Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation.
Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation.
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DOI：
10.7554/elife.24463
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发表时间：
2017-04-25
期刊：
影响因子：
7.7
通讯作者：
Ting AY
中科院分区：
文献类型：
--
作者：
Hung V;Lam SS;Udeshi ND;Svinkina T;Guzman G;Mootha VK;Carr SA;Ting AY
细胞器的面向胞质溶胶的膜含有能够进行信号转导、调节形态和运输、蛋白质输入和输出以及其他专门过程的蛋白质。通过传统的生物化学分级分离发现这些蛋白质可能受到污染物和关键组分损失的困扰。使用过氧化物酶介导的邻近生物素化，我们捕获并鉴定了活的人成纤维细胞的线粒体外膜（OMM）和内质网膜（ERM）上的内源性蛋白质。分别有137和634个蛋白质的蛋白质组具有高度特异性，并突出了94个潜在的新线粒体或ER蛋白。数据集交叉鉴定了可能定位于Escheria-ER接触位点的蛋白候选物。我们发现，一个候选人，尾锚定，PDZ结构域包含OMM蛋白SYNJ 2BP，显着增加线粒体接触粗糙ER过表达时。免疫沉淀-质谱鉴定核糖体结合蛋白1（RRBP 1）为SYNJ 2BP的ERM结合伴侣。我们的研究结果突出了接近生物素化的力量，以深入了解细胞内膜的分子组成和功能。
The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BP’s ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes.