Full-length wild-type Parkin is activated by ubiquitinPhospho−Ser65, and ΔUbl-Parkin is activated by ubiquitinPhospho−Ser65 and UblPhospho−Ser65
(A) Full-length wild-type (WT) and ΔUbl-Parkin are activated by ubiquitinPhospho−Ser65. A 2 μg amount of full-length (WT), S65A or ΔUbl-Parkin was incubated with the ubiquitylation assay components (E1 and UbcH7) in the presence of 0.05 mM ubiquitin. The ubiquitin used in these assays was a mixture of FLAG–ubiquitin and untagged phospho- or non-phospho-ubiquitin as indicated (total of 25 μg per reaction). Reactions were terminated after 60 min by the addition of SDS loading buffer and products were analysed by SDS/PAGE. Miro1, ubiquitin and Parkin were detected using anti-SUMO, anti-FLAG and anti-Parkin antibodies respectively. Formation of short, intermediate and long polyubiquitylated species is indicated by square brackets. P-ubiquitin, ubiquitinPhospho−Ser65. (B) UblPhospho−Ser65 activates ΔUbl-Parkin, but not full-length Parkin. Reactions were carried out as described in (A), but the 0.05 mM FLAG–ubiquitin was used in all reactions and phospho- or non-phospho-Ubl domain (Parkin residues 1–76) was added as indicated. P-Ubl, UblPhospho−Ser65. The molecular mass in kDa is indicated.