Figure 5. Monoubiquitination of USP30 by Parkin does not affect its catalytic activity.

a, In vitro reconstitution of USP30^c13 ubiquitination by phosphorylated Parkin, visualised by western blotting for USP30. Active USP30 can auto-deubiquitinate in trans. b, USP30 ubiquitination sites found by mass spectrometry (Supplementary Data Table 2) match annotated sites in cell lines²⁹,³¹ and cluster at the tip of the Fingers subdomain. c, Active USP30^c13 was modified by Parkin with ubiquitin F4R that prevents auto-deubiquitination. Free and modified forms were separated by ion-exchange chromatography, quantified by densitometry and their activities determined in ubiquitin-KG-TAMRA assays (triplicate measurements, values represent mean with standard error). See also Supplementary Fig. 10. Uncropped images for gels are shown in Supplementary Data Set 1.