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

Area of Science:

• Mitochondrial biology
• Protein import and processing
• Neurodegenerative disease mechanisms

Background:

• Nuclear-encoded mitochondrial proteins require N-terminal targeting sequences (N-MTS) for import.
• Mitochondrial processing peptidase (MPP) cleaves N-MTSs, a crucial step for protein maturation.
• Phosphatase and tensin homolog-induced putative kinase 1 (PINK1) is implicated in Parkinson's disease and its processing is linked to mitochondrial damage sensing.

Purpose of the Study:

• To elucidate the mitochondrial processing peptidase (MPP) cleavage site on phosphatase and tensin homolog-induced putative kinase 1 (PINK1).
• To investigate the role of MPP in PINK1 processing and signaling.
• To understand the substrate recognition mechanisms of human MPP.

Main Methods:

• Biochemical assays to define the MPP cleavage site on PINK1.
• Cellular assays to assess the functional relevance of MPP cleavage in PINK1 processing and function.
• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map substrate binding sites on MPP.

Main Results:

• The MPP cleavage site on PINK1 was identified between Ala28 and Tyr29, with inefficient processing compared to canonical N-MTSs.
• MPP cleavage of PINK1 is dispensable for subsequent PARL processing and PINK1's role in mitochondrial damage sensing in cells.
• In vitro, the PINK1 N-MTS potently binds MPP, inhibits other substrates, and reveals a two-step binding mechanism involving MPPα lid rearrangement.

Conclusions:

• PINK1 import and mitochondrial damage sensing are decoupled from N-MTS removal by MPP.
• PINK1 acts as a mechanistic probe to reveal fundamental aspects of MPP substrate recognition and processing.
• This study provides critical insights into the unique import pathway of PINK1 and the molecular mechanisms of MPP.