title: Regulation of mitophagy by the Gp78 E3 ubiquitin ligase.
abstract: Glycoprotein 78 (Gp78) is a critical E3 ubiquitin ligase in endoplasmic reticulum-associated degradation. Overexpression of Flag-tagged Gp78 (Flag-gp78), but not Flag-gp78 mutated in its RING-finger domain (Flag-RINGmut) with deficient ubiquitin ligase activity, induces mitochondrial fragmentation and ubiquitination and proteasome-dependent degradation of the mitofusin (Mfn) mitochondrial fusion factors Mfn1/Mfn2. After mitochondrial depolarization with carbonyl cyanide m-chlorophenylhydrazone (CCCP), Flag-gp78 induced a threefold loss of depolarized mitochondria and significant loss of the inner mitochondrial protein OxPhosV. Flag-gp78-dependent loss of OxPhosV, but not Mfn1 or Mfn2, was prevented by small interfering RNA (siRNA) knockdown of the autophagy protein Atg5 in CCCP-treated cells. Gp78-induced mitophagy required ubiquitin ligase activity, as it is not observed upon transfection of Flag-RINGmut or cotransfection of Flag-gp78 with ubiquitin mutated at three critical lysine residues (K29, 48, 63R) involved in polyubiquitin chain elongation. Short hairpin RNA knockdown of Gp78 in HT-1080 fibrosarcoma cells increased mitofusin levels and reduced depolarization-induced mitophagy, whereas siRNA knockdown showed that Mfn1, but not Mfn2, was required for Gp78-dependent depolarization-induced mitophagy. Mitochondrial depolarization induced Gp78-dependent expression of the autophagic marker LC3II and recruitment of enhanced green fluorescent protein-LC3 to the Gp78- and calnexin-labeled, mitochondria-associated ER. Finally, Gp78-induced mitophagy is Parkin independent, as it occurs in Parkin-null HeLa cells and upon siRNA-mediated Parkin knockdown in HEK293 cells. This study therefore describes a novel role for the ER-associated Gp78 ubiquitin ligase and the Mfn1 mitochondrial fusion factor in mitophagy.
authors: Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
authors: Min
authors: M
authors: Fu
authors: Pascal
authors: P
authors: St-Pierre
authors: Jay
authors: J
authors: Shankar
authors: Peter T C
authors: PT
authors: Wang
authors: Bharat
authors: B
authors: Joshi
authors: Ivan R
authors: IR
authors: Nabi
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008928:Mitochondria / Q000502:physiology; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D063306:Mitophagy; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000235:genetics; D060285:Receptors, Autocrine Motility Factor / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
chemical_list: C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C555000:AMFR protein, human; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D046988:Proteasome Endopeptidase Complex; D020558:GTP Phosphohydrolases; C470018:MFN2 protein, human; C470017:Mfn1 protein, human