br Conclusions Mulan is a mitochondrial E ubiquitin ligase w

Conclusions Mulan is a mitochondrial E3 ubiquitin ligase with diverse roles in cell growth, cell death, and mitophagy. To better understand the mechanism of Mulan\'s function we used a modified yeast two-hybrid system to isolate proteins that interact with its cytoplasmic RING domain (amino acids 259–352). Four different E2 ubiquitin conjugating Spironolactone mg were isolated as specific interactors. We constructed fusion baits between Mulan259–352 and each one of the E2 proteins. These fusion baits were used in a secondary screen to isolate interactors that bound to the Mulan259–352–E2 fusion but not to Mulan259–352 or the E2 alone. One such interactor was the GABARAP protein known to be involved in mitophagy. GABARAP was able to interact with Mulan259–352–Ube2E3 though an LIR motif present in the Mulan RING domain. In addition, the presence of Ube2E3 in the complex with Mulan was also required. Our data provide a plausible mechanism by which Mulan when in complex with Ube2E3 participates in mitophagy. This mechanism is different, potentially independent, and could be operating in parallel with Parkin, another E3 ubiquitin ligase with a well-characterized role in mitophagy and Parkinson\'s disease.
Authors\' contributions
Acknowledgements This work was supported by grant 2KB05 to A.S.Z. by James and Esther King Biomedical Research Program, Florida Department of Health. We thank all members of Dr. Zervos\' lab for their comments and suggestions.
Introduction The post-translational modification of proteins with ubiquitin (Ub) or poly-Ub chains regulates many cellular processes, including protein degradation, DNA repair, and cellular trafficking [1], [2], [3], [4]. Ubiquitination involves a cascade of enzymatic reactions in which Ub is transferred from an E1 Ub-activating enzyme to an E2 Ub-conjugating enzyme, forming a thioester bond with a cysteine residue of E2 [5]. E2 subsequently associates with an E3 Ub-ligase, which recognizes a target protein and facilitates the transfer of Ub from E2 to a lysine residue of the substrate [5]. There are three classes of E3 Ub-ligases, defined by the presence of a HECT, RING, or U-box domain. HECT E3s form a catalytic Ub thioester intermediate before transferring Ub to the protein substrates, whereas RING and U-box E3s catalyze the direct transfer of Ub from E2 to target proteins [6]. Despite the very similar structures of the RING and U-box domains, the U-box does not coordinate Zn2+ ion, whereas the RING domain does [7], [8]. Higher plants contain a larger number of U-box proteins compared to that of yeasts and mammals. For example, Arabidopsis and rice, dicot and monocot model plants, respectively, have at least 64 and 77 U-box motif-containing E3 proteins, with the majority containing the protein–protein interacting ARM repeat motif [9], [10], [11], [12]. By contrast, yeasts and humans have 3 and 7 U-box E3s, respectively [13], [14]. Plant U-box E3s participate in many diverse plant-specific events, such as responses to phytohormones [15], [16], biotic stress [17], [18], and abiotic stress [19], [20], self-incompatibility [21], and control of flowering time [22]. These results are consistent with the proliferation of U-box proteins in plants relative to yeasts and mammals.