BAG3 mediates chaperone-based aggresome-targeting and selective autophagy of misfolded proteins

Abstract

Increasing evidence indicates the existence of selective autophagy pathways, but the manner in which substrates are recognized and targeted to the autophagy system is poorly understood. One strategy is transport of a particular substrate to the aggresome, a perinuclear compartment with high autophagic activity. In this paper, we identify a new cellular pathway that uses the specificity of heat-shock protein 70 (Hsp70) to misfolded proteins as the basis for aggresome-targeting and autophagic degradation. This pathway is regulated by the stress-induced co-chaperone Bcl-2-associated athanogene 3 (BAG3), which interacts with the microtubule-motor dynein and selectively directs Hsp70 substrates to the motor and thereby to the aggresome. Notably, aggresome-targeting by BAG3 is distinct from previously described mechanisms, as it does not depend on substrate ubiquitination.

Figure 1

BAG3 facilitates aggresome formation. (A) Upper panel: immunoblot analysis of COS7 cells treated for 12 h with MG132 (25 μM) and vinblastine (25 μM). Lower panel: real-time PCR analysis showing gene expression changes in COS7 cells following MG132 exposure (25 μM, 12 h). (B,C) Immunofluorescence staining of indicated proteins in COS7 cells treated as in (A). Scale bar, 20 μm. (D) COS7 cells were transfected for 24 h with nonsense or bag3 siRNA and treated with MG132 (25 μM, 12 h). Aggresomes were visualized by ubiquitin immunostaining (supplementary Fig S1D online). Diagram shows the percentage of aggresome-positive cells. (E) Aggresome quantification as in supplementary Fig S1D online from cells analysed in (H). Images are shown in supplementary Fig S1G online. Diagram shows the percentage of aggresome-positive cells at 4, 24 and 48 h after MG132 wash out. (F) Cells were treated as in (H); however, 4 h after MG132 wash out, BafA1 (5 μM) was added for 20 h to inhibit aggresome clearance. Diagrams show the percentage of aggresome-positive cells and the mean size of aggresomes (supplementary Fig S1H online). Images are shown in supplementary Fig S1G online. (G) Dot-blot (upper panel, quantification in supplementary Fig S1B online) and western blot (lower panel) analyses of 0.5% NP40-soluble and -insoluble proteins from cells shown in Fig 1D; supplementary Fig S1D online. (H) COS7 cells transfected with BAG3 or empty vector were treated with either MG132 (25 μM, 18 h) or DMSO as a control. After MG132 wash out, cells were allowed to recover for 4, 24 and 48 h and 0.5% NP40-soluble and -insoluble proteins were analysed by immunoblotting. (I) COS7 cells were transfected for 24 h with GST or GST fused to DIC (GST-DIC) and treated with MG132 (25 μM, 12 h). Indicated proteins in whole-cell lysates and in precipitates obtained by GST pull-down with glutathione beads were analysed by immunoblotting. BafA1, bafilomycin A1; ctrl, control; DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione-S-transferase; Hsp70, heat-shock protein 70; nons, nonsense; siRNA, short-interfering RNA; vinblast, vinblastine.

