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  • One of the isolated interactors against Mulan Ube E


    One of the isolated interactors against Mulan259–352–Ube2E3 fusion bait was found to be the GABARAP protein, a known member of the Atg8 family that includes LC3 [20]. The Atg8 family-of-proteins are major players in autophagy/mitophagy since they are part of the autophagosome [20], [21], [22]. Our studies show that the interaction of GABARAP with Mulan–Ube2E3 requires the presence of Ube2E3 as well as an LIR motif located in the RING finger domain of Mulan. Exogenous expressed GABARAP interacts and co-localizes with Mulan, especially during conditions that stimulate mitophagy. Whether the interaction with GABARAP defines the main role of Mulan in mitophagy is unclear. Previous studies suggested regulation of Mfn2 by Mulan, which also participates in the process of mitophagy [13], [23]. The significance of this new Mulan-mediated pathway to mitophagy is evident in the phenotype on mnd2 (motor neuron disease 2) mutant mice. These mice have a mutation in the Omi/HtrA2 gene that causes accumulation of the Mulan protein and excessive mitophagy. This results in motor neuron disease with a Parkinsonian phenotype and premature aging of the mutant mice [24], [25], [26], [27].
    Materials and methods
    Discussion Omi/HtrA2 is a serine protease with high homology to the bacterial HtrA protease, a protein known to remove damaged and misfolded proteins at high temperatures [30]. The mature form of Omi/HtrA2 is found in the IMS of the am580 and, under cellular stress it is released to the cytosol where it participates in caspase-dependent and caspase-independent apoptosis pathways [53], [54], [55]. While in the IMS, Omi/HtrA2 has a different and unique pro-survival function. Mice lacking the active form of the protease (known as mnd2 mice) show a Parkinsonian phenotype and die within 30days after birth [24]. Furthermore, mice lacking Omi/HtrA2 function in non-neuronal tissues are observed to undergo premature aging [27]. We have recently identified a novel function of Omi/HtrA2 in the mitochondria as a regulator of Mulan protein level under conditions of oxidative stress or during mitophagy [12]. Mulan is a mitochondrial protein and is exclusively localized in the OMM with its RING domain facing the cytosol [14]. Based on this topology, Mulan can provide a link between molecular events in the mitochondria and cytoplasm. Mulan has been implicated in various processes including: mitochondrial dynamics, apoptosis, mitochondrial fission and more recently mitophagy, through its interaction with NF-κB, JNK, p53, Akt and Mfn2 [13], [14], [15], [18], [19], [23]. There are five large families of ubiquitin ligases characterized by the presence of the E3 signature motifs: HECT, RING, U-box, PHD and LAP [56]. Mulan belongs to the RING family of E3 ubiquitin ligases. E3 ubiquitin ligases are involved in a three-step process of ubiquitination that targets specific substrates. Ubiquitination is carried out by three classes of enzymes: the activating (E1), conjugating (E2) and ligating (E3) enzymes [57]. There are two known E1s, 30 known E2s and over 1000 identified E3s [58]. It has also been shown that E2s with high homology can interact with the same E3, indicating that enzyme function is correlated to structural similarities [59]. Mulan has a RING finger domain which is a loop–helix–loop structure and uses eight histidines and cysteines to coordinate a central Zn ion [60]. RING finger domain can then engage in interaction with the UBC domain in the E2s, forming a complex for substrate modification through ubiquitin. Specifically, RING-type E3s can catalyze the transfer of the ubiquitin directly from the E2 to the substrate [61]. Many E3 ligases are part of a multi-subunit heteromeric complex that generally includes an E2, adaptor proteins, and the substrate. They could also contain anchor proteins for a particular compartment as well as regulatory proteins [62]. Here, we have used the yeast two-hybrid system to isolate and characterize proteins that specifically interact with the cytosolic portion of Mulan that contains the RING domain. Four different interactors were isolated including Ube2G2 and Ube2L3 that belong to class I E2 conjugating enzymes and Ube2E2 and Ube2E3 that belong to class III. E2s are classified according to the addition of an extension at the amino-terminus and/or carboxy-terminus to the catalytic core UBC domain. Class I E2s consist of only the catalytic domain, while Class II and Class III have the extension at either the amino- or carboxy-terminus, respectively, and Class IV has both extensions [63].