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Gp78 regulation of mitophagy by PINK1 and USP13 is mediated by its CUE domain

University of British Columbia

Mitophagy is an evolutionarily conserved process by which cells remove damaged mitochondria. Defects in mitophagy lead to mitochondrial dysfunction and are associated with pathological states such as cancer and neurodegenerative disorders. PINK1 protein accumulates in damaged mitochondria, recruits the Parkin ubiquitin ligase and induces mitophagy. We showed in the past that Gp78, an endoplasmic reticulum (ER) membrane E3 ligase localizes to ER-mitochondria contact sites. We also showed that Gp78 induces mitophagy independently of Parkin which is dependent on the presence of an intact Gp78 RING finger domain. We now show that Gp78 mitophagy of damaged mitochondria, induced by the mitochondrial poison CCCP, also requires PINK1. Further, the Gp78 cytoplasmic domain also contains a ubiquitin-binding Coupling of Ubiquitin to ER degradation (CUE) domain. Unlike wild-type Gp78 that induces mitophagy only upon mitochondrial damage, Gp78 with CUE-domain mutation constitutively performs mitophagy regardless of mitochondrial damage and independently of PINK1. This suggests that PINK1 acts upon the CUE domain of Gp78 to promote its mitophagy. The USP13 deubiquitinase has been shown to bind to Gp78 and by co-immunoprecipitation analysis, we found that the USP13-Gp78 interaction is CUE domain-dependent. USP13 knock-down (KD) enhances Gp78 ubiquitination of mitochondria, and like the CUE-mutant Gp78, promotes mitophagy even without induction of mitochondrial damage. We then tested the interdependence of USP13 and PINK1 for Gp78-dependent mitophagy. While PINK1 siRNA KD alone prevents Gp78-mediated mitophagy of damaged mitochondria, knockdown of both PINK1 (siRNA) and USP13 (shRNA), restores the Gp78-dependent mitophagy. This suggests that USP13 inhibition of Gp78-mediated mitophagy is relieved upon mitochondrial damage by PINK1.

Text

2020-08-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/66804

Cell and Developmental Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/