Functional characterization of the membrane bound E3 ligase MARCH5 and its substrate Mitofusin-2

Mitochondrial integrity relies on balanced fission and fusion events. The ubiquitination, mediated by the mitochondrial membrane-bound ubiquitin ligase MARCH5, is crucial for the regulation of these mitochondrial dynamics by targeting proteins of the fission and fusion machinery. One key protein in mitochondrial fusion is the membrane associated large dynamin like GTPase Mitofusin-2, which is targeted by MARCH5-mediated ubiquitination. However, the molecular mechanism of mitochondrial fusion and how MARCH-5 regulates Mitofusin-2 is poorly understood. Furthermore, mitochondrial dynamics are highly controlled by specific phospholipids in the mitochondrial membranes. However, it remains unknown if and how lipids engage in a regulatory role in MARCH5-mediated ubiquitination also in regards to Mitofusin-2 regulation.

The functional characterization of MARCH5 in the presence of different lipid classes, native to the mitochondrial membranes, reveals that the activity and stability of MARCH5 is dependent on the phospholipid in vitro. In particular, the lipid cardiolipin shows specific interaction with purified MARCH5 and induces as the only one of the tested lipids a significant reduction in thermal stability, while the interaction with phosphatidic acid increases the stability of MARCH5 the strongest. In vitro ubiquitination assay revealed that the addition of lipids to purified MARCH5 alters the ubiquitination pattern. Specifically, the presence of cardiolipin and phosphatidylinositol-4-phosphate enhances auto- ubiquitination of MARCH5.

Furthermore, co-purification experiments of MARCH5 and Mitofusin-2 show that MARCH5 forms a stable complex with Mitofusin-2 in vivo and can be co-purified from mammalian cells and when recombinantly expressed in S. cerevisiae. Ubiquitination experiments with the purified MARCH5:Mitofusin-2 complex revealed alternation in ubiquitination of Mitofusin-2 mediated by MARCH5 dependent on the phospholipid.

The work presented here highlights that phospholipids directly affect the activity of MARCH5 and suggests that the lipid composition in mitochondrial membranes could control ubiquitination-dependent mechanisms that regulate the dynamics and turnover of mitochondria. Furthermore, the formation of a stable complex of MARCH5 and Mitofusin-2 allowing for the first time functional and structural studies of a full length Mitofusin-2 to investigate the functional mechanism of Mitofusin-2 and MARCH5 in vitro.