Gp78 E3 ubiquitin ligase mediates both basal and damage-induced mitophagy

Abstract

Mitophagy, the elimination of mitochondria by the autophagy machinery, evolved to monitor mitochondrial health and maintain mitochondrial integrity. PINK1 is a sensor of mitochondrial health that recruits Parkin and other mitophagy-inducing ubiquitin ligases to depolarized mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis, basal mitophagy, remain poorly understood. The Gp78 E3 ubiquitin ligase, an endoplasmic reticulum membrane protein, induces mitochondrial fission, endoplasmic reticulummitochondria contacts and mitophagy of depolarized mitochondria. CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells results in reduced ER-mitochondria contacts, increased mitochondrial volume and resistance to CCCP-induced mitophagy. Knockdown (KD) of the essential autophagy protein ATG5 increased mitochondrial volume of wild-type cells but did not impact mitochondrial volume of Gp78 knockout cells. This suggests that endogenous Gp78 actively eliminates mitochondria by autophagy in wild-type HT-1080 cells. Damage-induced mitophagy of depolarized mitochondria, in the presence of CCCP, but not basal mitophagy was prevented by knockdown of PINK1. This suggests that endogenous Gp78 plays dual roles in mitophagy induction: 1) control of mitochondrial homeostasis through mitophagy of undamaged mitochondria; and 2) elimination of damaged mitochondria through PINK1.

Single molecule network analysis identifies structural changes to caveolae and scaffolds due to mutation of the caveolin-1 scaffolding domain

“…CAV1 knockout MDA-MB-231 cell line was generated using GeneArt-CRISPR/Cas9 Nuclease vector containing OFP (Orange Fluorescence Protein, A21174, Life Technologies, USA), as previously described 58 . Guided RNAs (gRNAs) were designed using http://crispr.mit.edu and off-targets checked using http://www.rgenome.net/cas-offinder/ (RGEN tools).…”