Parkin shows minimal ubiquitination activity with an isolated substrate.
(A) Schematic diagram showing the domains of human Miro1 and human CISD1. (B) Schematic diagram showing the minimal target constructs mG2 and CISD1 used in this study. Also indicated are the N-terminal fluorescent labels for mG2, CISD1 (Alexa Fluor 680, red circle) and Ub (DyLight 800, green circle). (C) Model assay demonstrating the expected outcomes of single-fluorescent autoubiquitination (left) and dual-fluorescent substrate (right) assays. (D) Parkin autoubiquitination and minimal substrate ubiquitination were monitored by fluorescently labelled 800Ub (green) and 680mG2 or 680CISD1 (red). Reactions were initiated by addition of Uba1 (E1) and quenched at the required timepoints with 3× sample buffer and DTT.