Manchester Institute of Biotechnology, Faculty of Biology, Medicine and Health, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
Division of Evolution and Genomic Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Oxford Road, Manchester M13 9PL, UK.
Int J Mol Sci. 2022 Aug 1;23(15):8532. doi: 10.3390/ijms23158532.
The quantification of low abundant membrane-binding proteins such as transcriptional factors and chaperones has proven difficult, even with the most sophisticated analytical technologies. Here, we exploit and optimise the non-invasive Fluorescence Correlation Spectroscopy (FCS) for the quantitation of low abundance proteins, and as proof of principle, we choose two interacting proteins involved in the fission of mitochondria in yeast, Fis1p and Mdv1p. In , the recruitment of Fis1p and Mdv1p to mitochondria is essential for the scission of the organelles and the retention of functional mitochondrial structures in the cell. We use FCS in single GFP-labelled live yeast cells to quantify the protein abundance in homozygote and heterozygote cells and to investigate the impact of the environments on protein copy number, bound/unbound protein state and mobility kinetics. Both proteins were observed to localise predominantly at mitochondrial structures, with the Mdv1p bound state increasing significantly in a strictly respiratory environment. Moreover, a compensatory mechanism that controls Fis1p abundance upon deletion of one allele was observed in Fis1p but not in Mdv1p, suggesting differential regulation of Fis1p and Mdv1p protein expression.
即使使用最先进的分析技术，也很难定量检测低丰度膜结合蛋白，如转录因子和伴侣蛋白。在这里，我们利用并优化了非侵入性荧光相关光谱（FCS）来定量检测低丰度蛋白质，作为原理验证，我们选择了两种相互作用的蛋白质，它们参与酵母中线粒体的裂变，即 Fis1p 和 Mdv1p。在酵母中，Fis1p 和 Mdv1p 招募到线粒体对于细胞器的分裂以及细胞中功能性线粒体结构的保留是必不可少的。我们使用 FCS 在单个 GFP 标记的活酵母细胞中定量检测纯合子和杂合子细胞中的蛋白质丰度，并研究环境对蛋白质拷贝数、结合/未结合蛋白状态和迁移动力学的影响。两种蛋白质都主要定位于线粒体结构，Mdv1p 的结合状态在严格的呼吸环境中显著增加。此外，在 Fis1p 缺失一个等位基因时观察到一种控制 Fis1p 丰度的补偿机制，但在 Mdv1p 中没有观察到，这表明 Fis1p 和 Mdv1p 蛋白表达的调控存在差异。
