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  • Mulan is a mitochondrial protein and is exclusively


    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 pituitary adenylate cyclase-activating peptide 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]. Since the interaction between the E2 and E3 is weak and transient, it has been difficult to identify novel interactions between specific E2/E3 complexes [59], [64]. Here we used a modified bait consisting of the Mulan RING domain fused to one of the four E2s isolated in our screen and expressed in yeast as a fusion Mulan259–352–E2 bait. We were able to identify eight novel and specific interactors against three Mulan259–352–E2 fusion heterodimer baits. The Mulan259–352–Ube2G2 could not be used in this system since it auto-activated the transcription of the reporter gene in yeast. The eight interactors can be divided into those that were only able to interact with the Mulan259–352–E2 heterodimer and those that interacted with both the heterodimer and the specific E2 alone. None of the interactors could bind to the Mulan RING domain alone. From the isolated interactors, three of them have been previously reported to bind the specific E2 that was also used here [35]. The interaction of these isolated proteins was stronger against the Mulan259–352–E2 fusion than against the E2 protein alone. The ability of Mulan to form complexes with various E2s and the unique specificity of each Mulan259–352–E2 heterodimer suggest a mechanism of how Mulan can be involved in multiple biological processes. The importance of the different Mulan interactions identified in our screen requires further investigation to verify how each one contributes to Mulan\'s known functions. We further investigated the unique interaction between Mulan259–352–Ube2E3 and GABARAP. GABARAP is a member of the Atg8 family (LC3, GABARAP/GATE-16) that plays a major role in autophagy/mitophagy [65]. Its interaction with Mulan259–352–Ube2E3 could explain, at least in part, the mechanism by which Mulan participates to mitophagy. The interaction of GABARAP with Mulan259–352–Ube2E3 was very specific. When the Ube2E2 was used to replace Ube2E3 in the complex with Mulan, the interaction with GABARAP was lost. The presence of Mulan259–352 was also required for the interaction of GABARAP with Mulan259–352–Ube2E3. The interaction of Mulan with GABARAP was mediated via an LIR-like motif present in the RING domain of Mulan. The presence of LIR strengthens the case of a physiological interaction between Mulan and GABARAP, since all known proteins that interact with members of the Atg8 family (including GABARAP) invariably carry an LIR-like motif [66]. This interaction is specific for GABARAP, when LC3B (another member of the Atg8 family) was used in the same assay, no interaction was observed with Mulan259–352.