HeLa cells. (D) Colocalization of GFP-tagged WDR81 truncations with mCherry-p62 (mCh-p62) in HeLa cells. Schematic representations of the WDR81 truncations are shown in the top panel; the colocalizations are shown in the bottom panel. (E–G) Co-IP of HA-p62 with Flag-WDR81 (E), of HA-p62 with Flag-WDR81(1–650) (F), and of HA-p62 with Flag-WDR81(WD40) (G) in HEK293 cells. IPs were performed using Flag antibody, and precipitated proteins were detected with the indicated antibodies. (H) Interaction of GST-p62 with Flag-WDR81. Purified GST and GST-p62 (left) immobilized on glutathione-Sepharose beads were incubated for 12–16 h at 4°C with lysates of HEK293 cells expressing Flag-WDR81, Flag-WDR81(1–650), or Flag-WDR81(WD40). After extensive washing, bound proteins were subjected to immunoblotting (IB) with antibodies to Flag (right). (I) In vitro interaction of GST-p62 with ³⁵S-labeled WDR81. (J) Colocalization of GFP-WDR81(1–650) with mCh-tagged p62 truncations in HeLa cells. Schematic representations of the p62 truncations are shown in the top panel and protein colocalizations are shown in the bottom panel. Bars, 10 µm. H, leucine-rich nuclear export signal; L, LC3-interaction region; PB1, Phox and Bem1p domain; UBA, ubiquitin-associated domain; ZZ, ZZ-type zinc-finger domain. (K) In vitro interaction of GST-p62, GST-p62(PB1) with ³⁵S-labeled WDR81(1–650). (L) Co-IP of mCh-tagged p62 truncations with Flag-WDR81(1–650) in HEK293 cells. IPs were performed using Flag antibody, and precipitated proteins were detected with antibodies to Flag and mCherry. Bars: (main images) 10 µm; (A, insets) 5 µm.

Figure 5.

WDR81 promotes the binding of p62 with Ub proteins. (A–C) IP of endogenous p62 in Ctrl or KO-81 cells with anti-p62 antibodies. Coprecipitated Ub proteins were detected with antibodies to Ub (A), K63-Ub (B), and K48-Ub (C). The ratios of Ub proteins/p62 in the precipitates are indicated at the bottom. (D) Immunostaining of p62 and K63-Ub in Ctrl or KO-81 cells treated without or with 10 µM MG132 (12 h). Insets show magnified (1.8×) views of the box in the merged images. Bars: (main images) 10 µm; (insets) 5 µm. Quantification of p62-positive Ub foci is shown in the right panel. Data (mean ± SEM) were derived from three independent experiments and analyzed using ANOVA. *, P < 0.05. (E–G) IP of endogenous p62 in HEK293 cells with or without Flag-WDR81 overexpression. Coprecipitated Ub proteins were detected with antibodies to ubiquitin (E), K63-Ub (F), and K48-Ub (G). The ratios of Ub proteins/p62 in the precipitates are indicated at the bottom. Expression of Flag-WDR81 is shown in the right in E. (H and I) IP of HA-p62 with Myc-Ub(K63) (H) or Myc-Ub(48) (I) proteins with or without coexpression of Flag-WDR81 in HEK293 cells. Coprecipitated Ub proteins and WDR81 were detected with antibodies to Myc or Flag. The ratios of Ub proteins/p62 in the precipitates are indicated at the bottom. (J) Phosphorylation of p62 at Ser 403 in control and KO-81 HeLa cells, and in HEK293 cells with Flag-WDR81 overexpression or BFA1 treatment. Western blot was performed with the indicated antibodies. (K) Flag-WDR81 was expressed in wild-type (p62^+/+) or p62^−/− cells and immunoprecipitated in regular IP buffer or RIPA buffer. Precipitated proteins were detected with antibodies to Flag and Ub. Fold changes of Ub/WDR81 in the precipitates were normalized to control and are indicated at the bottom.

Figure 6.

