Skip to main content
. 2011 Jan 21;12(2):149–156. doi: 10.1038/embor.2010.203

Figure 2.

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 SODG85R-GFP, along with the indicated FLAG-tagged BAG3 constructs (shown in A) and HA-tagged Hsp70 (HA-Hsp70). SODG85R-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) SODG85R-GFP are shown. Scale bar, 10 μm. (C) HEK cells transfected with SODG85R-GFP, BAG3 and Hsp70 were additionally transfected with FLAG-p50, as indicated. Diagrams show the percentages of cells with pre-aggresomal and aggresomal SODG85R-GFP. Right panel: representative image showing pre-aggresomal localization of FLAG-p50 and SODG85R-GFP. Scale bar, 5 μm. (D) GST pull-down analysis as in (A), but in HEK cells transfected for 18 h with SODWT-GFP or mutant SODG85R-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 SODG85R-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 SODG85R-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 SODG85R-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; SODWT, wild-type SOD.