As an electron shuttle in the respiratory chain, the haemoprotein Cytochrome c plays a vital role in mitochondrial bioenergetics. In response to cellular stress, mitochondrial outer membrane permeabilisation (MOMP) releases Cytochrome c from the intermembrane space into the cytosol, where it interacts with APAF‑1 to activate apoptotic caspases and signal the demise of the cell. However, loss of mitochondrial membrane integrity below the lethal threshold has wide-ranging implications for different cell fates, including inflammatory phenotypes, senescence, and malignant transformation.
We observed that Cytochrome c released during MOMP is degraded in both primary and transformed cell lines with different kinetics. By employing small molecule inhibitors, this turnover was found to be mediated by the ubiquitin-proteasome system (UPS) in a p97/VCP-dependent manner. In SV40-transformed mouse embryonic fibroblasts (MEFs), degradation of Cytochrome c post-MOMP was unaffected by APAF‑1 deficiency. Moreover, direct ubiquitination of endogenous Cytochrome c in BH3-mimetic treated MEFs was detectable in both ubiquitin (TUBE) and Cytochrome c (IP) pulldown experiments, with several lysine residues on Cytochrome c identified as ubiquitination sites in LC-MS/MS analysis. However, in these cells, deletion of Cul9/Parc, an E3 ubiquitin ligase previously reported to target Cytochrome c, did not prevent Cytochrome c ubiquitination or degradation, implicating alternative E3 ubiquitin ligases.
Using complementary approaches including intracellular crosslinking, proximity labelling, and genetic screening, we aim to identify the E3 ubiquitin ligases regulating Cytochrome c degradation after MOMP and determine the physiological relevance of this turnover as a key cell fate decision point between apoptosis and alternative MOMP-induced phenotypes.