Unresolved endoplasmic reticulum ( ER ) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase ( MITOL / MARCH 5) inhibits ER stress‐induced apoptosis through ubiquitylation of IRE 1α at the mitochondria‐associated ER membrane ( MAM ). MITOL promotes K63‐linked chain ubiquitination of IRE 1α at lysine 481 (K481), thereby preventing hyper‐oligomerization of IRE 1α and regulated IRE 1α‐dependent decay ( RIDD ). Therefore, under ER stress, MITOL depletion or the IRE 1α mutant (K481R) allows for IRE 1α hyper‐oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL ‐deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE 1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE 1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE 1α ubiquitylation by MITOL at the MAM .

Mitochondria are highly dynamic organelles that constantly fuse, divide, and move, and their function is regulated and maintained by their morphologic changes. Mitochondrial disease (MD) comprises a group of disorders involving mitochondrial dysfunction. However, it is not clear whether changes in mitochondrial morphology are related to MD. In this study, we examined mitochondrial morphology in fibroblasts from patients with MD (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and Leigh syndrome). We observed that MD fibroblasts exhibited significant mitochondrial fragmentation by upregulation of Drp1, which is responsible for mitochondrial fission. Interestingly, the inhibition of mitochondrial fragmentation by Drp1 knockdown enhanced cellular toxicity and led to cell death in MD fibroblasts. These results suggest that mitochondrial fission plays a critical role in the attenuation of mitochondrial damage in MD fibroblasts.Biomolecules 2020, 10, 450 2 of 13 respiratory chain complexes [7]. However, in multiple cases, the exact genetic cause of Leigh syndrome remains unknown [8]. Currently, there is no cure or effective treatment for MD, but recent research has shown the potential benefits of some approaches, at least in preclinical in vivo models [9][10][11]. Many researchers assume that both bulk and mitochondrial autophagy play protective roles in MD because the accumulation of damaged mitochondria and other toxic aggregates causes deterioration of the pathophysiology of the cell [12]. Preclinical studies suggest that a potential therapeutic target of disease progression is the mammalian target of rapamycin (mTOR) pathway, a biological route fundamental for regulating cell metabolism and physiology [13]. In mice models of Leigh syndrome, treatment with rapamycin, an mTOR inhibitor, extended lifespan and reduced disease progression and severity [14]. Civiletto et al. provided evidence that rapamycin induces improvements in mitochondrial function and ultrastructure, indicating powerful clearance of dysfunctional organelles via activation of autophagic flux in skeletal muscle.Mitochondria are highly dynamic organelles that continuously fuse, divide, and move, and mitochondrial function is controlled and maintained by these morphologic changes. Mitochondrial fission is specifically mediated by dynamin-related guanosine triphosphatase (GTPase) protein 1 (Drp1); in addition, dynamin-related GTPases mitofusin (Mfn) and optic atrophy 1 (OPA1) are associated with the outer and inner mitochondrial membranes, respectively, and mediate fusion of these membranes [15][16][17][18][19]. The most direct consequence of mitochondrial division and fusion is the change in size of the mitochondria [20][21][22]. Mitochondrial fission via Drp1 has been thought to regulate mitophagy by dividing mitochondria into fragments suitable for autophagosome engulfment [23][24][25] and/or separating damaged subdomains of mitochondria for elimination [26]. Mitochondrial fusion enables efficient mixing of m...

The molecular pathology of diseases seen from the mitochondrial axis has become more complex with the progression of research. A variety of factors, including the failure of mitochondrial dynamics and quality control, have made it extremely difficult to narrow down drug discovery targets. We have identified MITOL (mitochondrial ubiquitin ligase: also known as MARCH5) localized on the mitochondrial outer membrane and previously reported that it is an important regulator of mitochondrial dynamics and mitochondrial quality control. In this review, we describe the pathological aspects of MITOL revealed through functional analysis and its potential as a drug discovery target.

Mitochondrial pathophysiology is implicated in the development of Alzheimer’s disease (AD). An integrative database of gene dysregulation suggests that the mitochondrial ubiquitin ligase MITOL/MARCH5, a fine-tuner of mitochondrial dynamics and functions, is downregulated in patients with AD. Here, we report that the perturbation of mitochondrial dynamics by MITOL deletion triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aβ pathology. Notably, MITOL deletion in the brain enhanced the seeding effect of Aβ fibrils, but not the spontaneous formation of Aβ fibrils and plaques, leading to excessive secondary generation of toxic and dispersible Aβ oligomers. Consistent with this, MITOL-deficient mice with Aβ etiology exhibited worsening cognitive decline depending on Aβ oligomers rather than Aβ plaques themselves. Our findings suggest that alteration in mitochondrial morphology might be a key factor in AD due to directing the production of Aβ form, oligomers or plaques, responsible for disease development.

Parkin promotes cell survival by removing damaged mitochondria via mitophagy. However, although some studies have suggested that Parkin induces cell death, the regulatory mechanism underlying the dual role of Parkin remains unknown. Herein, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) regulates Parkin‐mediated cell death through the FKBP38‐dependent dynamic translocation from the mitochondria to the ER during mitophagy. Mechanistically, MITOL mediates ubiquitination of Parkin at lysine 220 residue, which promotes its proteasomal degradation, and thereby fine‐tunes mitophagy by controlling the quantity of Parkin. Deletion of MITOL leads to accumulation of the phosphorylated active form of Parkin in the ER, resulting in FKBP38 degradation and enhanced cell death. Thus, we have shown that MITOL blocks Parkin‐induced cell death, at least partially, by protecting FKBP38 from Parkin. Our findings unveil the regulation of the dual function of Parkin and provide a novel perspective on the pathogenesis of PD.

CRMP-5-associated GTPase (CRAG), a short splicing variant of centaurin-γ3/AGAP3, is predominantly expressed in the developing brain. We previously demonstrated that CRAG, but not centaurin-γ3, translocates to the nucleus and activates the serum response factor (SRF)-c-Fos pathway in cultured neuronal cells. However, the physiological relevance of CRAG in vivo is unknown. Here, we found that CRAG/centaurin-γ3–knockout mice showed intensively suppressed kainic acid-induced c-fos expression in the hippocampus. Analyses of molecular mechanisms underlying CRAG-mediated SRF activation revealed that CRAG has an essential role in GTPase activity, interacts with ELK1 (a co-activator of SRF), and activates SRF in an ELK1-dependent manner. Furthermore, CRAG and ELK1 interact with promyelocytic leukaemia bodies through SUMO-interacting motifs, which is required for SRF activation. These results suggest that CRAG plays a critical role in ELK1-dependent SRF-c-fos activation at promyelocytic leukaemia bodies in the developing brain.

The transfer of phospholipids from the endoplasmic reticulum to mitochondria via the mitochondria-endoplasmic reticulum (ER) contact site (MERCS) is essential for maintaining mitochondrial function and integrity. Here, we identified RMDN3/PTPIP51, possessing phosphatidic acid (PA)-transfer activity, as a neighboring protein of the mitochondrial E3 ubiquitin ligase MITOL/MARCH5 by proximity-dependent biotin labeling using APEX2. We found that MITOL interacts with and ubiquitinates RMDN3. Mutational analysis identified lysine residue 89 in RMDN3 as a site of ubiquitination by MITOL. Loss of MITOL or the substitution of lysine 89 to arginine in RMDN3 significantly reduced the PA-binding activity of RMDN3, suggesting that MITOL regulates the transport of PA to mitochondria by activating RMDN3. Our findings imply that ubiquitin signaling regulates phospholipid transport at the MERCS.

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