Mulan E3 ubiquitin ligase interacts with multiple E2 conjugating enzymes and participates in mitophagy by recruiting GABARAP

Mulan is an E3 ubiquitin ligase embedded in the outer mitochondrial membrane (OMM) with its RING finger facing the cytoplasm and a large domain located in the intermembrane space (IMS). Mulan is known to have an important role in cell growth, cell death, and more recently in mitophagy. The mechanism of its function is poorly understood; but as an E3 ligase it is expected to interact with specific E2 ubiquitin conjugating enzymes and these complexes will bind and ubiquitinate specific substrates. The unique topology of Mulan can provide a direct link of communicating mitochondrial signals to the cytoplasm. Our studies identified four different E2 conjugating enzymes (Ube2E2, Ube2E3, Ube2G2 and Ube2L3) as specific interactors of Mulan. Each of these E2 conjugating enzymes was fused to the RING finger domain of Mulan and used in a modified yeast two-hybrid screen. Several unique interactors for each Mulan-E2 complex were isolated. One such specific interactor of Mulan-Ube2E3 was the GABARAP (GABAA receptor-associated protein). GABARAP is a member of the Atg8 family of proteins that plays a major role in autophagy/mitophagy. The interaction of GABARAP with Mulan-Ube2E3 required an LC3-interacting region (LIR) located in the RING finger domain of Mulan as well as the presence of Ube2E3. The isolation of four different E2 conjugating enzymes, as specific partners of Mulan E3 ligase, suggests that Mulan is involved in multiple biological pathways. In addition, the interaction of GABARAP with Mulan-Ube2E3 supports the role of Mulan as an important regulator of mitophagy and provides a plausible mechanism for its function in this process.