Figure 5.

Heterogeneity of the effects displayed by Parkinson's disease-associated point mutations. (upper panel) A schematic of Parkin domain architecture showing the location of disease-associated Parkin mutants. (lower panel) Parkin mutants exhibit diverse effects on E3 ligase activity. Assays using wild-type (WT) and kinase-inactive (KI) PINK1 (D359A) in combination with WT and indicated mutants of Parkin and the substrate Miro1 were undertaken as described in §3. A kinase reaction including 0.1 mM [γ-³²P] ATP (approx. 500 cpm pmol⁻¹) was carried out in parallel for 60 min to confirm the phosphorylation as described in methods. Reactions were terminated after 60 min by addition of SDS loading buffer and resolved by SDS–PAGE. Miro1, Ubiquitin, Parkin and PINK1 were detected using anti-SUMO, anti-FLAG, anti-Parkin and anti-MBP antibodies, respectively. Representative of three independent experiments.