*3.1. Mitochondrial Fission*

Drp1, a dynamin-related GTPase, is the central player in mitochondrial fission [28–30]. Drp1 basically exists in the cytosol and can be recruited to the surface of mitochondria to wrap around mitochondrial tubules. Increasing Drp1 GTP hydrolysis activity leads to conformational changes [31,32] that increase the tightness of mitochondrial constriction and the scission of mitochondrial tubules. In terms of the chemical modifications of Drp1, phosphorylation, ubiquitination, and SUMOylation by several kinases and E3 ligase, including ubiquitin and SUMO ligase, have been particularly well studied [33–35]. Drp1 is ubiquitylated by APC/CCdh1 E3 ubiquitin ligase complex for degradation, the regulator of the M- to G1-phase transition, for regulating mitochondrial morphology during the G1/S phase [36]. Parkin, an E3 ligase mainly localized in the cytosol in a steady state, also induces the proteasomal degradation of Drp1 [37,38], suggesting that Parkin targets Drp1 as a substrate and plays an inhibitory role in mitochondrial fission. Controversial results have been reported on functions of MITOL in regulating mitochondrial dynamics. We and others showed that MITOL interacts with Drp1 and leads to its ubiquitylation and proteasome-dependent degradation to inhibit excessive mitochondrial fission [39,40]. However, the opposite role of MITOL in mitochondrial fission, namely, its positive regulation of Drp1, was observed in another study [41]. In a later study, Karbowski's group reported that MITOL ubiquitinates a Mid49, a Drp1 receptor, for degradation to control mitochondrial fission [42]. These findings sugges<sup>t</sup> that there may be some specificity regarding regulation of MITOL for its substrates, especially Drp1, such as relating to modifications, structural changes, and interactions with or without Drp1 receptors (Figure 3A).

**Figure 3.** Regulation of mitochondrial fission and fusion factors by MITOL. ( **A**) MITOL might have some specificity for its target; e.g., especially Drp1, such as relating to modifications, structural changes, and interactions with or without Drp1 receptors; (**B**) Alteration of substrate specificity for mitochondrial fusion factors in response to stress.

Recently, denitrosylase *S*-nitrosoglutathione reductase (GSNOR)-deficient mice were generated and it was reported that excessive *S*-nitrosylation resulted in the disruption of their mitochondrial dynamics and presented a typical senescence phenotype [43]. At the molecular level, it has also been reported that Drp1 can be in active form when subjected to *S*-nitrosylation by NO, leading to excessive mitochondrial division and the induction of neurotoxicity [44]. Similarly, MAP1B-LC1, a microtubule-associated factor that regulates retrograde mitochondrial transport, is also activated by *S*-nitrosylation, changing its steric structure [45], which in turn causes mitochondrial aggregation and cytotoxicity [46]. Thus, NO disrupts mitochondrial dynamics via the *S*-nitrosylation of Drp1 and MAP1B-LC1, even at the molecular level, and is thought to induce cellular damage as a result. We identified MAP1B-LC1 as a binding protein of MITOL using the yeas<sup>t</sup> two-hybrid method and found that MITOL specifically recognizes and induces the degradation of *S*-nitrosylation-modified MAP1B-LC1 [47]. We also confirmed similar results in Drp1, suggesting that MITOL has selectivity for recognizing its substrates and protecting against the collapse of mitochondrial dynamics partly by degrading active Drp1 and MAP1B-LC1.

Mitochondrion–organelle contacts are involved in mitochondrial fission [48–51]. In mammals, Fis1 is a mitochondrial recruitment factor for TBC1D15, which drives lysosomal Rab7 GTP hydrolysis [52,53] and indirectly regulates mitochondrial fission at the mitochondrion–lysosome contact sites [50]. It has been reported thatMITOL ubiquitinates Fis1 and promotesits degradation [39]. These findings sugges<sup>t</sup> that MITOL also regulates mitochondrial fission at the mitochondrion–lysosome contact sites via Fis1. Further work examining the mechanistic role of MITOL in mitochondrion–lysosome contacts is required.

Although MITOL is clearly involved in regulating the fission machinery, we need to comprehensively consider the involvement of multiple signaling platforms explained by the diverse roles in cellular biology, variations in tissue-specific expression, and the activity of fission members for the regulation of fission by MITOL.
