PINK1 Is Selectively Stabilized on Impaired Mitochondria to Activate Parkin
Figure 3
Parkin recruitment to depolarized mitochondria requires PINK1 and its mitochondrial targeting N-terminus.
(A) Primary MEFs from PINK1+/+ or PINK1−/− mice cotransfected with YFP-Parkin (green) and the indicated construct (vector, PINK1-V5, PINK1 kinase-deficient [KD]-V5, or PINK1 156–581 [ΔN]-V5) in a 1∶4 ratio were treated with DMSO or 20 µM CCCP in serum for 3 h. Mitochondria were immunostained for Tom20 (red). The images in the column on the right, which are merged images of the middle and left-hand columns, are expansions of the boxed regions in the middle column. (B) Colocalization between YFP-Parkin and mitochondria in (A) was scored for ≥100 cells/condition in three or more independent experiments. (C) Transformed MEFs from independently generated PINK1+/+ and PINK1−/− mice were transfected and treated as in (A) and were scored as in (B). (D) M17 human neuroblastoma cells stably transduced with control shRNA or PINK1 shRNA were treated with 10 µM CCCP in serum for 3 h and imaged as in (A). (E) Colocalization between YFP-Parkin and mitochondria in (D) was scored as described in (B). (F) Control shRNA and PINK1 shRNA M17 cells transfected and treated as in (D) were fractionated into mitochondria-rich membrane fraction (Memb) and supernatant (Sup). Fractions were run on SDS gels and immunoblotted with anti-Parkin and anti-VDAC antibodies. Loading was adjusted for approximately equal concentrations of YFP-Parkin in the postnuclear supernatants (PNS) between the two cell types. Scale bars in (A and D) represent 10 µm. Error bars in (B, C, and E) indicate standard deviation.