HSP60 interacts with FUS, mediating FUS mitochondrial localization.

<p>(A) FUS-HSP60 interaction was detected by co-immunoprecipitation assay. Western blotting (WB) was performed using corresponding specific antibodies following immunoprecipitation of cell lysates with anti-GFP.(B)FUS-HSP60 interaction was detected by co-immunoprecipitation assay in both mitochondrial and cytosolic fractions. WB was performed using corresponding specific antibodies following immunoprecipitation of mitochondrial or cytosolic fractions with an anti-Myc monoclonal antibody. It should be noted that the protein concentrations of purified mitochondrial fraction relative to the cytosolic fraction is 10 to 1 and that approximately 5% of total FUS protein is localized to mitochondria.(C) GST pull-down experiments using purified FUS and HSP60 proteins indicate that the full-length (FL) Wt- or P525L-mutant FUS or the carboxyl terminal fragment (aa371-526) of FUS protein interacts with HSP60. GST pull-down proteins were analyzed by WB using the anti-HSP60 antibody. The lines below the FUS protein domain diagram depict different truncation mutants of FUS as GST-tagged proteins, with the corresponding amino acid residues shown. The lower panel of the gel image shows Coommassie blue staining of corresponding purified GST-FUS fusion proteins used. (D) Detection of direct FUS-HSP60 interaction by cross-linking coupled immunoprecipitation in live cells expressing the P525L-mutant FUS as a 6XHis-Myc tagged protein. The 130kDa cross-linked FUS-HSP60 species was detected by WB using the anti-HSP60 antibody in the pull-down proteins (“pull down”) following tandem-affinity purification using His-resins and anti-Myc beads (lane 2) or using anti-FUS in the “pull down” proteins following further immunoaffinity purification by anti-HSP60 (lane 8). Briefly, following 16-hour tetracycline induction of FUS expression, the control orP525L-mutant FUS expressing cells were treated with a cross-linking agent, formaldehyde (1%) for 10 min. FUS proteins were tandem-affinity purified using His-resin and anti-myc antibody and then analyzed by WB. A band was detected by anti-HSP60 antibody at 130-kDa in the pull-down fraction as marked by the arrow, whereas multiple bands including 60- and 130-kDa were detected in the input cross-linked cell lysates (input). HSP60 and the tagged FUS were detected at a size of 60 and 70 kDa, respectively, consistent with the prediction that the HSP60 containing 130kDa band consists of HSP60 and 6XHis-Myc tagged FUS. (E) Down-regulating HSP60 leads to a reduction in FUS mitochondrial localization. HEK293 cells were transfected with the control or HSP60-specific siRNAs and harvested for mitochondrial purification 72-hr post-transfection. The mitochondrial purity was confirmed by the enrichment of mitochondrial TOM20 and the absence of cytoplasmic protein such as RhoA or nuclear protein PCNA. Triplicates of the experiments are shown. (F) Quantification of the HSP60 levels in the total cell extracts and in the mitochondrial fractions. (G) Quantification of FUS levels in total cell extracts and the mitochondrial fractions. All data represent 3 independent experiments with statistical analyses using one-way ANOVA with Bonferronipost-test (*: p<0.05; **: p<0.01).