Alteration of Parkin expression in cells affects mitochondrial morphology. A, HeLa cells were transfected with Mito-DsRed together with GFP-Parkin or GFP vector for 24 h, and cells were then stained with DAPI and analyzed by fluorescence microscopy. Cells with a dense cluster of mitochondria were classified as perinuclear mitochondrial clustering. B, experiments were performed as in A except that SH-SY5Y cells were used. C, experiments were performed as in A and B, and the percentage of cells with mitochondrial clustering was quantified. Cells with an intact network of tubular mitochondria were defined as normal, cells with disrupted and predominantly spherical mitochondria were defined as fragmented mitochondria, and cells with a dense cluster of mitochondria were classified as perinuclear mitochondrial clustering. D, Western blot analysis of the expression of Parkin and β-actin in SH-SY5Y cells transfected with two different Parkin siRNAs or a scramble siRNA control was performed. Parkin protein level was determined by dividing the intensity of Parkin with the intensity of β-actin on the blot. E, SH-SY5Y cells were transfected with control or Parkin siRNAs, stained with Mito-Tracker Red and an anti-Parkin antibody, and then analyzed by immunofluorescence microscopy. F, experiments were performed as in E, and the percentage of cells with mitochondrial truncation or fragmentation was quantified. G, experiments were performed as in E, and the length of mitochondria was measured with ImageJ. All immunofluorescence data shown in the bar graphs represent means ± S.E. (error bars) from three independent experiments, with at least 100 cells counted in a blinded manner. *, p < 0.01 and **, p < 0.001.

Parkin interacts with Drp1 in vivo and in vitro. A, 293T cells were transfected with Myc-Parkin or the empty vector (control), and immunoprecipitation (IP) and Western blotting were performed to examine the interaction between Myc-Parkin and endogenous Drp1. B, endogenous Parkin (kD) interacts with endogenous Drp1. SH-SY5Y cells were transfected with Drp1 or control shRNAs. Cell lysates were subjected to immunoprecipitation with an anti-Drp1 antibody or an IgG control, and the immunoprecipitates were examined by Western blotting using an anti-Parkin antibody. The middle and bottom panels show the expression of Drp1 and Parkin in the cell lysates (1/20 input). C, Parkin and Drp1 interact in vitro. In vitro translated Drp1 was incubated with bacterially purified MBP-Parkin or MBP immobilized on MBP beads. The presence of Drp1 in the pulldown preparation was examined by Western blotting. The last lane shows 1/20 input of Drp1. D, various truncated forms of GFP-Parkin are shown as a schematic. E, 293T cells were transfected with Myc-Drp1 together with a series of truncated forms of GFP-Parkin. Immunoprecipitation and Western blotting were performed to examine the critical domains in Parkin that mediate its interaction with Myc-Drp1. All data are representative of at least three independent experiments.

Parkin mutations affect its ability to ubiquitinate Drp1 and to cause Drp1 degradation. A, 293T cells were transfected with HA-UB or HA-UB-K0 together with Myc-Drp1 and various GFP-Parkin constructs as indicated. Cells were treated with MG132 before harvest. Cell lysates were then subjected to immunoprecipitation (IP) and Western blotting to examine the ubiquitination of Drp1. Uniquitinated Drp1 protein level was quantified according to the results of three independent blots. B, in vitro translated Drp1 was incubated with purified ubiquitin, E1, E2 (UbcH7), and various Parkin proteins. The reaction products were analyzed by Western blotting with anti-ubiquitin and anti-Drp1 antibodies. Uniquitinated Drp1 protein level was quantified according to the results of three independent blots. C, cells were transfected with various GFP-Parkin constructs, stained with Mito-Tracker Red and then analyzed by fluorescence microscopy. D, experiments were performed as in C, and the percentage of cells with mitochondrial clustering was quantified. For each experiment, at least 100 cells were counted in three independent experiments. E, cells were transfected with various GFP-Parkin constructs, and Western blotting was performed. Drp1 protein level was quantified according to the results of three independent blots. All immunofluorescence data shown in the bar graphs represent mean ± S.E. (error bars) with at least 100 cells counted in a blinded manner. *, p < 0.01 and **, p < 0.001.

Parkin induces the degradation of Drp1 through the proteasome. A, 293T cells were untransfected or transfected with GFP-Parkin or GFP vector, and Western blotting was performed to examine the expression of Drp1, Mfn1/2, Fis1, β-actin, and GFP proteins. Drp1 protein level was quantified according to the results of three independent blots. B, Western blot analysis of the expression of Parkin, Drp1, Mfn1/2, Fis1 and β-actin in SH-SY5Y cells transfected with control or Parkin siRNAs was performed. Parkin and Drp1 protein levels were quantified according to the results of three independent blots. C, Western blot analysis of the expression of Drp1, β-actin, and GFP proteins in 293T cells untransfected or transfected with GFP-Parkin or GFP vector, in the presence or absence of the proteasome inhibitor MG132, was performed. Drp1 protein level was quantified according to the results of three independent blots. D, 293T cells were untransfected, transfected with GFP vector, or transfected with GFP-Parkin in the presence of various inhibitors, including two proteasome inhibitors (MG132 and PS341), a lysosome inhibitor (chloroquine), or two protease inhibitors (PMSF and pepstain). Western blotting was then performed to examine the expression of Drp1, β-actin, and GFP proteins. E, CHX chase assay for the half-life of Drp1 is shown. 293T cells were transfected with GFP and GFP-Parkin for 24 h. Cells were then treated with CHX (100 μg/ml) for the indicated hours, and Western blotting was performed. The level of remaining Drp1 at different time points was quantified as the percentage of initial Drp1 level (0 h of CHX treatment). All data are representative of at least three independent experiments. *, p < 0.01 and **, p < 0.001.

Parkin promotes the ubiquitination of Drp1 in cells. A, SH-SY5Y cells were transfected with HA-UB together with control or Parkin siRNAs as indicated. Cells were treated with MG132 before harvest. Cell lysates were then subjected to immunoprecipitation (IP) and Western blotting to examine the ubiquitination of Drp1. Uniquitinated Drp1 protein level was quantified according to the results of three independent blots. B, 293T cells were transfected with HA-UB together with GFP-Parkin or GFP vector. Cells were treated with MG132 for 8 h before harvest. Cell lysates were then subjected to immunoprecipitation and Western blotting. Uniquitinated Drp1 protein level was quantified according to the results of three independent blots. C, 293T cells were transfected with various HA-UB constructs together with GFP-Parkin or GFP vector. Cells were treated with MG132 for 8 h before harvest. Cell lysates were then subjected to immunoprecipitation and Western blotting. All Western blot data shown in the bar graphs represent means ± S.E. (error bars) from three independent experiments. *, p < 0.01 and **, p < 0.001.