Fig 1: TRAP1 associates with the mitochondrial protein import machinery and favors localized translation. (A) Representative images of PLA positivity between TRAP1 and TOM40. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue), mitochondria are marked by the mitochondria-directed YFP (green). Scale bar: 10 µm. The graph shows the average number of PLA spots/cell, with a two-tailed p-value representing the statistical significance based on the Student’s t-test (n=5). (B) Immunoprecipitation of unfused GFP and TRAP1-GFP performed in HeLa cells following 24 hrs induction of GFP and TRAP1-GFP. Total lysates were incubated with GFP-trap beads to isolate the proteins and the resulting samples were immunoblotted with indicated antibodies. (C) Representative image of PLA showing the interaction of TRAP1 with EF-TuMT in HCT116 cells. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). Negative control has been obtained by hybridizing cells with TRAP1 antibody only. (D-E) Immunoprecipitation of unfused GFP and TRAP1-GFP performed in HeLa cells following 24 hrs induction of GFP and TRAP1-GFP. Where indicated, cells were treated for 15 min with emetine (100 µg /mL) or harringtonine (2 µg/mL), or for 1 hr with chloramphenicol (200 µg /mL) or Linezolid (30 µM). Total lysates were incubated with GFP-trap beads to isolate the proteins and the resulting samples were immunoblotted with indicated antibodies. (F) Representative image of PLA showing the interaction of TOM20 with phosphorylated (active) ribosomal protein eS6 in HeLa cells following 24hrs induction of TRAP1-GFP or unfused GFP. Positive signals of interaction are shown as red dots, nuclei are stained with DAPI (blue). Scale bar: 10 µm. The graph shows the average number of PLA spots/cell, with a p-value representing the statistical significance based on the Student’s t-test (n=3). (G) Cytosolic, MAM, ER, fractions isolated from HCT116 cells probed with the indicated antibodies.