Quantitative characterisation of low abundant yeast mitochondrial proteins reveals compensation for haplo-insufficiency in different environments
Quantitative characterisation of low abundant yeast mitochondrial proteins reveals compensation for haplo-insufficiency in different environments
复制标题
低丰度酵母线粒体蛋白的定量表征揭示了不同环境中单倍体不足的补偿
DOI：
10.1101/2021.07.09.451775
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Manousaki A
中科院分区：
文献类型：
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作者：
Manousaki A
即使使用最先进的分析技术，转录因子和伴侣蛋白等低丰度膜结合蛋白的定量也被证明是困难的。在这里，我们利用并优化了非侵入性荧光相关光谱（FCS）来定量低丰度蛋白质，并作为原理证明，我们选择了酵母中参与线粒体裂变的两个相互作用蛋白，Fis1p和Mdv1p。在酿酒酵母中，线粒体中的Fis1p和Mdv1p的募集对于细胞器的分裂和细胞中功能性线粒体结构的保留至关重要。我们在单个gfp标记的活酵母细胞中使用FCS来量化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/非结合蛋白状态和迁移动力学的影响。观察到这两种蛋白主要定位于线粒体结构，在严格的呼吸环境中，Mdv1p结合状态显著增加。此外，在Fis1p中观察到一种补偿机制，在一个等位基因缺失时控制Fis1p的丰度，而在Mdv1p中则没有，这表明Fis1p和Mdv1p蛋白表达的差异调节。
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. InSaccharomyces cerevisiae, 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.