Department of Physics, University of York, YO10 5DD York, UK.
Department of Chemistry and Molecular Biology, University of Gothenburg, 405 30 Gothenburg, Sweden.
Methods. 2021 Sep;193:46-53. doi: 10.1016/j.ymeth.2020.05.003. Epub 2020 May 6.
Membrane proteins play key roles at the interface between the cell and its environment by mediating selective import and export of molecules via plasma membrane channels. Despite a multitude of studies on transmembrane channels, understanding of their dynamics directly within living systems is limited. To address this, we correlated molecular scale information from living cells with real time changes to their microenvironment. We employed super-resolved millisecond fluorescence microscopy with a single-molecule sensitivity, to track labelled molecules of interest in real time. We use as example the aquaglyceroporin Fps1 in the yeast Saccharomyces cerevisiae to dissect and correlate its stoichiometry and molecular turnover kinetics with various extracellular conditions. We show that Fps1 resides in multi tetrameric clusters while hyperosmotic and oxidative stress conditions cause Fps1 reorganization. Moreover, we demonstrate that rapid exposure to hydrogen peroxide causes Fps1 degradation. In this way we shed new light on aspects of architecture and dynamics of glycerol-permeable plasma membrane channels.
膜蛋白在细胞与其环境的界面处发挥关键作用，通过质膜通道介导分子的选择性内外运输。尽管对跨膜通道进行了大量研究，但对其在活系统中的动力学的理解仍然有限。为了解决这个问题，我们将来自活细胞的分子尺度信息与它们微环境的实时变化相关联。我们采用具有单分子灵敏度的超分辨率毫秒荧光显微镜，实时跟踪标记的感兴趣分子。我们以酵母酿酒酵母中的水甘油通道蛋白 Fps1 为例，剖析并将其化学计量和分子周转率动力学与各种细胞外条件相关联。我们表明，Fps1 存在于多四聚体簇中，而高渗和氧化应激条件会导致 Fps1 重组。此外，我们证明快速暴露于过氧化氢会导致 Fps1 降解。通过这种方式，我们揭示了甘油通透性质膜通道的结构和动力学的新方面。
