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  • Since the interaction between the E and

    2019-10-22

    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 Wnt agonist 1 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. Mulan protein level is regulated with conditions that stimulate mitophagy such as treatment of cells with CCCP [12]. We found that exogenously expressed GFP-GABARAP and RFP-Mulan could co-precipitate and co-localize in mammalian cells and the degree of co-localization increased when the cells were treated with CCCP. In summary, we have identified four different E2 conjugating enzymes that interact with Mulan to form E2/E3 heterodimers. Each heterodimer in turn has unique specificity against various target proteins. In addition, our results suggest a mechanism of how Mulan participates in mitophagy. Mulan and Parkin are both E3 ubiquitin ligases but Mulan resides in the mitochondria whereas Parkin is recruited to the mitochondria by PINK1 during conditions of mitophagy [9], [10], [67]. Our present study shows that Mulan directly interacts with GABARAP that is different from the mechanism used by Parkin (Fig. 6B). In addition, both Mulan and Parkin are known to regulate Mfn2 whose downregulation is necessary for mitophagy to occur [13], [23]. Whether these two pathways leading to mitophagy (Mulan versus Parkin) work synergistically or are independent from one another needs to be determined. The Mulan pathway potentially compensates the absence of Parkin in knock-out (KO) animals that show only subtle phenotypes [68], [69], [70]. It will also be interesting to investigate if the Mulan pathway is compromised in Parkinson\'s disease or other neurodegenerative disorders. Our previous studies showed the Mulan pathway to mitophagy is overactive when Omi/HtrA2 protease is absent or inactive in mutant mice [12]. This results in increased mitophagy that causes neurodegeneration, premature aging, and a plethora of defects in the mutant mice [25], [27]. These observations suggest that, not only loss of mitophagy but also uncontrolled and excessive mitophagy can lead to neurodegeneration and potentially several other disorders.