Departments of Biology, Genetics, and Chemistry, Stanford University, Stanford, CA 94305, USA.
The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Cell. 2023 Jul 20;186(15):3307-3324.e30. doi: 10.1016/j.cell.2023.05.044. Epub 2023 Jun 28.
The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.
能够一次性在活细胞中绘制数千种内源性蛋白质的运输图谱，将揭示目前显微镜和质谱都无法看到的生物学现象。在这里，我们报告了 TransitID，这是一种在活细胞中以纳米空间分辨率进行内源性蛋白质组运输无偏映射的方法。两种邻近标记（PL）酶，TurboID 和 APEX，被靶向到源和目的地隔室，并且通过顺序添加它们的小分子底物来串联进行两种酶的 PL。质谱鉴定出两种酶标记的蛋白质。使用 TransitID，我们绘制了细胞质和线粒体、细胞质和细胞核以及核仁与应激颗粒（SGs）之间的蛋白质组运输图谱，揭示了 SGs 在保护转录因子 JUN 免受氧化应激中的作用。TransitID 还鉴定了在巨噬细胞和癌细胞之间进行细胞间信号传递的蛋白质。TransitID 提供了一种强大的方法，可以根据细胞器或细胞类型的来源来区分蛋白质群体。