WDR81 promotes LC3C recruitment to Ub proteins. (A) Immunostaining of LC3 and Ub in Ctrl or KO-81 HeLa cells treated without or with 10 µM MG132 (12 h; left). The LC3 antibody reacts with LC3A, B, and C. Insets show images of LC3 foci (green) and Ub proteins (red) indicated by arrows in the main panels. Bars: (main images) 10 µm; (insets) 5 µm. Quantification of LC3-positive Ub foci (right). Data (mean ± SEM) were derived from three independent experiments. Comparisons were made using ANOVA. **, P < 0.01. (B) Co-IP of Flag-WDR81 with LC3B in HEK293 cells without or with 10 µM MG132 treatment (12 h). IPs were performed with Flag antibody, and precipitated proteins were detected with antibodies to mCherry and Flag. (C) Co-IP of Flag-WDR81 with individual members of the LC3 or GABARAP subfamilies in HEK293 cells. IPs were performed with Flag antibody, and precipitated proteins were detected with antibodies to mCherry and Flag. (D–F) Colocalization of Myc-Ub with GFP-LC3B (D) or GFP-LC3C (E) in Ctrl and KO-81 cells treated without or with 10 µM MG132 (12 h). Insets show images of GFP-LC3B or GFP-LC3C foci (green) and Myc-Ub foci (red) in the boxes in the main panels. Bars: (main images) 10 µm; (insets) 5 µm. Quantification of Myc-Ub foci positive for GFP-LC3B or GFP-LC3C is shown in F. Data (mean ± SEM) were derived from three independent experiments. Comparisons were made using ANOVA. **, P < 0.01. (G) Quantitative RT-PCR analysis of the specificity and efficiency of siLC3C. The mRNA levels of LC3A, LC3B, and LC3C were normalized to siCtrl treatment; error bars represent SEM derived from three independent experiments. Comparisons were made using ANOVA. **, P < 0.01. (H) Western blot analysis of LC3C and LC3A in HeLa cells treated with siCtrl or siLC3C. (I) Colocalization of endogenous Ub proteins with p62 or WDR81 in HeLa cells treated with siCtrl or siLC3C. (J) Quantifications of enlarged Ub protein foci positive for p62 or WDR81 (left) and the intensity of Ub protein or WDR81 staining (right) in HeLa cells treated with siCtrl or siLC3C. ≥100 cells were analyzed for each treatment. Data representing mean ± SEM were derived from three independent experiments. Comparisons were made using ANOVA. **, P < 0.01. (K) Immunoblotting of U proteins (top) and p62 (bottom) in siCtrl- or siLC3C- treated HeLa cells. Fold changes in Ub proteins or p62 were determined by normalizing to the control and are indicated at the bottom. (L) Immunoblotting of Flag-WDR81 that was stably expressed in HeLa cells treated with siCtrl or siLC3C. The fold change in Flag-WDR81 was determined by normalizing to the control and is indicated at the bottom.

Figure 7.

WDR81 interacts with LC3C through two LIRs to facilitate aggrephagy. (A and B) Co-IP of GFP-tagged WDR81 fragments with mCh-LC3C. Individual GFP-WDR81 fragments were coexpressed with mCh-LC3C in HEK293 cells. IPs were performed with mCherry antibody, and precipitated proteins were detected with antibodies to GFP and mCherry. (C) Alignment of canonical and noncanonical LIRs in WDR81 with those in p62, NBR1, FUNDC1, or NDP52. Conserved residues are shown in red. (D) Co-IP of mCh-LC3C with wild-type (WT), LIR, or CLIR mutants of GFP-WDR81(341–650) in HEK293 cells. WDR81 mutations are shown in the box. IPs were performed with mCherry antibody and precipitated proteins were detected with antibodies to GFP and mCherry. (E) Co-IP of mCh-LC3C with WT or the indicated LIR mutants of Flag-WDR81 full-length protein in HEK293 cells. IPs were performed as in A. (F) Pull-down of Flag-WDR81 by GST-LC3C. Purified recombinant GST and GST-LC3C immobilized on glutathione-Sepharose beads were incubated overnight at 4°C with lysates of HEK293 cells expressing Flag-WDR81 or the indicated Flag-WDR81 mutants. After extensive washing, bound proteins were subjected to Western blot with Flag antibody. (G and H) KO-81 HeLa cells were transfected with Flag-WDR81 or the indicated Flag-WDR81 mutants. 48 h later, cells were subjected to immunostaining (G) or immunoblotting (H) with p62 antibody. Quantification (mean ± SEM) of p62 in (G) is shown in the right panel. ***, P < 0.001. Bars, 10 µm. (I) Co-IP of Flag-WDR81 with WT or the indicated hydrophobic pocket mutants of LC3C in HEK293 cells. IPs were performed with Flag antibody, and precipitated proteins were detected with antibodies to Flag and mCherry. (J) Immunoblotting of p62 in siCtrl or siLC3C cells expressing RNAi-resistant LC3C (WT) or the LC3C (K32Q/F33H/L64V/F69) mutant in HEK293 cells. In H and J, fold changes in p62 levels were normalized to the control and are indicated at the bottom.

