Fig 2: 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 3: TRAP1 binds both cytosolic and mitochondrial translation elongation factors and slows down elongation rate. (A) Fluorescent confocal microscopy analysis of TRAP1-cy3 (acceptor) and eEF1G-cy2 (donor) in HeLa cells. Dipole-dipole energy transfer from the fluorescent donor to the fluorescent acceptor allowed calculating FRET efficiency (EFRET %) as described in Materials and Methods section. The overlay images show the InSet area in which FRET has been analyzed. Scale bar: 10 µm. tns values are expressed as mean ± SEM. The two-tailed p-value represents the statistical significance based on the Student’s t-test. (B) Fluorescent confocal microscopy analysis of TRAP1-GFP (donor) and EF-TuMT-Cy3 (acceptor) in TRAP1-GFP inducible HeLa cells. Dipole-dipole energy transfer from the fluorescent donor to the fluorescent acceptor allowed calculating FRET efficiency (EFRET %) as described in Materials and Methods section. The overlay images show the InSet area in which FRET has been analyzed. Scale bar: 10 µm. tns values are expressed (C) TRAP1 inhibits EF-Tu release from 70S initiation complex (70SIC) in stopped-flow assays. TRAP1 recombinant protein was preincubated with a Ternary Complex (TC) in which EF-Tu is labeled with the QSY9 fluorescence quencher (QSY-TC, purple trace) and the resulting solution was rapidly mixed with a Cy3-labeled 70S Initiation Complex (Cy3-70SIC, blue trace). The change in Cy3 fluorescence was monitored using a stopped-flow fluorometer. Upon entering in the A site, the quencher-labeled EF-Tu decreases the Cy3-labeled ribosome fluorescence, whereas its dissociation from the ribosome allows Cy3 fluorescence recovery. Black trace, negative control; red trace, positive control. (D) 72 hrs after tet-induction shGFP-directed (control) or TRAP1-directed shRNAs, HeLa cells were treated with harringtonine (2 µg/mL) for the indicated times (0, 1, 2, 3 and 5 min) and subsequently treated with puromycin (10 µg/mL) for 10 min. Cells were lysed and subjected to immunoblotting with anti-puromycin antibody. The graph shows densitometric intensity of the puromycin labeling, normalized on the total protein content (quantified by no-stain labeling, see Methods). Data are represented as mean ± SEM from 6 independent experiments, with trend lines showing exponential one-phase decay analysis.