*3.2. Mitochondrial Fusion*

Mitochondria are fused through a few steps, termed mitochondrial fusion. First, mitochondria tether together via mitofusin (MFN), a large GTPase called Fzo1 in yeast, and MFN1/2 in mammals. Then, following MFN activation and conformational changes induced by GTP hydrolysis, each OMM attaches and fuses [54–56]. In recent years, various E3 ligases have been shown to regulate mitochondrial fusion via the modulation of one or both MFNs in response to various physiological or stress-induced conditions. Glycoprotein 78 (Gp78), an ER membrane-anchored E3 ubiquitin ligase, interacts with both MFNs, and Gp78 overexpression induces mitochondrial fragmentation [57]. Moreover, autocrine motility factor (AMF) prevents the Gp78-induced degradation of both MFNs [58]. MGRN1, an E3 ligase located in the cytoplasm, plasma membrane, endosomes, and nucleus, was reported to promote mitochondrial fusion via the non-degradative ubiquitylation of MFN1, consistent with previous observations [59,60]. The OMM E3 ligase MAPL/Mul1 leads to the specific ubiquitylation and degradation of MFN2 to regulate mitochondrial morphology [61]. HUWE1, a cytoplasmic E3 HECT family ubiquitin ligase also termed Mule/ARF-BP1/HectH9/E3Histone/Lasu12, ubiquitinated MFN2 associated with genotoxic stress to regulate mitochondrial fusion [62]. MITOL-mediated ubiquitylation and degradation of MFN1, but not of MFN2, lead to mitochondrial fragmentation in various inducible stresses and situations. In prostate cancer cells, the induction of cell death with CGP, an inhibitor of mitochondrial calcium e fflux, led to ubiquitylation and degradation of MFN1 by MITOL [63]. It was also shown that MITOL ubiquitylates and degrades MFN1 at G2/M, the notable phase of mitochondrial fragmentation before cellular division [64]. However, under hypoxic stress induced by deferoxamine (DFO), MITOL interacts with MFN2 and is responsible for the ubiquitylation and degradation of MFN2 in cells lacking HDAC6 [65]. In conclusion, although mitochondrial fission and fusion were clearly regulated by ubiquitylation, further studies are required to understand how MITOL divides substrate-specificity according to its surroundings in order to regulate mitochondrial dynamics

(Figure 3B). In addition, mitochondrial dysfunction is suspected to be one of the causes of many neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, so investigating the involvement of MITOL in these diseases may lead to future therapies.

### **4. Relation between MITOL and Membrane Bontact Site with the Endoplasmic Reticulum**

### *4.1. Membrane Contact Site with the Endoplasmic Reticulum*

The mitochondrial surface also represents the signal hub where a host of metabolic systems cross-talk through inter-organelle communication. Mitochondria indeed have a unique microdomain physically and functionally connecting to other organelles such as the endoplasmic reticulum (ER). The membrane contact site (MCS) between the ER and mitochondria is maintained by some tethering or spacer proteins such as PDZD8, Fis1-BAP31, VDAC-IP3R, PTPIP51-VAPB, and MFN2 (the tethering function of Mfn2 appears to still be controversial) in mammals [66,67]. Mitochondria and the ER can exchange lipids and calcium ion through their MCS [68,69]. The proximal domain between the ER and mitochondria is also available as a membrane sca ffold for signal transmission including autophagy, inflammation, and the unfolded protein response (UPR) due to the raft-like membrane structure [70]. Taking these findings together, the ER and mitochondria complement each other through inter-organelle communication such as membrane contacts.
