Thanks to you and the structural biology platform at @umontreal.ca !

Deeply grateful to NSERC for funding this work, as well as the Canada Research Chair program and CFI, which helped create and sustain the SPR-MS Facility and HDX-MS platform. 7/7

What does it mean for PINK1's function? Mutation of the MPP cleavage site in PINK1 does not affect accumulation on damaged mitochondria, nor processing by PARL under steady-state. However, a.a. 2-34, which binds to MPP, is required for sensing loss of membrane potential. 6/n

Thus, for MPP to turn over multiple substrates, the alpha and beta subunits must dissociate. Structural modelling shows how MPPalpha binding guides (or misguides in the case of PINK1) the cleavage site of the MTS in the catalytic subunit. 5/n

Using mass photometry and hydrogen-deuterium exchange mass spectrometry, we show that the PINK1 MTS acts like a molecular glue by interacting extensively with the regulatory MPPalpha subunit and with the catalytic MPPbeta subunit. 4/n

Intriguingly, we found that while MPP cleaves PINK1's MTS between A28-Y29, it cleaves it much more slowly than other substrates. Using a FRET-based cleavage assay based on MDH2, we find that the PINK1-MTS is a potent (IC50 ca. 500 nM) competitive inhibitor of MPP. 3/n

We first established a bacterial expression system for human MPP alpha and beta. The enzyme is active in vitro and readily cleaves mitochondrial targeting sequence (MTS) from multiple mitochondrial proteins. 2/n