Faculty of Biology, Department of Biochemistry and Functional Proteomics, Institute of Biology II, University of Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany.
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
Nat Commun. 2017 May 9;8:15272. doi: 10.1038/ncomms15272.
Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here we introduce a method that enables charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins with dual or multiple locations and the substrates of distinct protein import pathways. We demonstrate the specificity and versatility of this ImportOmics method by targeting import factors in mitochondria and glycosomes, which demonstrates its potential for globally studying protein import and inventories of organelles.
蛋白质向细胞器的输入对于所有真核生物都是必不可少的，这是由多蛋白易位子机器协助完成的。分析蛋白质是否被运输到细胞器主要依赖于单个成分。这使得关于输入蛋白质的知识不完整，限制了我们对细胞器发生和功能的理解。在这里，我们介绍了一种能够绘制细胞器输入组图谱的方法。该方法依赖于诱导型 RNAi 介导的易位子必需亚基的敲低，以损害输入并进行定量质谱分析。为了突出其潜力，我们建立了布氏锥虫的线粒体输入组图谱，其中包含 1120 种蛋白质，包括 331 种新的候选蛋白质。此外，该方法还允许鉴定具有双重或多重位置的蛋白质和不同蛋白输入途径的底物。我们通过靶向线粒体和糖体中的输入因子来证明这种 ImportOmics 方法的特异性和多功能性，这表明它具有用于全面研究蛋白质输入和细胞器目录的潜力。
