Knocking-out the Siah2 E3 ubiquitin ligase prevents mitochondrial NCX3 degradation, regulates mitochondrial fission and fusion, and restores mitochondrial function in hypoxic neurons

MARCH5, an OMM-associated E3 ubiquitin ligase, controls mitochondrial function. Despite its importance, the mechanism and factors controlling MARCH5 activity are largely unknown. Here we report that the MARCH5 C-terminal domain plays a critical role in degradation of MARCH5 substrates, likely by facilitating release of ubiquitinated proteins from the OMM. We also found that the mitochondrial fission proteins Drp1 and Mff negatively regulate MARCH5’s activity toward MiD49 and Mcl1. Knockouts of either Drp1 or Mff led to reduced expression, shorter half-lives, and increased ubiquitination of MiD49 and Mcl1. Effects of Mff and Drp1 depletion on degradation rates and ubiquitination of Mcl1 and MiD49 were eliminated in Drp1−/−/MARCH5−/− and Mff−/−/MARCH5−/− cells. Our data show that it is not mitochondrial morphology per se but rather Mff and Drp1 that directly control MARCH5. Consistently, we find that Mff is an integral component of the MARCH5/p97/Npl4 complex, which is also controlled by MARCH5’s C-terminal domain. Furthermore, not only mitochondrial fission but also fusion is regulated through Mff and Drp1 protein activities. Thus, in addition to their canonical roles in mitochondrial fission, Mff and Drp1 also act as regulatory factors that control mitochondrial fission and fusion.

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The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division

The Journal of Cell Biology ◽

10.1083/jcb.200611064 ◽

2007 ◽

Vol 178 (1) ◽

pp. 71-84 ◽

Cited By ~ 314

Author(s):

Mariusz Karbowski ◽

Albert Neutzner ◽

Richard J. Youle

Keyword(s):

Rna Interference ◽

Ubiquitin Ligase ◽

Molecular Interactions ◽

Mitochondrial Fission ◽

Ectopic Expression ◽

E3 Ubiquitin Ligase ◽

Dependent Manner ◽

Wild Type ◽

Subcellular Trafficking ◽

Mitochondrial Division

We identify a mitochondrial E3 ubiquitin ligase, MARCH5, as a critical regulator of mitochondrial fission. MARCH5 RING mutants and MARCH5 RNA interference induce an abnormal elongation and interconnection of mitochondria indicative of an inhibition of mitochondrial division. The aberrant mitochondrial phenotypes in MARCH5 RING mutant–expressing cells are reversed by ectopic expression of Drp1, but not another mitochondrial fission protein Fis1. Moreover, as indicated by abnormal clustering and mitochondrial accumulation of Drp1, as well as decreased cellular mobility of YFP-Drp1 in cells expressing MARCH5 RING mutants, MARCH5 activity regulates the subcellular trafficking of Drp1, likely by impacting the correct assembly at scission sites or the disassembly step of fission complexes. Loss of this activity may account for the observed mitochondrial division defects. Finally, MARCH5 RING mutants and endogenous Drp1, but not wild-type MARCH5 or Fis1, co-assemble into abnormally enlarged clusters in a Drp1 GTPase-dependent manner, suggesting molecular interactions among these proteins. Collectively, our data suggest a model in which mitochondrial division is regulated by a MARCH5 ubiquitin-dependent switch.

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Abstract 1699: Suppressor of cytokine signaling 6 (SOCS6) promotes mitochondrial fission through E3 ubiquitin ligase complex activity.

10.1158/1538-7445.am2013-1699 ◽

2013 ◽

Author(s):

Huan-Yu Lin ◽

Shiu-Ting Lin ◽

Mei-June Wang ◽

Jeou-Yuan Chen

Keyword(s):

Ubiquitin Ligase ◽

Mitochondrial Fission ◽

E3 Ubiquitin Ligase ◽

Cytokine Signaling ◽

Suppressor Of Cytokine Signaling ◽

Complex Activity ◽

Ubiquitin Ligase Complex

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Study of an FBXO7 patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria

10.1101/2021.12.22.473884 ◽

2021 ◽

Author(s):

Sara Al Rawi ◽

Lorna Simpson ◽

Neil Q McDonald ◽

Veronika Chernuha ◽

Orly Elpeleg ◽

...

