FIGURE 1.

The loss of Ubc13 activity impairs the accumulation of Lys-63-linked ubiquitin chains during Parkin-mediated mitophagy. A, MEFs retrovirally introduced with GFP-Parkin were treated with Dox to remove Ubc13 genes and then treated with 30 μm CCCP for 6 h. Parkin and mitochondria were visualized with GFP fluorescence (green) and anti-Tom20 (red), respectively. B, the mitochondrial translocation efficiency of Parkin treated as in A was graphed. The values represent the means ± S.E. of the percentages of cells exhibiting mitochondrial recruitment in three independent experiments. The translocation efficiency was similar in Ubc13^+/+ and Ubc13^−/− (3 h, p < 0.8024; 6 h, p < 0.1309 by Student's t test). C, ubiquitin accumulation was detected with anti-polyubiquitin (red) in cells treated as in A. D, accumulation of a Lys-63-linked ubiquitin (K63-Ubiquitin) chain was detected with anti-Lys-63 linkage-specific ubiquitin antibody (red) in cells treated as in A. Scale bars = 10 μm. E, accumulation of Lys-63-linked polyubiquitin (K63-Ub) but not of Lys-48-linked polyubiquitin (K48-Ub) was reduced in the absence of Ubc13 activity. Crude mitochondrial fractions from MEFs expressing GFP-Parkin (1 × 10⁶) treated with (+) or without (−) 30 μm valinomycin for 6 h were prepared. Polyubiquitin purified with TUBE1-agarose in the mitochondrial fractions was detected by Western blot. poly-Ub, polyubiquitin. All experiments were repeated at least three times in A–D and two times in E, and representative results were shown.