Objective: To disect the role of macroautophagy in PINK1-/Parkin-dependent removal of depolarized mitochondria.

Background: The ubiquitin ligase Parkin and mitochondrial kinase PINK1 function together in the clearance of damaged mitochondria. Upon mitochondrial depolarization, Parkin translocates to mitochondria in a PINK1-dependent manner to ubiquitinate outer mitochondrial membrane proteins. According to the current model, the ubiquitin- and LC3-binding adaptor protein p62 is recruited to mitochondria, followed by their selective degradation through autophagy (mitophagy). Although these findings are highly reproducible across the >70 published studies, in order to see effects, they are based on an artificial experimental set-up using overexpression of Parkin and the use of agents inducing complete loss of the mitochondrial membrane potential. For the latter, the field has been employing almost exclusively one type of respiratory chain uncoupler, i.e. CCCP or FCCP.

Methods: We here further extend these investigations by applying another depolarizing agent, the potassium ionophore Valinomycin in PINK1-deficient human fibroblasts and isogenic neuroblastoma cell lines generated by CRISPR/Cas9.

Results: Although identical to CCCP/FCCP in terms of dissipating the mitochondrial membrane potential and triggering complete removal of mitochondria, Valinomycin did not induce macroautophagy as evidenced by lack of LC3 conversion to its autophagy-related form. Moreover, FCCP-induced conversion of LC3 occurred even in mitophagy-incompetent, PINK1-deficient cell lines. While both stressors required a functional ubiquitin proteasome system (UPS), the removal of depolarized mitochondria persisted in cells depleted of LC3.

Conclusions: We here propose an alternative mechanism of PINK1-/Parkin-dependent mitochondrial clearance, i.e. ‘mitolysis’, by which the UPS destabilizes mitochondrial membranes that subsequently disrupt due to intramitochondrial pressure triggered by the uncouplers and followed by lysosomal-mediated removal of mitochondrial debris.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/pink1-dependent-clearance-of-depolarized-mitochondria-is-driven-by-the-ups-and-can-occur-independently-of-macroautophagy/