Biophysical Chemistry, Institute of Chemistry, Charles-Tanford Protein Center, University of Halle, Kurt-Mothes-Str. 3 A, 06120, Halle, Germany.
Sci Rep. 2020 Feb 20;10(1):3100. doi: 10.1038/s41598-020-59700-1.
Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies. They feature accessibility for manipulation and detection, but lack the compositional complexity needed to reconstitute complicated cellular processes. For the plasma membrane (PM), this gap was bridged by the establishment of giant PM vesicles (GPMVs). These native membranes have facilitated studies of protein and lipid diffusion, protein interactions, electrophysiology, fluorescence analysis of lateral domain formation and protein and lipid partitioning as well as mechanical membrane properties and remodeling. The endoplasmic reticulum (ER) is key to a plethora of biological processes in any eukaryotic cell. However, its intracellular location and dynamic and intricate tubular morphology makes it experimentally even less accessible than the PM. A model membrane, which will allow the afore-mentioned types of studies on GPMVs to be performed on ER membranes outside the cell, is therefore genuinely needed. Here, we introduce the formation of giant ER vesicles, termed GERVs, as a new tool for biochemistry and biophysics. To obtain GERVs, we have isolated ER membranes from Saccharomyces cerevisiae and fused them by exploiting the atlastin-like fusion protein Sey1p. We demonstrate the production of GERVs and their utility for further studies.
人工巨型囊泡已被证明在各种生物物理和生物化学研究中作为膜模型非常有用。它们具有可操作性和可检测性，但缺乏重新构建复杂细胞过程所需的组成复杂性。对于质膜（PM），通过建立巨型 PM 囊泡（GPMV）来弥合这一差距。这些天然膜促进了蛋白质和脂质扩散、蛋白质相互作用、电生理学、侧向域形成的荧光分析以及蛋白质和脂质分配以及机械膜特性和重塑的研究。内质网（ER）是真核细胞中众多生物过程的关键。然而，其细胞内位置和动态复杂的管状形态使其在实验中比 PM 更难以接近。因此，确实需要一种模型膜，使细胞外 ER 膜上能够进行上述类型的 GPMV 研究。在这里，我们介绍了内质网巨型囊泡（GERV）的形成，作为生物化学和生物物理学的新工具。为了获得 GERV，我们从酿酒酵母中分离出 ER 膜，并通过利用类星状融合蛋白 Sey1p 使其融合。我们证明了 GERV 的产生及其在进一步研究中的应用。
