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. 2013 Jul;23(7):886-97.
doi: 10.1038/cr.2013.66. Epub 2013 May 14.

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

Xinde Zheng  1 , Tony Hunter
Affiliations

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

Xinde Zheng et al. Cell Res. 2013 Jul.

Abstract

Pink1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pink1 accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-linked chain ubiquitination of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine∼ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin's in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteine-mediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine (Parkin C431S) traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-linked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin translocation is achieved through a novel catalytic activity coupled mechanism.

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Figures

Figure 1
Figure 1
Mutation of the putative catalytic cysteine to serine in the Parkin RBR domain blocks mitochondrial translocation and leads to ubiquitin trapping. (A) Mutation of the putative catalytic cysteine 431 to serine in the Parkin RBR domain abrogated its mitochondrial translocation. HeLa cells transfected with Flag-tagged Parkin WT or Parkin C431S were treated with CCCP. The cells were stained with anti-Flag (green), anti-Hsp60 (red) and Hoechst 33342 (blue). Scale bars, 10 μm. The graph corresponds to means ± SD of percent of cells exhibiting mitochondrial recruitment in three independent experiments, with 100 anti-Flag staining-positive cells being counted per sample. (B) Interaction between Pink1 and Parkin was not affected by C431S mutation. HeLa cells were transfected with C-terminally HA-tagged Pink1ΔMTS (mitochondrial translocation signal) and Flag-Parkin WT or Flag-Parkin C431S. Parkins were immunoprecipitated using anti-Flag M2 antibody conjugated beads; the immunoprecipitated Parkin was detected with anti-Flag antibody (rabbit) and the co-precipitated Pink1ΔMTS-HA was detected with anti-HA antibody (rat) by immunoblotting. An extra higher molecular weight species, ∼8 kDa above the main band, was detected for Parkin C431S but not Parkin WT as indicated by arrows. (C) Co-expression of Pink1ΔMTS-HA or CCCP treatment led to an extra higher molecular weight band of Parkin C431S. HeLa cells were transfected with Pink1ΔMTS-HA and Flag-Parkin C431S or Flag-Parkin C431S alone. HeLa cells transfected with Flag-Parkin C431S were treated with CCCP for 3 h or untreated. Flag-Parkin C431S was detected with anti-Flag antibody (mouse) by immunoblotting. (D) The extra band in Parkin C431S was serine dependent. HeLa cells were transfected with Flag-tagged Parkin WT, Parkin C431S or Parkin C431A, and then treated with CCCP for 3 h. Flag-Parkins were detected by immunoblotting using anti-Flag antibody. (E) The extra band of Parkin C431S is caused by serine-trapped ubiquitin. HeLa cells were transfected with HA-ubiquitin and Flag-Parkin WT or Flag-Parkin C431S. Flag-Parkins were immunoprecipitated by anti-Flag M2 antibody conjugated beads. The co-precipitated HA-ubiquitin conjugates were detected by immunoblotting with anti-HA rat antibody (left panel). The immunoprecipitated Flag-Parkin C431S containing the serine-trapped ubiquitin was treated with sodium hydroxylamine as described in Materials and Methods, and detected by anti-Flag rabbit antibody (right panel). (F) Pink1 was essential for ubiquitin trap of Parkin C431S. Pink1-null and WT mouse fibroblasts were transfected with Flag-tagged Parkin WT or Parkin C431S, and treated with CCCP for 3 h. Parkin was detected by immunoblotting with anti-Flag antibody.
Figure 2
Figure 2
