Structural analysis of dynamins involved in mitochondrial morphology

除动力蛋白外，其他动力蛋白家族成员还参与多种基本的细胞过程，包括线粒体分裂和融合、抗病毒活性、植物细胞板形成和叶绿体生物发生。在这些蛋白质中，自组装和寡聚化成有序结构是一个共同的特征，对大多数蛋白质来说，这对它们的功能至关重要。虽然关于动力蛋白的信息非常丰富，但对动力蛋白相关蛋白的结构特性知之甚少。近年来，我们研究了其他动力蛋白家族成员，一个参与线粒体裂变（Dnm1），两个参与线粒体融合，Mfn1和Opa1。我们正在测试一个假设，即动力家族成员有共同的作用机制。我们的工作提供了明确的证据，表明动力蛋白相关蛋白具有相似的组装和GTPase特性。然而，结构限制仍然必须允许它们广泛多样的生物功能。

In addition to dynamin, other dynamin family members have been implicated in a variety of fundamental cellular processes, including mitochondrial fission and fusion, anti-viral activity, plant cell plate formation and chloroplast biogenesis. Among these proteins, self-assembly and oligomerization into ordered structures is a common characteristic and, for the majority, is essential for their function. Although there is a wealth of information regarding dynamin, little is known about the structural properties of dynamin-related proteins. In recent years, we have examined other dynamin family members, one involved in mitochondria fission (Dnm1) and the two in mitochondrial fusion, Mfn1 & Opa1. We are testing the hypothesis that dynamin family members share a common mechanism of action. Our work provides clear evidence that dynamin related proteins have similar assembly and GTPase properties. However, structural constraints must still allow for their widely diverse biological functions. The dynamin family of proteins consists of unique GTPases involved in membrane fission and fusion events throughout the cell. Our goal is to understand the dynamic structural properties of these proteins and correlate them with their diverse cellular functions. Dynamin is essential for endocytosis and vesiculation events in the cell. Additional dynamin family members have been implicated in a variety of fundamental cellular processes, including mitochondrial fission and fusion, anti-viral activity, cell plate formation and chloroplast biogenesis. Among these proteins, self-assembly and oligomerization into ordered structures is a common characteristic and, for the majority, is essential for their function. Although there is a wealth of information regarding dynamin, little is known about the structural properties of dynamin-related proteins. To determine if a common mechanism of action exists among the dynamin family members, we examined the structure and function of Dnm1, a yeast dynamin family member involved in mitochondria fission and human dynamins involved in mitochondria fusion (Opa1 & Mfn1). A balance between mitochondrial membrane fusion and fission is required for normal mitochondrial morphology and function. Mitofusins have been shown to mediate mitochondrial outer membrane fusion in mammalian cells while Opa1 has been shown to play a role in the fusion of the inner mitochondrial membrane. Both mammalian mitofusins contain a large cytosolic GTPase domain at their N-terminus followed by two transmembrane domains and a short C-terminal domain. Similar to other dynamin family members, it is predicted that GTP binding and hydrolysis drive mitofusin conformational changes that mediate membrane fusion. However, the precise mechanism for mitofusin-mediated membrane fusion remains unclear. It is still unknown if mitochondrial outer membrane fusion proceeds through the canonical steps of tethering, docking, fusion, and disassembly. Similar to the SNARE protein complex, we expect that mitofusins will interact with each other on opposing membranes, undergo a conformational change that drives the membranes close enough to overcome the activation energy barrier for fusion, and after fusion disassemble to be available for the next round of fusion. To gain insight into the conformational changes that lead to membrane fusion we are examining the structure of mitofusins in a lipid bilayer. As with other transmembrane proteins, mitofusins are difficult to express and purify in their full-length state and are poor candidates for crystallography. Nevertheless, we have optimized a purification and liposome-incorporation strategy for full-length mitofusin 1, which can be visualized for the first time by cryo-EM. These electron micrographs show networks of tightly tethered proteoliposomes with electron dense seams. This suggests that mitofusin can tether proteoliposomes by forming oligomers that interact in trans between synthetic membranes. In addition, we have established a fusion assay wherein fluorescent lipids can be dequenched upon lipid mixing as a result of the fusion of two populations of proteoliposomes. This preliminarily data suggests that mitofusin 1 alone is sufficient to drive membrane tethering, docking, and lipid mixing between distinct liposomes Previously, in collaboration with Dr. David Chan from Cal Tech, we examined the structure of OPA1 by negative stain and cryo electron microscopy. Mutations in OPA1 (autosomal dominant optic atrophy) can lead to an inherited neuropathy of the retinal ganglion cells. In the cell, OPA1 has been shown to be essential for the fusion of the inner mitochondrial membranes, but its mechanism of action remains poorly understood. Addition of OPA1 to liposomes containing cardiolipin results in enhanced GTP hydrolysis rate and promotes OPA1 to self-assemble into helical arrays around the lipid, forming protein-lipid tubes. Currently we analyzing the structure of OPA1 by biochemistry and cryo-EM methods and exploring different constructs and nucleotide states of the protein.