Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA.
Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA.
Nat Chem Biol. 2019 Aug;15(8):830-837. doi: 10.1038/s41589-019-0325-3. Epub 2019 Jul 18.
Non-vesicular lipid transport between bilayers at membrane contact sites plays important physiological roles. Mechanistic insight into the action of lipid-transport proteins localized at these sites requires determination of the distance between bilayers at which this transport can occur. Here we developed DNA-origami nanostructures to organize size-defined liposomes at precise distances and used them to study lipid transfer by the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain of extended synaptotagmin 1 (E-Syt1). Pairs of DNA-ring-templated donor and acceptor liposomes were docked through DNA pillars, which determined their distance. The SMP domain was anchored to donor liposomes via an unstructured linker, and lipid transfer was assessed via a Förster resonance energy transfer (FRET)-based assay. We show that lipid transfer can occur over distances that exceed the length of an SMP dimer, which is compatible with the shuttle model of lipid transport. The DNA nanostructures developed here can also be adapted to study other processes occurring where two membranes are closely apposed to each other.
双层膜之间的非囊泡脂质转运在膜接触位点发挥着重要的生理作用。要了解定位于这些位点的脂质转运蛋白的作用机制，就需要确定能够发生这种转运的双层膜之间的距离。在这里，我们开发了 DNA 折纸纳米结构，将大小确定的脂质体在精确的距离处进行组织，并用它们来研究通过延伸突触融合蛋白 1（E-Syt1）的突触融合蛋白样线粒体脂质结合蛋白（SMP）结构域进行的脂质转运。通过 DNA 柱将 DNA 环模板化的供体和受体脂质体对接，从而确定它们的距离。SMP 结构域通过无规连接子锚定在供体脂质体上，并通过荧光共振能量转移（FRET）测定法评估脂质转移。我们表明，脂质转移可以在超过 SMP 二聚体长度的距离上发生，这与脂质转运的穿梭模型是兼容的。这里开发的 DNA 纳米结构也可以适应于研究其他在两个膜紧密贴合的情况下发生的过程。
