Molecular analysis of lipid transporters at the single vesicle level

脂类转运蛋白是一种完整的膜蛋白，在脂类跨细胞膜运输中发挥核心作用，从而控制包括囊泡生物发生、细胞信号转导、形态发生和迁移在内的众多细胞过程。它们可以分为两类：(I)能量非依赖性转运蛋白，称为扰乱酶，它使脂类在整个双层中的分布随机化；(Ii)由ATP驱动的载体转运蛋白，包括保守的P型ATPase亚家族成员，它主动地将特定的脂类从一个膜小叶转移到另一个膜小叶。目前的概念认为，脂类转运蛋白的活性受脂类双层组成的调节，并且脂类转运的动态过程可能有助于驱动膜弯曲和囊泡萌发。然而，这些概念缺乏直接的实验证据。一方面，这可以归因于处理完整的膜蛋白的困难，以及它们在生产、纯化和与膜脂组装的特性方面的微妙。另一方面，在分子水平上研究膜蛋白需要复杂的技术。我们的方法将从这两个方面解决这一问题，方法是建立基于巨大单层囊泡的明确的重组系统和最先进的成像技术，以实现对不同组成复杂程度(从简单的脂类混合物到细胞状脂类混合物)的脂类转运体的分子分析。将纯化的转运蛋白重组为巨大的单层囊泡，将使我们能够通过时间分辨荧光显微镜研究它们在特定脂质环境中的活性。我们期望这些在单个囊泡水平上的研究将揭示(I)脂组成对脂转运蛋白活性的影响，以及(Ii)依赖于ATP的脂转运蛋白在膜弯曲和囊泡形成中的作用。总之，本文提出的膜蛋白质化学和成像技术的强大结合将为理解作为细胞重要组成部分的脂转运蛋白的分子特征、调控和工作机制提供重要的新见解。

Lipid transporters are integral membrane proteins that play a central role in moving lipids across cellular membranes, thereby controlling numerous cellular processes including vesicle biogenesis, cell signaling, morphogenesis and migration. They can be classified into two categories: (i) energy-independent transporters, termed scramblases, which randomize the distribution of lipids across the bilayer and (ii) ATP-driven, vectorial transporters, including members of a conserved subfamily of P-type ATPases, that actively translocate specific lipids from one membrane leaflet to the other. Current concepts suggests that the activity of lipid transporters is regulated by the lipid bilayer composition and that the dynamic process of lipid translocation may help driving membrane bending and vesicle budding. However, direct experimental evidence for these concepts is lacking. On one hand, this can be attributed to difficulties in handling of integral membrane proteins, their delicacy in production, purification, and characterization of their assembly with membrane lipids. On the other hand, sophisticated techniques are required for studying membrane proteins on the molecular level. Our approach will tackle this problem from both these sides by establishing defined reconstituted systems based on giant unilamellar vesicles and state-of-the-art imaging techniques to enable the molecular analysis of lipid transporters at different levels of compositional complexity (from simple lipid mixtures to cell-like lipid mixtures). Reconstitution of the purified transporters into giant unilamellar vesicles will allow us to study their activity in defined lipid environments by time-resolved fluorescence microscopy. We expect that these studies at the single vesicle level should reveal (i) the impact of lipid composition on the activity of the lipid transporters, and (ii) the role of ATP-dependent lipid translocation in membrane bending and vesiculation. In conclusion, the powerful combination of membrane protein chemistry and imaging techniques as proposed here should provide important new insight into the molecular characteristics, regulation and working mechanism of lipid transporters as vital components of cells.