Extended Data Figure 7: The Ile44 patch is essential for PINK1-mediated phosphorylation of Ub and PARKIN Ubl.
From: Mechanism of phospho-ubiquitin-induced PARKIN activation
All assays were performed three times with consistent results. a, b, Coomassie-stained PhosTag gels comparing the phosphorylation of (a) the HsPARKIN Ubl domain (amino acids 1–72) and (b) ubiquitin. In both cases, the wild-type form is compared with the I44A mutant form of the protein. GST–TcPINK1 does not efficiently phosphorylate the I44A mutants of ubiquitin or of the HsPARKIN Ubl domain. This is important since the Ile44 patch in the PARKIN Ubl domain is inaccessible and binds to RING1 in the structure of full-length RnPARKIN (PDB 4K95 (ref. 16)) (see Fig. 4a). c, d, Controls for Fig. 4c. Coomassie-stained gel with proteins labelled (c), and full-size blot (d) for Fig. 4c. Molecular weight markers are in kDa for all panels.
