(A) Mitochondrial targeting of the Parkin RBR domain generates polyUb chain at the mitochondria in an E3 activity-dependent manner. Mitochondrial Ub chains (green) and mitochondria (red) were visualized with anti-polyUb (FK2) and anti-Tom20, respectively, in HeLa cells transfected with Tom70MTS-RBR or Tom70MTS-RBR C431S. The polyUb and Tom20 (mitochondria) signals are also shown as monochrome images. (B) Tom70MTS-RBR synthesizes both K48 and K63 Ub linkages. HeLa cells transfected with Tom70MTS or Tom70MTS-RBR along with 3xMyc-Parkin C431S were treated as in Fig. 2. Mitochondrial Ub chains (green) were visualized with anti-polyUb (FK2), anti-K48-linkeage specific (K48) and anti-K63-linkeage specific (K63) polyUb. Parkin C431S was visualized with anti-Myc (red). (C) HeLa cells transfected with Tom70MTS or Tom70MTS-RBR along with 3xMyc-Parkin C431S were treated with or without 10 µM valinomycin for 2 hr. 3xMyc-Parkin C431S, polyUb and the mitochondria were visualized with anti-Myc (green), anti-polyUb (red), and anti-Tom20 (blue), respectively. (D, E) Mitochondrial phospho-polyUb generated by Tom70MTS-RBR accelerates Parkin translocation. HeLa cells transfected with 3xMyc-Parkin WT along with E3 active Tom70MTS-RBR WT or E3-dead Tom70MTS-RBR C431S were treated with 10 µM valinomycin for 40 min. (D) Parkin (green) and mitochondria (red) were visualized with anti-Myc and anti-Tom20, respectively. Each signal is also shown as monochrome images. (E) Quantification of the mitochondrial translocation efficiency of Parkin as in (D). The graph shows the means ±SEM of the percentages of cells exhibiting mitochondrial recruitment in three independent experiments, with ∼100 anti-Myc staining-positive cells counted per sample. ** p<0.01 (two-tailed unpaired Student's t-test). Scale bars = 10 µm in (A–D).