Mitochondria are made up of over 1000 proteins, most of which are synthesised on cytosolic ribosomes and then imported as precursor proteins into their destined mitochondrial subcompartment. Precursor proteins enter first via the translocase of the outer membrane (TOM complex) and the majority are then relayed onto the translocase of the inner membrane (TIM23 complex) for insertion into the inner membrane or translocation into the matrix. This import process is essential for mitochondria to maintain the machinery needed for ATP production and other metabolic functions. However, it has remained a decades-long challenge to understand how an imported protein traverses these translocases and how the TOM and TIM23 complexes come together to enable precursor handover.
The ubiquitin kinase PINK1, a protein linked to early onset Parkinson’s disease, is a known substrate of the TOM and TIM23 translocases. In healthy mitochondria, PINK1 is imported into the mitochondrial inner membrane where it is then cleaved and degraded. However, a unique aspect of PINK1 function is its ability to sense mitochondrial damage, upon which it becomes stabilised at the TOM complex, allowing its kinase domain to fold and activate. PINK1 then phosphorylates ubiquitin on the mitochondrial surface, which signals the initiation of mitophagy. We have been able to show that PINK1 can trap the TOM and TIM23 complexes in a super-complex state, and in this talk, I will explain how we are using PINK1 as a proxy to structurally elucidate how precursor proteins are imported into mitochondria.