Differential effects of ubiquitinPhospho−Ser65 and UblPhospho−Ser65 on ubiquitin discharge by full-length wild-type, S65A and ΔUbl-Parkin
(A) UbiquitinPhospho−Ser65 leads to activation of full-length wild-type, S65A and ΔUbl-Parkin and increased ubiquitin discharge. An E2-discharge assay was established by incubation of full-length (WT), S65A or ΔUbl-Parkin in the presence or absence of phospho- or non-phospho-ubiquitin (1 μg) as indicated with 2 μg of UbcH7 that had been pre-incubated with E1 and FLAG–ubiquitin in the presence of Mg2+-ATP for 60 min. Reactions were allowed to continue for 15 min and were stopped using SDS loading buffer in the absence of reducing agent. The reaction products were resolved using SDS/PAGE and the proteins were visualised by Colloidal Coomassie Blue staining. (B) UblPhospho−Ser65 leads to activation and increased ubiquitin discharge by ΔUbl-Parkin, but does not affect the full-length wild-type and S65A Parkin. The assays were carried out as described above, but phospho- and non-phospho-Ubl were added to the reactions as indicated. P-ubiquitin, ubiquitinPhospho−Ser65; P-Ubl, UblPhospho−Ser65. The molecular mass in kDa is indicated.