Instituto de Investigaciones Bioquímicas de Bahía Blanca, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional del Sur, Bahía Blanca B8000, Argentina.
Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, Bahía Blanca B8000, Argentina.
Proc Natl Acad Sci U S A. 2024 Jul 23;121(30):e2313609121. doi: 10.1073/pnas.2313609121. Epub 2024 Jul 16.
Mitofusins (Mfn1 and Mfn2) are the mitochondrial outer-membrane fusion proteins in mammals and belong to the dynamin superfamily of multidomain GTPases. Recent structural studies of truncated variants lacking alpha helical transmembrane domains suggested that Mfns dimerize to promote the approximation and the fusion of the mitochondrial outer membranes upon the hydrolysis of guanine 5'-triphosphate disodium salt (GTP). However, next to the presence of GTP, the fusion activity seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion through the formation of Mfn1-Mfn2 heterodimers. Here, we purified and reconstituted the full-length murine Mfn2 protein into giant unilamellar vesicles (GUVs) with different lipid compositions. The incubation with GTP resulted in the fusion of Mfn2-GUVs. High-speed video-microscopy showed that the Mfn2-dependent membrane fusion pathway progressed through a zipper mechanism where the formation and growth of an adhesion patch eventually led to the formation of a membrane opening at the rim of the septum. The presence of physiological concentration (up to 30 mol%) of dioleoyl-phosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP-induced Mfn2-dependent fusion. Our observations show that Mfn2 alone can promote the fusion of micron-sized DOPE-enriched vesicles without the requirement of regulatory cofactors, such as membrane curvature, or the assistance of other proteins.
线粒体融合蛋白 1 和 2（Mfn1 和 Mfn2）是哺乳动物线粒体外膜融合蛋白，属于具有多个结构域的 GTP 酶的动力蛋白超家族。最近对缺乏 α 螺旋跨膜结构域的截短变体的结构研究表明，Mfns 通过二聚化促进线粒体外膜的接近和融合，在鸟嘌呤 5'-三磷酸二钠盐（GTP）水解的过程中。然而，除了 GTP 的存在外，融合活性似乎还需要多个调节因子，这些调节因子通过形成 Mfn1-Mfn2 异二聚体来控制线粒体融合的动力学和动力学。在这里，我们将全长的鼠源 Mfn2 蛋白纯化并重组到具有不同脂质组成的巨大单层囊泡（GUVs）中。用 GTP 孵育导致 Mfn2-GUVs 的融合。高速视频显微镜显示，Mfn2 依赖性的膜融合途径通过拉链机制进行，其中粘附斑的形成和生长最终导致隔膜边缘形成膜开口。实验表明，生理浓度（高达 30mol%）的二油酰基磷脂酰乙醇胺（DOPE）的存在是观察 GTP 诱导的 Mfn2 依赖性融合所必需的。我们的观察表明，Mfn2 本身可以促进富含 DOPE 的微米大小囊泡的融合，而不需要调节因子（如膜曲率）或其他蛋白质的协助。