Figure 2

BAG3 specifically directs Hsp70 substrates to the aggresome by loading misfolded proteins on to dynein. (A) GST pull-down analysis as in Fig 1I, but performed in cells transfected for 36 h with full-length FLAG-BAG3 (I) or the BAG3 deletion mutants FLAG–(Δ421–498)–BAG3 (II), FLAG–BAG-domain (III), and FLAG–PxxP–BAG-domain (IV). Asterisk, nonspecific band. (B) HEK cells were transfected for 18 h with SOD^G85R-GFP, along with the indicated FLAG-tagged BAG3 constructs (shown in A) and HA-tagged Hsp70 (HA-Hsp70). SOD^G85R-GFP showed three distinct localization patterns: cytoplasmic, pre-aggresomal and aggresomal (see representative pictures on the right and supplementary Fig S2F online). The percentages of cells bearing pre-aggresomal (upper panel) and aggresomal (lower panel) SOD^G85R-GFP are shown. Scale bar, 10 μm. (C) HEK cells transfected with SOD^G85R-GFP, BAG3 and Hsp70 were additionally transfected with FLAG-p50, as indicated. Diagrams show the percentages of cells with pre-aggresomal and aggresomal SOD^G85R-GFP. Right panel: representative image showing pre-aggresomal localization of FLAG-p50 and SOD^G85R-GFP. Scale bar, 5 μm. (D) GST pull-down analysis as in (A), but in HEK cells transfected for 18 h with SOD^WT-GFP or mutant SOD^G85R-GFP, along with FLAG-BAG3. (E) HEK cells were transfected with the indicated plasmids with nonsense or bag3 siRNA. At 6 h after transfection, lactacystin (10 μM) or DMSO as vehicle control was added for 12 h and GST pull-down analysis was performed as in (D). (F) Upper panel: sucrose-density gradient analysis of extracts from HEK cells transfected with SOD^G85R-GFP and GST-DIC, along with FLAG-BAG3 or vector control. Note the immunoreactive bands in high-density fraction 14 on BAG3 overexpression (arrow). Migration of a 20S marker protein is indicated. Lower panel: GST-DIC pull-down analyses of dynein complexes present in sucrose fraction 6 and 14. (G) GST pull-down analysis of SOD^G85R-GFP as in (D) but in cells transfected with FLAG-tagged BAG3 constructs (I–IV; indicated as in A). (H) GST pull-down analysis of SOD^G85R-GFP as in (D) but in cells treated with PYR41 (10 μM; 12 h), as indicated. DIC, dynein intermediate chain; DMSO, dimethyl sulphoxide; GST, glutathione-S-transferase; HA, haemagglutinin; Hsp70, heat-shock protein 70; NS, not significant; nons, nonsense; PxxP, proline-rich repeat; siRNA, short-interfering RNA; SOD, superoxide dismutase; SOD^WT, wild-type SOD.

Figure 3

Aggresome-targeted proteins are degraded by autophagy. (A) Analysis of SOD^G85R-GFP (green) localization with aggresome marker vimentin, the autophagic receptors p62 and NBR1, the autophagosome marker LC3 and the lysosomal marker LAMP2 (all in red) in BAG3/Hsp70-transfected HEK cells. Representative merged images of cells with cytoplasmic, pre-aggresomal and aggresomal SOD distribution are shown. Arrows indicate LC3-positive autophagosomes. Scale bar, 5 μm. (B) Transmission electron microscope analysis of SOD^G85R-GFP aggresome in cells transfected as in (A). Marked area in (a) is shown magnified in (b). Arrows, autophagosomes. Arrowheads, autolysosomes. Scale bar, 1 μm. (C) HEK cells transfected for 8 h with SOD^G85R-GFP along with FLAG-BAG3 and HA-Hsp70, as indicated, were treated for 24 h with BafA1 (0.1 μM). Indicated proteins were analysed by immunoblotting. Diagram shows the ratio of SOD^G85R-GFP levels in BafA1-treated cells to those in untreated cells. (D) Same analysis as in (C) but with SOD^WT-GFP. (E) Similar analysis as in (C) but in cells transfected 24 h before with nonsense or atg7 siRNA. (F) HEK cells were transfected with SOD^G85R-GFP and indicated FLAG-tagged BAG3 constructs together with HA-Hsp70, and lysosomal degradation of SOD^G85R-GFP was examined as in (C). Corresponding immunoblots are shown in supplementary Fig S3F online. Ag, aggresome; BafA1, bafilomycin A1; GFP, green fluorescent protein; HA, haemagglutinin; Hsp70, heat-shock protein 70; N, nucleus; NS, not significant; nons, nonsense; PxxP, proline-rich repeat; siRNA, short interfering RNA; SOD, superoxide dismutase; SOD^WT, wild-type SOD.

Figure 4

BAG3 associates with aggresomes in vivo. (A) Spinal cord sections of mice overexpressing SOD^WT and SOD^G85R (disease end-stage) were immunostained for BAG3 and SOD1. Arrows indicate SOD1- and BAG3-positive aggregates in the neuropil. Scale bar, 20 μm. (B) Localization analysis of BAG3 and SOD^G85R in surviving spinal cord motor neurons from SOD^G85R mice at the time of disease symptom onset. Arrows indicate perinuclear aggresomes containing BAG3 and SOD^G85R. Scale bar, 10 μm. Magnific., magnification; SOD, superoxide dismutase; SOD^WT, wild-type SOD.