Substrate recognition by the human mitochondrial processing peptidase and its processing of PINK1

Nuclear-encoded mitochondrial proteins rely on N-terminal targeting sequences (N-MTS) for their import. These N-MTSs interact with the translocation machinery and are cleaved in the matrix by the mitochondrial processing peptidase (MPP), a heterodimeric zinc metalloprotease which is essential for the maturation of imported proteins. Import and cleavage of PINK1, a kinase implicated in Parkinson’s disease, govern its ability to sense mitochondrial damage, but the MPP cleavage site and its role in PINK1’s function remains cryptic. MPP typically cleaves a unique motif in N-MTSs with an arginine in the P2 position, but how MPP recognizes this motif is unclear. Here, we show that recombinant human MPP cleaves PINK1 between Ala28 and Tyr29 yet is turned over slowly compared to other canonical N-MTSs. While MPP cleavage is not required for downstream PARL processing or PINK1 accumulation in cells, the PINK1 N-MTS binds potently to MPP and inhibits the cleavage of other N-MTSs by glueing the regulatory (α) and catalytic (β) subunits. Finally, we utilize hydrogen-deuterium exchange mass spectrometry to reveal the binding site of the PINK1 N-MTS on MPP. Taken together, our work provides key insight into both the PINK1 import pathway and the mechanisms of MPP processing.