Co-expression of WT Parkin restores Parkin C431S mitochondrial translocation.(A) Parkin C431S localizes to damaged mitochondria when co-expressed with WT Parkin. HeLa cells transfected with N-terminal GFP-Parkin C431S and Flag-Parkin WT, and then treated with CCCP. The cells were stained with anti-Flag antibody (red), anti-Hsp60 antibody (white) and Hoechst 33342 (blue). The relative levels of GFP- and Flag-tagged Parkin were determined by immunoblotting using anti-Parkin antibody; untransfected HeLa cells were used as control. The blot was also probed by anti-GFP and anti-Flag antibody, respectively. (B) A chimeric fusion of Pink1ΔKinase and Parkin RBR domain was sufficient to restore Parkin C431S mitochondrial localization. HeLa cells were transfected with GFP-Parkin C431S and Pink1ΔKinase-Flag-RBR WT or Pink1ΔKinase-Flag-RBR C431S, and then treated with CCCP and stained with anti-Flag antibody (red), anti-Hsp60 antibody (white) and Hoechst 33342 (blue). Scale bars, 10 μm. The graph shows means ± SD of percent of cells showing mitchondrial recruitment in three independent experiments, with 100 anti-Flag staining- and GFP-positive cells being counted per sample.
Figure 3
Figure 3
Parkin binds K63-linked ubiquitin chains. (A) Recombinant Parkin can pull down K63-linked polyubiquitin chains. GST-p62UBA was used as a positive control for K63-linked chain binding. The bound K63-linked ubiquitin chains were detected by anti-ubiquitin antibody (left panel); K48-linked ubiquitin chains were detected by anti-ubiquitin chain antibody (right panel). The amount of recombinant proteins was determined by Ponceau S staining. (B) Expression of a chimeric Pink1 whose kinase domain was replaced by a linear ubiquitin chain comprising four copies of G76V can restore Parkin C431S mitochondria localization. HeLa cells were transfected with GFP-Parkin C431S and Pink1ΔKinase-Flag-1×ubiquitinG76V or Pink1ΔKinase-Flag-4×ubiquitinG76V, and then treated with CCCP. The cells were stained with anti-Flag antibody (red), anti-Hsp60 antibody (white) and Hoechst 33342 (blue). Scale bars, 10 μm. The graph shows means ± SD of percent of cells exhibiting mitochondrial recruitment in three independent experiments, with 100 anti-Flag staining- and GFP-positive cells being counted per sample.
Figure 4
Figure 4
Pink1 activates ubiquitin chain binding ability of Parkin. (A) Mitochondrially-targeted linear ubiquitin chains alone are not sufficient to recruit Parkin C431S to mitochondria without CCCP treatment. HeLa cells were transfected with GFP-Parkin C431S and Tom70-Flag-4×ubiquitinG76V or Tom70-Flag, and then treated with CCCP. The cells were stained with anti-Flag antibody (red), anti-Hsp60 (white) antibody and Hoechst 33342 (blue). (B) Expression of Pink1ΔMTS can stimulate Parkin C431S localization to mitochondria carrying linear ubiquitin chains. HeLa cells were transfected with GFP-Parkin C431S/Tom70-Flag-4×ubiquitinG76V/Pink1ΔMTS-HA; GFP-Parkin C431S/Tom70-Flag-4×ubiquitinG76V/Pink1ΔMTS KD-HA; or GFP-Parkin C431S/Tom70-Flag/Pink1ΔMTS-HA, respectively. 12 h after transfection, cells were stained with anti-Flag antibody (red), anti-Hsp60 antibody (white) and Hoechst 33342 (blue). The graphs show means ± SD of percent of cells exhibiting mitchondrial recruitment in three independent experiments, with 100 anti-Flag staining- and GFP-positive cells being counted per sample. (C) Pink1 might enhance Parkin ubiquitin chain binding. HeLa cells were transfected with Flag-Parkin WT together with Pink1ΔMTS-HA or Pink1ΔMTS KD-HA. Parkins were immunoprecipitated and then incubated with 6× His-tagged K63-linked polyubquitin chain for 2 h. The bound ubiquitin chains were detected by anti-His antibody (mouse); Parkin was detected by anti-Flag rabbit antibody. 200 ng of 6× His-tagged K63-linked polyubquitin chains were loaded as a positive control for immunobloting. The total intensities of bound ubiquitin chains and the corresponding immunoprecipitated Parkin were determined by normalizing to the levels of Flag-Parkin without co-expression of Pink1ΔMTS using Odyssey software. (D) A model for catalytic activity coupled Parkin mitochondrial recruitment. Details are in the main text.
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