Molecular analysis of lipid transporters at the single vesicle level

脂质转运蛋白是一种完整的膜蛋白，在脂质跨细胞膜移动中起核心作用，从而控制许多细胞过程，包括囊泡生物发生、细胞信号传导、形态发生和迁移。它们可以分为两类：(i)能量无关的转运蛋白，称为超扰酶，它使脂质在双分子层上的分布随机化；（ii） atp驱动的矢量转运蛋白，包括p型atp酶的保守亚家族成员，它们主动地将特定的脂质从一个膜叶转移到另一个膜叶。目前的观点认为，脂质转运体的活性受脂质双分子层组成的调节，脂质转运的动态过程可能有助于驱动膜弯曲和囊泡出芽。然而，这些概念缺乏直接的实验证据。一方面，这可以归因于处理整体膜蛋白的困难，它们在生产、纯化和表征它们与膜脂组装时的精细性。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个问题，通过建立基于巨大单层囊泡和最先进的成像技术的定义重构系统，使脂质转运体的分子分析能够在不同的组成复杂性水平（从简单的脂质混合物到细胞样脂质混合物）。将纯化的转运蛋白重组成巨大的单层囊泡，将使我们能够通过时间分辨荧光显微镜研究它们在确定的脂质环境中的活性。我们希望这些单囊泡水平的研究能够揭示(i)脂质成分对脂质转运体活性的影响，以及（ii） atp依赖性脂质转运在膜弯曲和囊泡中的作用。综上所述，本文所提出的膜蛋白化学和成像技术的有力结合，将为了解作为细胞重要组成部分的脂质转运体的分子特征、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.