Mechanism of secretory cargo sorting at the trans-Golgi Network

分选用于运输至细胞表面或用于从细胞分泌的蛋白质对于蛋白质如胰岛素、胶原蛋白、抗体、表面受体、分泌的蛋白酶和GPCR（仅举几例）的细胞外活性是必不可少的。细胞内蛋白质分布的主要调节器是反式高尔基体网络（trans-Golgi network，TGN），其将分泌蛋白质分类到特定载体中以将它们运输到其最终目的地。通过甘露糖-6-磷酸受体对TGN的溶酶体水解酶进行分选是很好理解的。然而，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，将细胞质肌动蛋白细胞骨架连接到膜锚定的Ca 2 + ATP酶SPCA 1和管腔Ca 2+结合蛋白Cab 45，以分类TGN的分泌蛋白亚群。然而，调节这些组成部分之间相互作用的基本机制尚不清楚。因此，我们建议采取一种办法来处理这一具有根本重要性的问题。质谱和人类细胞中的现代基因组编辑技术将用于识别这种分选途径的其他组分。超分辨率显微镜和DNA-PAINT为基础的多路复用将被用来映射这些组件的TGN组织。此外，活细胞显微镜将确定时空组织及其与复合物的Ca 2+依赖性分选的联系。最后，我们将在体外重组的无细胞系统和模型膜，以确定所需的最小机械的分泌货物的排序。总而言之，我们的研究结果将揭示TGN将蛋白质分类并靶向其各自目的地的机制，这是一个对细胞比较，组织和功能至关重要的过程。

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.