A small molecule probe elucidates the role of mitochondrial translocase TIMM44 in PINK1/Parkin regulated mitophagy

Neurodegenerative diseases have been linked to a dysfunctional mitochondrial quality control system that is partially maintained by proteins PINK1 and Parkin. Whereas mitophagy pathways are becoming well-characterized, less is known about the molecular mechanisms of PINK1 trafficking in mitochondria. Accordingly, we have used a small molecule probe (MitoBloCK-10/MB-10) that modulates the activity of TIMM44, an essential component of the protein associated motor (PAM) complex for the mitochondrial inner membrane (TIM23) translocase, to characterize PINK1 import. MB-10 did not inhibit the import or degradation of PINK1 in energized mitochondria. However, when mitophagy was induced by the addition of an uncoupler or respiratory inhibitor, MB-10 treatment altered PINK1 trafficking by inhibiting association with the TOM complex and impairing Parkin recruitment and subsequent mitophagy. MB-10 analogs that did not inhibit TIMM44 activity failed to impair mitophagy, thereby assigning specificity to MB-10. Because PINK1 undergoes lateral release from the TIM23 translocon to interact with inner membrane (IM) modulators, our studies support that TIMM44 may be a key regulator and that the PAM complex has a central role in regulating PINK1-dependent mitophagy. Our studies also provide a probe for dissecting PINK1/Parkin events for mitochondria as well as studying PINK1-dependent mitophagy in cell and animal models. ### Competing Interest Statement The authors have declared no competing interest. * CCCP : carbonyl cyanide 3-chlorophenylhydrazone IM : inner membrane IMS : intermembrane space MitoBloCK/MB : Mitochondrial protein import blockers from the Carla Koehler laboratory MTS : mitochondrial targeting sequence OM : outer membrane OMS : outer mitochondrial membrane localization signal PAM : protein associated motor PINK1 : PTEN-induced putative kinase 1 SAR : structure-activity relationship TIM : translocase of inner membrane TOM : translocase of outer membrane