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.

分泌和内吞途径的区室之间的细胞内交通是由基于囊泡的载体介导的。用于许多细胞器的载体的蛋白质组是不明确的，因为囊泡中间体是短暂的、低丰度且难以纯化。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，以确定不同内体衍生的囊泡的内容，这些囊泡将进入跨高尔基体网络（TGN）。先前显示高尔金卷曲螺旋蛋白 golgin-97 和 GCC88 在 TGN 捕获内体衍生的囊泡，分别重新定位到线粒体，并通过细胞器蛋白质组学确定随后重新定位的囊泡的内容。我们的研究结果揭示了 45 个整合膜蛋白和 51 个外周膜蛋白被 golgin-97 重新路由，证据表明 golgin-97 和 GCC88 共享一类不同的囊泡，以及各个 golgins 特有的各种货物。这些结果说明了通过将急性细胞重连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。