Mechanism of secretory cargo sorting at the trans-Golgi Network

蛋白质转运到细胞表面或从细胞分泌是必不可少的蛋白质的细胞外活动，如胰岛素，胶原蛋白，抗体，表面受体，分泌蛋白酶和gpcr等等。细胞内蛋白质分布的一个主要调节因子是反式高尔基网络（TGN），它将分泌蛋白分类到特定的载体中，并将它们运送到最终目的地。甘露糖-6-磷酸受体在TGN上对溶酶体水解酶的分选是很清楚的。然而，对分泌蛋白的分选机制仍然知之甚少。我们最近发现了一个新的过程，将细胞质肌动蛋白骨架与膜锚定的Ca2+ atp酶SPCA1和腔内Ca2+结合蛋白Cab45连接到TGN的分泌蛋白子集。然而，调节这些成分之间相互作用的潜在机制尚不清楚。因此，我们建议采取一种方法来解决这一具有根本重要性的问题。质谱法和人类细胞中的现代基因组编辑技术将被应用于鉴定这一分选途径的其他成分。超分辨率显微镜和基于DNA-PAINT的多路复用将用于绘制这些成分的TGN组织。此外，活细胞显微镜将确定时空组织及其与Ca2+依赖的复合物分类的联系。最后，我们将在体外对无细胞系统和模型膜中鉴定的成分进行重构，以确定分选分泌货物所需的最小机械。总之，我们的研究结果将揭示TGN分类和靶向蛋白的机制，这一过程对细胞比较、组织组织和功能至关重要。

Sorting of proteins for transport to the cells surface or for secretion from cells is essential for the extracellular activities of proteins like insulin, collagens, antibodies, surface receptors, secreted proteases, and GPCRs to name a few. A major regulator of intracellular protein distribution is the trans-Golgi network (TGN) that sorts secretory proteins into specific carriers to transport them to their final destination. The sorting of lysosomal hydrolases at the TGN by the mannose-6-phosphate receptor is well understood. However, the sorting mechanism for secreted proteins remains poorly understood. We recently revealed a novel process that links the cytoplasmic actin cytoskeleton to the membrane anchored Ca2+ ATPase SPCA1 and the luminal Ca2+ binding protein Cab45 to sort of a subset of secretory proteins at the TGN. The underlying mechanism regulating the interplay between these components, however, is not clear. Thus we suggest an approach to address this issue of fundamental importance. Mass spectrometry and modern genome editing techniques in human cells, will be applied to identify other components of this sorting pathway. Super-resolution microscopy and DNA-PAINT based multiplexing will be used to map TGN organization of these components. Furthermore live cell microscopy will determine the spatio-temporal organization and its link to Ca2+ dependent sorting of the complexes. Finally we will apply in vitro reconstitution of identified components in cell-free systems and model membranes to identify the minimal machinery required for the sorting of secretory cargoes. Altogether, our findings will reveal the mechanism by which TGN sorts and targets proteins to their respective destination, a process that is essential for cell compartimentation, tissue organization and function.