Figure 4.
Figure 4. Phospho-ubiquitin activates Parkin. (A) YFP-ParkinWT translocates to damaged mitochondria in cells expressing high levels of WT HA-Ub (red, HA immunostaining, 10 µM CCCP for 1 h). Cells expressing high levels of HA-UbS65A have impaired YFP-Parkin translocation. Bars, 10 µm. (B) Quantification of A shown as averages ± SD from n = 3 experiments, 100 cells/experiment. (C) Immunoprecipitation of HA-Ub WT, S65D, S65E, and S65A from cells stably expressing YFP-Parkin revealed a stronger interaction between activated YFP-Parkin and HA-UbS65D and HA-UbS65E than WT or S65A. In the absence of HA-Ub, some YFP-Parkin bound to the HA IP beads (†), and this was consistent in all immunoprecipitations. The higher molecular weight form of YFP-Parkin was found only in the HA-Ub and was bound to a much larger extent to the HA-UbS65D and S65E. (D) Quantification of C shown as averages ± SD from n = 3 experiments. (E) ParkinC431S forms an oxyester-bound Ub intermediate upon activation with CCCP, which is cleaved by NaOH (lanes 1 and 2). The oxyester was not detected in cells without CCCP, with overexpressing HA-UbWT (lane 9), but was detected with overexpression of HA-UbS65D for both YFP-ParkinC431S and YFP-ParkinS65A/C431S (lanes 13 and 15). (F and G) HA-Ub was in vitro phosphorylated by (F) incubation with mitochondria from control or CCCP treated cells or (G) recombinant MBP-TcPINK1 WT or KD using the same protocol as used for the Phos-tag gels in Fig. 3 A. This HA-Ub was then added to an in vitro reaction (at 4 ng and 20 ng) with recombinant Parkin, E1, E2, untreated Ub (to a total of 1 µg), and ATP. In both cases the addition of phospho-Ub to the reaction caused an increase in Parkin activity (Ubn). (H) To ensure activation in F was due to S65 phospho-Ub, the experiment was repeated with WT, S65A, and no Ub (−). Activation was only observed with WT His-Ub (top). MBP-TcPINK1 was removed from the His-Ub phosphorylation reaction by binding to amylose beads (bottom). The “†” symbol in G and H represents a nonspecific band. *, P < 0.01; ***, P < 0.001.

Phospho-ubiquitin activates Parkin. (A) YFP-ParkinWT translocates to damaged mitochondria in cells expressing high levels of WT HA-Ub (red, HA immunostaining, 10 µM CCCP for 1 h). Cells expressing high levels of HA-UbS65A have impaired YFP-Parkin translocation. Bars, 10 µm. (B) Quantification of A shown as averages ± SD from n = 3 experiments, 100 cells/experiment. (C) Immunoprecipitation of HA-Ub WT, S65D, S65E, and S65A from cells stably expressing YFP-Parkin revealed a stronger interaction between activated YFP-Parkin and HA-UbS65D and HA-UbS65E than WT or S65A. In the absence of HA-Ub, some YFP-Parkin bound to the HA IP beads (†), and this was consistent in all immunoprecipitations. The higher molecular weight form of YFP-Parkin was found only in the HA-Ub and was bound to a much larger extent to the HA-UbS65D and S65E. (D) Quantification of C shown as averages ± SD from n = 3 experiments. (E) ParkinC431S forms an oxyester-bound Ub intermediate upon activation with CCCP, which is cleaved by NaOH (lanes 1 and 2). The oxyester was not detected in cells without CCCP, with overexpressing HA-UbWT (lane 9), but was detected with overexpression of HA-UbS65D for both YFP-ParkinC431S and YFP-ParkinS65A/C431S (lanes 13 and 15). (F and G) HA-Ub was in vitro phosphorylated by (F) incubation with mitochondria from control or CCCP treated cells or (G) recombinant MBP-TcPINK1 WT or KD using the same protocol as used for the Phos-tag gels in Fig. 3 A. This HA-Ub was then added to an in vitro reaction (at 4 ng and 20 ng) with recombinant Parkin, E1, E2, untreated Ub (to a total of 1 µg), and ATP. In both cases the addition of phospho-Ub to the reaction caused an increase in Parkin activity (Ubn). (H) To ensure activation in F was due to S65 phospho-Ub, the experiment was repeated with WT, S65A, and no Ub (−). Activation was only observed with WT His-Ub (top). MBP-TcPINK1 was removed from the His-Ub phosphorylation reaction by binding to amylose beads (bottom). The “†” symbol in G and H represents a nonspecific band. *, P < 0.01; ***, P < 0.001.

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