A mitochondrial FUNDC1-HSC70 interaction organizes the proteostatic stress response at the risk of cell morbidity (2018)
Figure 1

Figure 1 - FUNDC1 interacts with HSC70 and promotes the mitochondrial recruitment of the proteasomal clients

HeLa cells were co-transfected with Myc empty vector (Myc), Myc-tagged FUNDC1 (F1-Myc) or Myc-tagged FUNDC1-ΔLIR mutant (ΔLIR-Myc) and Mito-OM-cherry, which localizes to the mitochondrial outer membrane, and immunostained with fluorescent anti-p62 and anti-Myc antibodies. Scale bar=10 μm.

HeLa cells were co-transfected with Myc, F1-Myc or ΔLIR-Myc and Mito-OM-cherry and immunostained with anti-Ubiquitin (Ub) and anti-Myc antibodies. Scale bar=10 µm.

Fractionation analysis of HeLa cells transfected with Myc, F1-Myc or ΔLIR-Myc. The loading controls (L.C.) were α-Tubulin for total cell lysate (TCL) and cytosolic fraction (Cyto), and HSP60 for mitochondrial fraction (Mito).

HeLa cells transfected with Myc or Myc/His6 double-tagged F1-Myc-His were lysed and precipitated with Ni-NTA beads. Samples were then subjected to SDS-PAGE. The differential band (upper arrow) was isolated and subjected to mass spectral analysis. The identified proteins with a score higher than 50 are listed.

Co-IP of endogenous HSC70 with F1-Myc or ΔLIR-Myc. α-Myc, anti-Myc antibody

Co-IP of HSC70 with FUNDC1. α-F1, anti-FUNDC1 antibody. rIgG, control rabbit IgG.

Co-IP analysis of endogenous FUNDC1 with Flag-HSP70 or Flag-HSC70. α-Flag, anti-Flag antibody. Flag，empty vector.

HeLa cells were co-transfected with Myc, F1-Myc or ΔLIR-Myc and Mito-OM-cherry and immunostained with anti-HSC70 and anti-Myc antibodies. Scale bar=10 µm.

Fractionation analysis of HeLa cells transfected with Myc or F1-Myc.

HeLa cells were co-transfected with Ub-R-GFP and Myc or F1-Myc and immunostained with fluorescent anti-Myc and anti-GFP (α-GFP) antibodies. The α-GFP signals in F1-Myc transfected cells were lowered to avoid the overexposure of the mitochondrially localized R-GFP, making such signals of the equally distributed R-GFP invisible. Green arrowheads show the mitochondrially localized R-GFP proteins, which were more abundant than the equally distributed R-GFP and showed stronger R-GFP fluorescence, white arrowheads show the mitochondrially localized R-GFP proteins without stronger R-GFP fluorescence, although abundant. Scale bar=10 µm.

Fractionation analysis of HeLa cells co-transfected with Ub-R-GFP and Myc or F1-Myc.

HeLa cells were co-transfected with Ub-R-GFP and F1-Myc. Mitochondria were isolated and then treated with or without proteinase K (Pro.K), or proteinase K in combination with digitonin or Triton X-100 (TX-100). Samples were then subjected to Immunoblot analysis. HSP60, TIM23 and TOM20 are representatives of mitochondrial matrix-, mitochondrial inner membrane- and mitochondrial outer membrane-localized proteins, respectively.

Fractionation analysis of HeLa cells stably transfected with control (Ctrl) shRNA vector or HSC70 shRNA and transiently co-transfected with Ub-R-GFP, Flag or Flag-HSC70 (shRNA-resistant) and Myc, or F1-Myc, as indicated.

Data information: In panels C and I, 3 replicates were analyzed using Student's 2-tailed t-test and are presented as the means ± SEM. * p<0.05; ** p<0.01; NS, nonsignificant.