Hung Victoria, Lam Stephanie S, Udeshi Namrata D, Svinkina Tanya, Guzman Gaelen, Mootha Vamsi K, Carr Steven A, Ting Alice Y
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, United States.
Broad Institute of MIT and Harvard, Cambridge, United States.
Elife. 2017 Apr 25;6:e24463. doi: 10.7554/eLife.24463.
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.
细胞器面向胞质溶胶的膜含有能够实现信号转导、形态调控和运输、蛋白质进出口及其他特殊过程的蛋白质。通过传统生化分级分离法发现这些蛋白质可能会受到污染物的影响以及关键成分的丢失。利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线粒体外膜（OMM）和内质网膜（ERM）上的内源性蛋白质。分别有137种和634种蛋白质的蛋白质组具有高度特异性，并突出显示了94种潜在的新型线粒体或内质网蛋白质。数据集交叉分析确定了可能定位于线粒体-内质网接触位点的蛋白质候选物。我们发现一个候选物，即尾部锚定的、含PDZ结构域的线粒体外膜蛋白SYNJ2BP，过表达时会显著增加线粒体与粗面内质网的接触。免疫沉淀-质谱分析确定核糖体结合蛋白1（RRBP1）为SYNJ2BP的内质网膜结合伴侣。我们的结果突出了邻近生物素化在深入了解细胞内膜的分子组成和功能方面的作用。
