School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan.
Department of Applied Chemistry and Biotechnology, Chiba University, Chiba, Japan.
Biochem Biophys Res Commun. 2019 Dec 3;520(2):237-242. doi: 10.1016/j.bbrc.2019.09.066. Epub 2019 Oct 5.
In the living cells, the majority of proteins does not work alone, but interact with other proteins or other biomolecules to maintain the cellular function, constituting a "protein community". Previous efforts on mass spectroscopy-based protein interaction networks, interactomes, have provided a picture on the protein community. However, these were static information after cells were disrupted. For a better understanding of the protein community in cells, it is important to know the properties of intracellular dynamics and interactions. Since hydrodynamic size and mobility of proteins are related into such properties, direct measurement of diffusional motion of proteins in single living cells will be helpful for uncovering the properties. Here we completed measurement of the diffusion and homo-oligomeric properties of 369 cytoplasmic GFP-fusion proteins in living yeast Saccharomyces cerevisiae cells using fluorescence correlation spectroscopy (FCS). The large-scale analysis showed that the motions of majority of proteins obeyed a two-component (i.e. slow and fast components) diffusion model. Remarkably, both of the two components diffused more slowly than expected monomeric states. In addition, further analysis suggested that more proteins existed as homo-oligomeric states in living cells than previously expected. Our study, which characterizes the dynamics of proteins in living cells on a large-scale, provided a global view on intracellular protein dynamics to understand the protein community.
在活细胞中，大多数蛋白质不是单独发挥作用，而是与其他蛋白质或其他生物分子相互作用，以维持细胞功能，构成一个“蛋白质群落”。基于质谱的蛋白质相互作用网络（互作组）的前期研究已经提供了蛋白质群落的图谱。然而，这些都是细胞被破坏后的静态信息。为了更好地了解细胞内的蛋白质群落，了解细胞内动力学和相互作用的性质非常重要。由于蛋白质的流体力学大小和迁移率与这些性质有关，因此直接测量单个活细胞中蛋白质的扩散运动将有助于揭示这些性质。在这里，我们使用荧光相关光谱（FCS）完成了对 369 个细胞质 GFP 融合蛋白在活酵母酿酒酵母细胞中的扩散和同型寡聚性质的测量。大规模分析表明，大多数蛋白质的运动符合双组分（即慢组分和快组分）扩散模型。值得注意的是，这两个组分的扩散速度都比预期的单体状态慢。此外，进一步的分析表明，在活细胞中，同型寡聚状态的蛋白质比以前预期的要多。我们的研究对活细胞中蛋白质的动力学进行了大规模的描述，为理解蛋白质群落提供了细胞内蛋白质动力学的整体视图。
