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Regulation of mitophagy by the Gp78 E3 ubiquitin ligase

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 a...

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Autores principales: Fu, Min, St-Pierre, Pascal, Shankar, Jay, Wang, Peter T. C., Joshi, Bharat, Nabi, Ivan R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623636/
https://www.ncbi.nlm.nih.gov/pubmed/23427266
http://dx.doi.org/10.1091/mbc.E12-08-0607
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author Fu, Min
St-Pierre, Pascal
Shankar, Jay
Wang, Peter T. C.
Joshi, Bharat
Nabi, Ivan R.
author_facet Fu, Min
St-Pierre, Pascal
Shankar, Jay
Wang, Peter T. C.
Joshi, Bharat
Nabi, Ivan R.
author_sort Fu, Min
collection PubMed
description 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.
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spelling pubmed-36236362013-06-30 Regulation of mitophagy by the Gp78 E3 ubiquitin ligase Fu, Min St-Pierre, Pascal Shankar, Jay Wang, Peter T. C. Joshi, Bharat Nabi, Ivan R. Mol Biol Cell Articles 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. The American Society for Cell Biology 2013-04-15 /pmc/articles/PMC3623636/ /pubmed/23427266 http://dx.doi.org/10.1091/mbc.E12-08-0607 Text en © 2013 Fu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell BD; are registered trademarks of The American Society of Cell Biology.
spellingShingle Articles
Fu, Min
St-Pierre, Pascal
Shankar, Jay
Wang, Peter T. C.
Joshi, Bharat
Nabi, Ivan R.
Regulation of mitophagy by the Gp78 E3 ubiquitin ligase
title Regulation of mitophagy by the Gp78 E3 ubiquitin ligase
title_full Regulation of mitophagy by the Gp78 E3 ubiquitin ligase
title_fullStr Regulation of mitophagy by the Gp78 E3 ubiquitin ligase
title_full_unstemmed Regulation of mitophagy by the Gp78 E3 ubiquitin ligase
title_short Regulation of mitophagy by the Gp78 E3 ubiquitin ligase
title_sort regulation of mitophagy by the gp78 e3 ubiquitin ligase
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623636/
https://www.ncbi.nlm.nih.gov/pubmed/23427266
http://dx.doi.org/10.1091/mbc.E12-08-0607
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