Fig 2: 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.

Fig 3: TRAP1 is coexpressed with the mitochondrial translational machinery. (A) Network analysis of the top 100 TRAP1-coexpressed genes, generated by STRING using a Markov Cluster (MCL) algorithm. Network nodes represent proteins; edges represent protein-protein associations, by co-expression (black line), experimentally-determined association (pink line), or database-curated association (blue line). Edges between clusters are represented by dotted lines. The red cluster is enriched in mitochondrial electron transport components; pink and yellow clusters are constituted by structural component of the mitochondrial ribosome (small and large subunit, respectively); the green cluster contains the mitochondrial prohibitin complex; the purple cluster is constituted by the association between the mitochondria protein import inner membrane translocase subunit PAM16 and the mitochondrial-processing peptidase subunit alpha PMPCA. (B) Gene set enrichment analysis on the list of genes significantly coexpressed with TRAP1 in human tissues. (C) Co-expression analysis performed with COXPRESdb between TRAP1 and EF-TuMT (gene name: TUFM). (D) Co-expression analysis between TRAP1 and EF-TuMT at protein level according to the Provisional database. (E) Co-expression analysis performed with COXPRESdb between TRAP1 and Cluh.

Fig 4: TRAP1 is coexpressed with the mitochondrial translational machinery. (A) Network analysis of the top 100 TRAP1-coexpressed genes, generated by STRING using a Markov cluster (MCL) algorithm. Network nodes represent proteins; edges represent protein–protein associations, by coexpression (black line), experimentally determined association (pink line), or database-curated association (blue line). Edges between clusters are represented by dotted lines. The red cluster is enriched in mitochondrial electron transport components; pink and yellow clusters are constituted by structural component of the mitochondrial ribosome (small and large subunit, respectively); the green cluster contains the mitochondrial prohibitin complex; and the purple cluster is constituted by the association between the mitochondria protein import inner membrane translocase subunit PAM16 and the mitochondrial-processing peptidase subunit alpha PMPCA. (B) Gene set enrichment analysis on the list of genes significantly coexpressed with TRAP1 in human tissues. (C) Coexpression analysis performed with COXPRESdb between TRAP1 and TUFM. (D) Coexpression analysis between TRAP1 and TUFM at protein level according to the Provisional database. (E) Coexpression analysis performed with COXPRESdb between TRAP1 and CLUH.