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. 2014 Aug 15;127(16):3488–3504. doi: 10.1242/jcs.147520

Fig. 7.

Fig. 7.

UBE2D, UBE2L3 and UBE2N are utilized by Parkin during mitophagy. (A) HeLa cells were transfected with Flag–Parkin C431S upon knockdown of UBE2 enzymes. Cells were treated with CCCP for 4 h and harvested. Western blotting was used to monitor the formation of a Parkin-C431S–ubiquitin oxyester (indicated by an arrow), which appears as a band shift and is sensitive to NaOH treatment. Densitometric analysis of western blots is shown in B. Knockdown of UBE2D2/3 significantly reduced the formation of the Parkin C431S–ubiquitin oxyester. Consistent with the stronger effects of two siRNAs, combined knockdown of UBE2D2/3 and UBE2L3 reduced the formation, similar to siRNA against PINK1. Silencing of UBE2N had no effect on oxyester formation (n≧6, one-way ANOVA, Tukey's post-hoc, P<0.0001, F = 9.911). (C) Parental HeLa cells and HeLa cells that stably overexpressed 3×Flag-tagged Parkin wild type (WT) or C431S were left untreated or were treated with 10 µM CCCP for 2 h. Cells were lysed in low pH MES buffer and subjected to SDS-PAGE under reducing and non-reducing conditions. The resulting western blots were probed with antibodies against endogenous UBE2 enzymes to monitor unmodified UBE2 enzymes (closed arrowheads) and 8-kDa shifted ‘charged’ ubiquitin-bound UBE2 enzymes (open arrowheads). HeLa 3×Flag–Parkin WT cells showed a decrease of ubiquitin ‘charged’ species for UBE2D3, UBE2L3 and UBE2N. By contrast, parental HeLa cells and HeLa cells that expressed non-functional C431S Parkin did not show altered thioester levels upon treatment with CCCP. No obvious changes in this ubiquitin-discharge were found for the other E2 enzymes analyzed. (D) Co-immunoprecipitation of Parkin with UBE2 enzymes after treatment with CCCP. HeLa cells that stably expressed 3×FLAG–Parkin wild type were transfected with Myc-tagged E2 enzymes (Myc–UBE2D3, Myc–UBE2L3, Myc–UBE2N) or an empty vector control (Ø). Cells were left untreated or treated with CCCP for 3 h followed by immunoprecipitation for Myc. Upon CCCP treatment, Parkin specifically and strongly co-immunoprecipitated with Myc–UBE2L3 and Myc–UBE2N. ***P<0.0005; ns, not significant.