Proximity labelling of mitochondria and endosomes upon mtDNA replication stress.

This project aims to identify novel regulators of mtDNA turnover, for what we used TurboID. For specific detection of the mitochondria-endosome proteome, we fused the N-terminal part of Turbo ID to RAB5C (TurboID aa1-aa72; RAB5C-SplitTurboNt-V5) and, the C-terminal part to SAMM50 (TurboID aa73–aa246; SAMM50-SplitTurboCt-HA). Proximity biotinylation was performed in transduced HEK293 cells expressing Split TurboID constructs. Cells expressing only SAMM50-SplitC-HA were used as a negative control. For protein isolation, HEK293 cells were incubated for 4 h with 0,25 mM biotin. For inducing mtDNA damage, cells were previously transfected with Twinkle K319E-Cherry 24 h before the biotin incubation.