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  • Mulan is one of three E ubiquitin ligases that are


    Mulan is one of three E3 ubiquitin ligases that are present in the mitochondria along with MARCH5/MITOL (membrane associated RING-CH5) and RNF185 [14], [15], [16], [17]. Mulan is a 352-residue polypeptide that crosses the mitochondrial outer membrane (OMM) twice, its amino-terminus, which includes the RING finger domain, is located in the old town and a large domain is present in the inter-membrane space (IMS) of the mitochondria. Both the mitochondrial localization signal as well as, the RING-finger domain have been shown to be necessary for Mulan's function [15]. Mulan's location suggests that its substrates will be proteins that are either integral or associated with the mitochondrial membrane. Mulan has been shown to directly or indirectly regulate various proteins including NF-κB, JNK, p53, Akt and Mfn2, suggesting a potential role in cell growth, apoptosis and mitophagy [13], [14], [15], [18], [19]. In order to investigate the mechanism by which Mulan performs its normal function, we set out to identify E2 ubiquitin conjugating enzymes that form specific complexes with Mulan. We used the cytoplasmic domain of Mulan (amino acids 259–352) that includes its RING finger domain in a yeast two-hybrid screen, and identified four E2 conjugating enzymes, namely Ube2E2, Ube2E3, Ube2G2 and Ube2L3, as specific interactors. We then used fusion baits consisting of Mulan's RING finger domain linked to the full length of each of the four specific E2's that were identified. These fused baits were used to screen for interactors of the heterodimeric Mulan259–352–E2 recombinant protein. In this screen, several distinct interactors were identified for each of the Mulan259–352–E2 fusion baits. The identity of these interactors confirms Mulan's diverse functions that are determined by the nature of the E2 conjugating enzyme. 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 mitochondria 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].