The PARKIN E3 UB ligase is mutated in Parkinson’s disease (PD) and controls mitochondrial autophagy. PARKIN functions via a so-called RING-HECT hybrid mechanism wherein a catalytic Cys residue in the RING2 domain receives UB from an E2 and transfers it to substrate. Here we use quantitative proteomics and live-cell imaging to dissect individual steps in the PINK1 kinase-PARKIN UB ligase mitochondrial control pathway abnormal in Parkinson’s disease.
PARKIN activation by PINK1 produces UB chains on mitochondria, and PARKIN-dependent chain assembly is required for accumulation of poly-phospho-UB on mitochondria. A ten-plex tandem mass tagging quantitative proteomic experiment revealed an increase of 3-fold in p-S65 UB at 30 min after depolarization in PINK1+/+ mouse embryonic ﬁbroblasts (MEFs).
PINK1 promotes PARKIN association with poly-UB chains by phosphorylating both PARKIN and poly-UB.
UB can undergo chain extension on seven lysines and the N-terminal methionine, with different fates for different linkage types. PARKIN promotes the synthesis of canonical and non-canonical poly-UB chains on depolarized mitochondria in a manner that depends on its catalytic Cys residue and S65 within its UBL domain.
To summary, we take the power of proteomics to analysis individual steps in complex phosphorylation-driven UB cascades. It will be useful for understanding signaling-ubiquitylation pathways in the future.