Abstract
The kinase PINK1 and the E3 ubiquitin (Ub) ligase Parkin participate in mitochondrial quality control. The phosphorylation of Ser65 in Parkin's ubiquitin-like (UBl) domain by PINK1 stimulates Parkin activation and translocation to damaged mitochondria, which induces mitophagy generating polyUb chain. However, Parkin Ser65 phosphorylation is insufficient for Parkin mitochondrial translocation. Here we report that Ser65 in polyUb chain is also phosphorylated by PINK1, and that phosphorylated polyUb chain on mitochondria tethers Parkin at mitochondria. The expression of Tom70MTS-4xUb SE, which mimics phospho-Ser65 polyUb chains on the mitochondria, activated Parkin E3 activity and its mitochondrial translocation. An E3-dead form of Parkin translocated to mitochondria with reduced membrane potential in the presence of Tom70MTS-4xUb SE, whereas non-phospho-polyUb mutant Tom70MTS-4xUb SA abrogated Parkin translocation. Parkin binds to the phospho-polyUb chain through its RING1-In-Between-RING (IBR) domains, but its RING0-linker is also required for mitochondrial translocation. Moreover, the expression of Tom70MTS-4xUb SE improved mitochondrial degeneration in PINK1-deficient, but not Parkin-deficient, Drosophila. Our study suggests that the phosphorylation of mitochondrial polyUb by PINK1 is implicated in both Parkin activation and mitochondrial translocation, predicting a chain reaction mechanism of mitochondrial phospho-polyUb production by which rapid translocation of Parkin is achieved.
Highlights
Parkin (Gene ID: 5071) is an RBR (RING-in-between-RING) E3 Ub ligase with a Ubl domain at its N-terminus, and an atypical RING domain, RING0, has been newly identified in the linker region between the Ubl and the RBR domains [1,2]
We describe that the phosphorylation of polyUb chain by PINK1 is a key event to recruit Parkin on the mitochondria
Our study suggests the existence of an amplification cascade of Parkin activation and mitochondrial translocation, in which a ‘seed’ of phosphorylated polyUb on the mitochondria, generated by PINK1 and Parkin, triggers a chain reaction of Parkin recruitment and activation
Summary
Parkin (Gene ID: 5071) is an RBR (RING-in-between-RING) E3 Ub ligase with a Ubl domain at its N-terminus, and an atypical RING domain, RING0, has been newly identified in the linker region between the Ubl and the RBR domains [1,2]. The activated PINK1 recruits Parkin from the cytosol to the mitochondria upon decreased membrane potential, which stimulates Parkin E3 activity, promoting mitochondrial degradation via an autophagic event known as mitophagy [7,8,9,10,11]. The recruitment of cytosolic Parkin to the mitochondria upon disruption of DYm is thought to be the first step of mitophagy for the removal of damaged mitochondria. Translocated Parkin leads to polyUb accumulation on the mitochondria [9], which further recruits Ub-proteasome- and autophagy-related proteins for mitochondrial elimination, including the 26S proteasome, p97/VCP, p62/SQSTM1, LC3, ATG5 and ATG7 [8,9,10,17,18,19,20]
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