Figure 1.
Parkin mediates self ubiquitination in the presence or absence of E2.
(A) In vitro ubiquitination reaction products generated by MBP-parkin, C441R or IBR-R2 in the absence of E1, E2 or parkin (i.e. “-E3”), or in the presence of all three components (Full) were subjected to immunoblotting with anti-parkin, anti-FK1 and anti-FK2, as indicated. Notice the ladders of immunoreactivities observed in MBP-parkin and IBR-R2-catalyzed reactions but not in C441R-containing reactions. (B) Reaction products generated by His-tagged parkin (His-parkin) purified from insect cells in the presence or absence of UbcH7 at various time points were subjected to immunoblotting with anti-parkin, anti-FK2 and anti-E1, as indicated. Arrows indicate His-parkin. (C) Reaction products generated by MBP-parkin or IBR-R2 in the absence of E2 at various time points were subjected to immunoblotting with anti-parkin, anti-FK1 and anti-FK2. Control refers to full reactions. (D & E) MBP-parkin autoubiquitination assay was performed in the presence of different doses of E1 (i.e. 1.6, 5 or 10 µg/ml), in the presence or absence of E2 (as indicated) and visualized by means of anti-parkin and anti-FK2 immunoblotting.
Figure 2.
E2-independent activity is specific to parkin and is dependent on RING2 integrity.
(A) In vitro ubiquitination reaction products generated by MBP-TRAF6, Rma or Momo in the absence of E1, E2 or E3, or in the presence of all three components including their respective cognate E2 (Full) were subjected to immunoblotting with anti-MBP and anti-FK2, as indicated. Unlike parkin, none of these E3 ligase could catalyze E2-independent ubiquitination. (B) Reaction products generated by disease-associated MBP-parkin K211N and G430D mutants in the absence of E1, E2 or E3, or in the presence of all three components (Full) were subjected to immunoblotting with anti-parkin and anti-FK2, as indicated. Reaction products catalyzed by wild type MBP-parkin in the presence or absence of UbcH7 were immunoblotted alongside for comparison.
Figure 3.
MS analysis of reaction products catalyzed by MBP-parkin or IBR-R2.
(A) CBB-stained gel showing the reaction products produced by MBP-parkin or IBR-R2 under different conditions, as indicated. (B) Schematic diagrams of b and y ion assignments of K48- and K63-linkage ubiquitin peptides (C-E) MS/MS analysis of K48- or K63-linked ubiquitin chains catalyzed by IBR-R2. Precursor peaks estimated as K48- and K63-linkages were subsequently confirmed by MS/MS analysis.
Table 1.
Summary of MS/MS results.
Figure 4.
N-terminal region of parkin represses its intrinsic polyubquitination activity.
(A) Schematic depiction of full length parkin protein and the deletion mutants Δ152 and Δ237. (B) In vitro ubiquitination reaction products generated by MBP-parkin, Δ152, Δ237 or IBR-R2 in the absence of E2 were subjected to immunoblotting with anti-parkin, anti-FK1 and anti-FK2, as indicated. (C) As in (B) except that reactions were conducted in the presence of UbcH7.
Figure 5.
Parkin S/T-D phospho-mimetic mutants display similar ubiquitination profile as the wild type protein.
(A) Schematic depiction of full length parkin protein with the position of known S/T phosphorylation sites indicated by arrows (B) In vitro ubiquitination reaction products generated by MBP-parkin, MBP-parkin T175D or IBR-R2 in the presence of UbcH7 or Ubc13 were subjected to immunoblotting with anti-parkin, anti-FK1 and anti-FK2, as indicated. (C) In vitro ubiquitination reaction products generated by various parkin phospomimetic mutants in the absence or presence of E2 (UbcH7) were subjected to immunoblotting with anti-parkin and anti-FK1.
Figure 6.
IBR-R2 but not full length parkin promotes synphilin-1ubiquitination.
In vitro ubiquitination reaction was carried out with synphilin-1 as a substrate in the absence of parkin (i.e. –E3) or in the presence of MBP-parkin (FL) or IBR-R2 in combination with either UbcH7, Ubc13 or no E2 (as indicated). Parkin-mediated ubiquitination of synphilin-1 was analyzed by means of anti-synphilin-1 immunoblotting (top panel) whereas parkin-mediated self ubiquitination was analyzed by means of anti-parkin immunoblotting (bottom panel).