MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
The Milner Therapeutics Institute, University of Cambridge, Cambridge, CB2 0AW, UK.
Nat Commun. 2020 Nov 25;11(1):5987. doi: 10.1038/s41467-020-19840-4.
Intracellular traffic between compartments of the secretory and endocytic pathways is mediated by vesicle-based carriers. The proteomes of carriers destined for many organelles are ill-defined because the vesicular intermediates are transient, low-abundance and difficult to purify. Here, we combine vesicle relocalisation with organelle proteomics and Bayesian analysis to define the content of different endosome-derived vesicles destined for the trans-Golgi network (TGN). The golgin coiled-coil proteins golgin-97 and GCC88, shown previously to capture endosome-derived vesicles at the TGN, were individually relocalised to mitochondria and the content of the subsequently re-routed vesicles was determined by organelle proteomics. Our findings reveal 45 integral and 51 peripheral membrane proteins re-routed by golgin-97, evidence for a distinct class of vesicles shared by golgin-97 and GCC88, and various cargoes specific to individual golgins. These results illustrate a general strategy for analysing intracellular sub-proteomes by combining acute cellular re-wiring with high-resolution spatial proteomics.
细胞内的分泌和内吞途径的隔室之间的物质交换是通过基于囊泡的载体进行介导的。许多细胞器的载体蛋白质组都还没有被明确界定，因为囊泡中间产物是短暂的、低丰度的，并且难以纯化。在这里，我们将囊泡重定位与细胞器蛋白质组学和贝叶斯分析相结合，以定义不同的内体衍生的囊泡的内容物，这些囊泡被运往高尔基体网络（TGN）。先前已经显示卷曲螺旋蛋白 golgin-97 和 GCC88 可以捕获内体衍生的囊泡在 TGN 处，我们将这两种蛋白分别重新定位到线粒体，然后通过细胞器蛋白质组学来确定随后重新路由的囊泡的内容物。我们的研究结果揭示了 45 种完整的和 51 种外周膜蛋白被 golgin-97 重新路由，这为 golgin-97 和 GCC88 共享的一类独特的囊泡提供了证据，以及各种特定于单个 golgin 的货物。这些结果说明了通过急性细胞重新布线与高分辨率空间蛋白质组学相结合来分析细胞内亚蛋白质组的一般策略。
