Figure 7. PINK1-dependent phosphorylation of PARKIN’s UBL domain promotes association with PINK1-phosphorylated poly-UB chains.

(A) Scheme for examining phosphorylation-dependent binding of PARKIN to UB chains.

(B) Stoichiometry of PINK1Tc-dependent phosphorylation of K48 and K63 UB^(2–7) chains used in panel C and D measured by AQUA proteomics.

(C) Phosphorylation dependent binding of PARKIN to phosphorylated UB chains. p-WT and p-S65A PARKIN were incubated with the indicated poly-UB chains as shown in panel A and UB chains captured with Ni-NTA beads prior to immunoblotting. The p-WT PARKIN used in this experiment was 99% phosphorylated on S65 as determined by AQUA (Figure S5C).

(D) Binding of a panel of PD patient and structure-based mutants of PARKIN to poly-p-UB chains. PARKIN proteins were phosphorylated with PINK1Tc, the PINK1Tc removed, and the proteins incubated with K48 and K63 UB^(2–7) chains previously phosphorylated with PINK1Tc and the PINK1Tc removed (panel A). UB chains were captured and association with PARKIN examined by immunoblotting.

(E) Phosphorylation of WT PARKIN and PARKIN^C431S on S65 in response to depolarization was measured by AQUA proteomics.

(F) Absolute levels of UB and p-S65 UB on mitochondria from HeLa Flp-In T-REx cells expressing WT PARKIN or PARKIN^C431S, in the presence or absence of depolarization measured by AQUA proteomics (derived from primary data in Figure 1K).

(G) A feed forward model for PARKIN activation and retention on the mitochondrial surface in response to depolarization. See text for details.

(H) Effect of p-UB (99% phosphorylated) on UB chain synthesis by p-WT PARKIN (99% phosphorylated). The total UB concentration was 30 µM.