Keyword(s):

Paediatric Patient ◽

Ubiquitin Ligase ◽

Mitochondrial Function ◽

E3 Ubiquitin Ligase ◽

Adaptor Proteins ◽

Atypical Parkinsonism ◽

Mitochondrial Network ◽

Dimerization Domain ◽

Proteasome Subunits ◽

Patient Mutation

Mutations in FBXO7 have been discovered associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerization domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, L250P patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts directly with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts as an adaptor enabling SCFFbxo7 ligase to ubiquitinate MiD49. Thus, the L250P mutation changes the function of Fbxo7 by altering its substrate repertoire. Although MiD49/51 expression was reduced in L250P patient cells, there was no effect on the mitochondrial network. However, patient cells had higher levels of ROS and reduced viability under stress. Our study shows that Fbxo7 and PI31 affect each other's functions in regulating both proteasomal and mitochondrial function and demonstrate a new function for PI31, as an adaptor for the SCFFbxo7 E3 ubiquitin ligase.

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

10.1101/407593 ◽

2018 ◽

Author(s):

Bharat Joshi ◽

Yayha Mohammadzadeh ◽

Guang Gao ◽

Ivan R. Nabi

Keyword(s):

Endoplasmic Reticulum ◽

Ubiquitin Ligase ◽

Mitochondrial Fission ◽

E3 Ubiquitin Ligase ◽

Endoplasmic Reticulum Membrane ◽

Wild Type ◽

Dual Roles ◽

Mitochondrial Homeostasis ◽

Fibrosarcoma Cells ◽

Mitochondrial Volume

AbstractMitophagy, 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 reticulum-mitochondria 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.

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p38 MAP kinase–dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER–mitochondria association and mitochondria motility

Molecular Biology of the Cell ◽

10.1091/mbc.e15-02-0120 ◽

2015 ◽

Vol 26 (21) ◽

pp. 3828-3840 ◽

Cited By ~ 21

Author(s):

Lei Li ◽

Guang Gao ◽

Jay Shankar ◽

Bharat Joshi ◽

Leonard J. Foster ◽

...

Keyword(s):

Amino Acid ◽

Map Kinase ◽

P38 Mapk ◽

Ubiquitin Ligase ◽

Mitochondrial Fission ◽

Phosphorylation Site ◽

E3 Ubiquitin Ligase ◽

P38 Map Kinase ◽

Serum Starvation ◽

Mapk Phosphorylation

Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8–amino acid motif (533–541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41–amino acid region containing 14-3-3– and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER–mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER–mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER–mitochondria association and mitochondria motility.

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Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein

Molecular Biology of the Cell ◽

10.1091/mbc.e15-09-0678 ◽

2016 ◽

Vol 27 (2) ◽

pp. 349-359 ◽

Cited By ~ 62

Author(s):

Shan Xu ◽

Edward Cherok ◽

Shweta Das ◽

Sunan Li ◽

Brian A. Roelofs ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Cellular Response ◽

Molecular Mechanisms ◽

Mitochondrial Fission ◽

E3 Ubiquitin Ligase ◽

Negative Regulator ◽

Mitochondrial Fragmentation ◽

Cell Sensitivity ◽

Response To Stress ◽

Induced Apoptosis

Ubiquitin- and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5−/− cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5−/− cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress.

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Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

The Journal of Cell Biology ◽

10.1083/jcb.201007013 ◽

2010 ◽

Vol 191 (7) ◽

pp. 1367-1380 ◽

Cited By ~ 806

Author(s):

Atsushi Tanaka ◽

Megan M. Cleland ◽

Shan Xu ◽

Derek P. Narendra ◽

Der-Fen Suen ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Mitochondrial Fission ◽

E3 Ubiquitin Ligase ◽

Membrane Depolarization ◽

Dependent Manner ◽

Inner Mitochondrial Membrane ◽

Aaa Atpase ◽

Selective Autophagy ◽

Selective Elimination ◽

Large Gtpases

Damage to mitochondria can lead to the depolarization of the inner mitochondrial membrane, thereby sensitizing impaired mitochondria for selective elimination by autophagy. However, fusion of uncoupled mitochondria with polarized mitochondria can compensate for damage, reverse membrane depolarization, and obviate mitophagy. Parkin, an E3 ubiquitin ligase that is mutated in monogenic forms of Parkinson’s disease, was recently found to induce selective autophagy of damaged mitochondria. Here we show that ubiquitination of mitofusins Mfn1 and Mfn2, large GTPases that mediate mitochondrial fusion, is induced by Parkin upon membrane depolarization and leads to their degradation in a proteasome- and p97-dependent manner. p97, a AAA+ ATPase, accumulates on mitochondria upon uncoupling of Parkin-expressing cells, and both p97 and proteasome activity are required for Parkin-mediated mitophagy. After mitochondrial fission upon depolarization, Parkin prevents or delays refusion of mitochondria, likely by the elimination of mitofusins. Inhibition of Drp1-mediated mitochondrial fission, the proteasome, or p97 prevents Parkin-induced mitophagy.

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