Figure 3.

Parkin ubiquitylates Miro1 at multiple sites in a PINK1-dependent manner. (a) A schematic of Miro1 domain architecture showing the identified ubiquitylation sites and truncation site (red dotted line) used in this paper. (b) Identification of Lys¹⁵³, Lys²³⁰, Lys²³⁵, Lys³³⁰ and Lys⁵⁷² ubiquitylation sites on Miro1. Ubiquitylation assays using wild-type (WT) and kinase-inactive (KI) PINK1 (D359A) in combination with WT Parkin and the substrate Miro1 were undertaken as described in §3. The SDS–PAGE bands were subjected to in-gel tryptic digestion and analysis by LTQ-Orbitrap mass spectrometry. Ubiquitin isopeptides were identified by Mascot (www.matrixscience.com), and spectra were manually validated to ensure peptide fragmentation gave good sequence coverage (*417 ppm error equates to a −1.81 ppm error around the C13 isotope). (c) Sequence alignment of residues around Lys¹⁵³, Lys²³⁰ and Lys²³⁵ (left), Lys³³⁰ and Lys⁵⁷² (right), respectively, in human Miro1 and a variety of lower organisms, showing high degree of conservation.