Figure 8.

WDR81 deficiency causes accumulation of p62-positive aggregates in mouse brain. (A) RT-PCR analysis of WDR81 expression in different mouse tissues. (B) Graphic representation of the scheme for generating WDR81 conditional knockout (WDR81^f/fcKO) mice by crossing WDR81^f/f mice with Nestin-Cre mice. (C) WDR81^f/f cKO mice display perinatal lethality at P0. (D, left) Immunostaining of p62 in brain sections from WDR81^f/f and WDR81^f/f cKO mice at P0 (left). Nuclei are stained with DAPI. Boxed cortex regions in each image of whole brain sections (10x objective, top row) were observed under 60× objective and are shown in the bottom row (bars, 10 µm). (right) p62 intensities in the cortex were quantified and normalized to WDR81^f/f. 20 sections from four mice for each genotype were examined. Data (mean ± SEM) were analyzed using ANOVA. **, P < 0.01; ***, P < 0.001. (E) Immunostaining of p62 and Ub in brain sections from WDR81^f/f and WDR81^f/f cKO mice at P0 (left). Nuclei are stained with DAPI. Insets show magnified (2×) views of the dotted (Ub-positive) or solid (Ub-negative) box in the merged images. Bars: (main images) 10 µm; (insets) 5 µm. Quantification of p62 foci positive or negative for ubiquitin (right). 30 sections from four mice for each genotype were examined. Data (mean ± SEM) were analyzed using ANOVA. ***, P < 0.001. For views of whole-brain sections in D, F, and H, multiple images (10x) were stitched together to generate the composite images. (F) p62 immunostaining and DAPI labeling in brain sections from WDR81^f/f and WDR81^f/f:Nestin-Cre mice at embryonic day 14.5 (left) and 18.5 (right). The indicated cortex regions, F1, F2, F3, and F4, in each image of whole-brain sections (10× objective, top rows) were observed under a 60× objective and are shown in the middle columns (bars, 10 µm). Magnifications (4×) of the boxed regions in F1, F2, F3, and F4 are shown in the right columns (bars, 5 µm). (G) Quantification of p62 foci in brain sections shown in F. 10 sections from two mice were examined for each genotype at each embryonic stage. Data (mean ± SEM) were analyzed using ANOVA. ***, P < 0.001. (H) p62 immunostaining and DAPI labeling in lateral brain sections from WDR81^f/f and WDR81^f/f cKO mice at P180 (left). Boxed regions in the whole brain sections (top) were observed under 60× objective and are shown in the bottom row (bars, 10 µm). H1 and H3 indicate cortex regions. H2 and H4 indicate striatum regions. Right: >500 cells in 10 sections from two mice for each genotype were examined for p62 foci, and data (mean ± SEM) were analyzed using ANOVA. ***, P < 0.001.

Figure 9.

WDR81 is essential for preventing accumulation of p62 foci in neuron cells in mouse brain. (A–C) Immunohistological costaining of WDR81 with NeuN or GFAP in the mouse brain (A). Bars, 10 µm. Quantification of WDR81 expression and intensity in NeuN⁺ or GFAP⁺ cells is shown in B and C. 25 sections from three mice were examined. (D and E) Immunostaining (D) and quantification (E) of p62 and NeuN or GFAP in brain sections from WDR81^f/f (top) and WDR81^f/f cKO (bottom) mice at postnatal day 180. Bars, 10 µm. More than 500 cells in 10 sections from two mice for each genotype were examined for p62 foci. (F) Representative images of WDR81 and DAPI staining in brain cells infected with lentivirus expressing ΔCre-GFP or Cre-GFP (left). Bars, 10 µm. Graphic depiction of lentivirus grafting (right). Lentiviruses expressing ΔCre-GFP or Cre-GFP were grafted into contralateral sides of the same animals. (G and H) Immunostaining (left) and quantification (right) of p62 and NeuN (G) or GFAP (H) in brain sections from WDR81^f/f mice infected by lentivirus expressing ΔCre-GFP or Cre-GFP. The merged images also include DAPI staining. Bars, 10 µm. >300 cells in 16 sections from four mice were examined for each treatment. For all quantifications, data (mean ± SEM) were analyzed using ANOVA, except for data in B and C (t test). **, P < 0.01; ***, P < 0.001; NS, not significant.