Fusion of micron-size vesicles: interplay between the mitochondrial Mfn2 protein and lipids

PEÑALVA, D.A.; MONNAPPA, A.K.; NATALE, P.; LOPEZ MONTERO, I.

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Researc; 2022

Membrane fusion is crucial for the coordination of mitochondrial dynamics. An imbalanced mitochondrial dynamic leads to the formation of fragmented mitochondria and a decrease in intracellular ATP levels, contributing to the development of important diseases, including neurodegenerative, cardiac or cancer conditions. The fusion process is energetically unfavorable, thereby requiring specialized proteins. In mammals, Mitofusins (Mfn) 1 and 2 are responsible for mitochondrial outer-membrane (OMM) fusion. They belong to the dynamin superfamily of multi-domain GTPases. Recent structural studies suggest that, upon GTP hydrolysis, Mfns dimerize to promote the approaching and fusion of OMM. However, the OMM fusion seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion throught the formation of heterotypic Mfn1-Mfn2 dimers. In this study, we purified and functionally reconstituted the full-length mouse Mfn2 in large and giant unilamellar vesicles (LUVs and GUVs, respectively). Vesicles were prepared with POPC alone or with 30 % of plasmalogen-PC or DOPE. Unlike GDP, after incubation with GTP, vesicles underwent fusion. Fast video microscopy imaged the Mfn2-dependent membrane fusion pathway which involves the formation and expansion of a membrane diaphragm and the opening of a fusion pore. The incorporation of DOPE (30% mol) in the lipid composition did not alter the fusion sequence but enhanced the fusion kinetics significantly, as revealed by a lipid-mixing assay. Our observations show that Mfn2 alone can promote the fusion of micron-sized vesicles, without the presence of other proteins in the membrane. In addition, the lipid environment is an important factor in the modulation of Mfn2-dependent membrane fusion, a process that seems to require topological lipid intermediates with negative curvature.