HeLa shGFP and shTRAP1 cells were seeded in four 15 cm plates. L-Homopropargylglycine (HPG – Thermo Scientific) labeling was performed as follows. Following silencing of TRAP1 for 72 hrs upon induction with 1 μg/mL tetracycline, cells were incubated in cysteine/methionine-free medium (Sigma-Aldrich) for 45 min at 37°C and then treated with 100 μg/mL of cycloheximide and 100 μg/mL of emetine for 15 min at 37°C to inhibit cytosolic translation. Subsequently, 100 μM of HPG was added to the media for 2 hrs at 37°C. After harvesting the cells, mitochondria were isolated by differential centrifugation, as previously described (23 ). Freshly isolated mitochondria were resuspended in 50 μl of click-it lysis buffer (50 mM TRIS-HCl pH 8, 1% SDS, 250 U/mL Universal Nuclease [Thermo Fisher]) and incubated on ice for 15 min. Mitochondrial extracts were centrifuged at 18’000 rcf for 5 min and protein concentrations were determined by BCA assay (Thermo Fisher). 180 μg of mitochondrial proteins were subjected to a click reaction using a commercial kit (Click-iT Cell Reaction Buffer Kit; Thermo Fisher), with 40 μM Tetramethylrhodamine (TAMRA)-azide (Sigma). According to the manufacturer’s protocol, proteins were purified from the mixture using a MeOH/chloroform approach, after the end of the click reaction. The extracted pellet was dissolved in 20 ul of click-it lysis buffer containing 3% SDS and protein concentrations were determined by BCA assay. Equal amounts of proteins were loaded on a 15% Tris-Glycine gel. Fluorescent signals in the gel were analyzed using a Chemidoc MP imaging system (Bio-Rad). Total protein levels were detected using the No-Stain Protein Labeling Reagent (Thermo Scientific). Click chemistry and immunostaining of fixed cells have been performed as follows. After HPG labeling, coverslips were fixed in 4% paraformaldehyde and pre-permeabilized with a 0.0005% digitonin solution. Then cells were fully permeabilized with 0.1% Triton X-100 for 5 min at room temperature and subsequently incubated with the click reaction buffer for 30 min according to the Clicki-T Cell Reaction Buffer Kit (Thermo Fisher Scientific). After washing with 2% BSA in PBS solution, mitochondria were immunostained with anti-TOM20 primary antibody (Santa Cruz Biotechnology, sc-17764) and then cells were subsequently labelled with Alexa Fluor488-conjugated secondary antibody (Invitrogen). The coverslips were mounted using mowiol, and images were acquired using confocal microscope Zeiss LSM700.