Fig. 1. NEDD8 phosphorylated at S65 activates Parkin.

a Protein sequence alignment of Ub and NEDD8. Identical amino acids are indicated by a black background, similar amino acids by a grey background. The serine residue at position 65 is highlighted by a red background. b PhPINK1 phosphorylates NEDD8. NEDD8 or Ub were incubated with PhPINK1 for the times indicated. Reactions were subjected to Phos-tag SDS-PAGE followed by Coomassie blue staining. c pNEDD8 enhances Parkin phosphorylation by PhPINK1. GST-Parkin was incubated with PhPINK1 in the absence (−) or presence of either pUb, pNEDD8, or NEDD8 for the times indicated. Reactions were subjected to Phos-tag SDS-PAGE followed by Coomassie blue staining. *, the reaction in the absence of ATP; **, the reaction in the absence of PhPINK1. d, e Parkin/Parkin H302A autoubiquitylation was performed as described in Methods in the presence or absence of the Ub/NEDD8 variants indicated. The ratios of the different Ub/NEDD8 variants present in the reactions are indicated in percent (in d, 90% corresponds to 35 µM Ub, 10% correspond to 4 µM of the Ub/NEDD8 variants indicated; in e, 100% corresponds to 39 µM of Ub or 30 µM of pUb-His/pNEDD8-His). Reactions were stopped after 60 min and analyzed by SDS-PAGE followed by Western blot analysis with an anti-Ub antibody (upper panels) or by Coomassie blue staining (lower panels). d Levels of the ubiquitylated forms of Parkin (upper panel) and of the non-modified form (lower panel) were quantified with respective levels in the presence of Ub set to 1. The fold change is indicated below the panels. d, e Running positions of molecular mass markers, unmodified Parkin, ubiquitylated forms of Parkin, UBA1, UbcH7, and Ub/NEDD8 variants are indicated. f The different Ub/NEDD8 variants were used as affinity matrices and incubated with GST fusion proteins of Parkin, a Parkin variant devoid of the Ub-like domain (∆Ubl) or the H302A mutant. Upon affinity enrichment, 25% of the respective elution fractions were subjected to SDS-PAGE followed by Western blot analysis with an anti-Parkin antibody. Input represents 5% of the respective Parkin variant. g, h Phosphorylation of S65 of NEDD8 under mitochondrial stress. g Schematic overview of the workflow for the identification of S65-phosphorylated NEDD8 peptides under normal growth conditions (DMSO) or upon mitochondrial stress (CCCP). h 80% of the HA-His-NEDD8 elution fraction were digested with trypsin, subjected to targeted LC-MS/MS and the normalized relative intensity of the pS65-containing NEDD8 peptide plotted. Shown are five biological replicates measured in technical duplicates (1, 2) or triplicates (3-5). See also Supplementary Fig. 1; source data are provided as a Source Data file.