Figure 4.

Parkin can interact with multiple different E2 conjugating enzymes to catalyse Miro1 ubiquitylation with or without free ubiquitin chain formation. An E2 scan of 25 different E2 conjugating enzymes was undertaken. Two micrograms of wild-type Parkin was incubated with 1 μg of wild-type (WT) or kinase-inactive (KI) (D359A) MBP-TcPINK in a kinase reaction for 60 min as described in §3. Activated Parkin was then added into pre-assembled ubiquitylation reactions containing 1 μg of the E2 conjugating enzyme as indicated. Reactions were terminated after 60 min by addition of SDS–PAGE loading buffer and resolved by SDS–PAGE. Miro1, ubiquitin and PINK1 were detected by immunoblotting using anti-SUMO, anti-FLAG and anti-MBP antibodies, respectively.