Mechanism of secretory cargo export from the TGN

项目摘要 可溶性蛋白质的分选对于运输到细胞内区室和从细胞中分泌是必不可少的 维持细胞和组织的稳态细胞内蛋白质分布的主要调节器是反式高尔基体 TGN网络将分泌蛋白分类到特定的载体中，以将它们运送到最终目的地。 通过甘露糖-6-磷酸受体对TGN的溶酶体水解酶进行分选是公认的。 然而，分泌蛋白的分选机制仍然知之甚少。最近发现的 TGN分泌物的基于Ca 2+的分选开始揭示细胞 从高尔基体的居民和货物分配到其他细胞隔室的分泌货物排序。这 基于Ca 2+的分选涉及细胞质肌动蛋白细胞骨架，它通过膜锚定Ca 2 + ATP酶SPCA 1和腔Ca 2+结合蛋白Cab 45是一个分泌蛋白的子集， TGN。然而，调节这些组成部分之间相互作用的基本机制并不是 清楚我们采取跨学科和高度创新的方法来解决这一基本问题， 重要性我们的目标包括在模型膜中体外重构这些组分，以鉴定 分泌物分类所需的最小机械。现代基因组编辑技术 人类细胞加上荧光和超分辨率显微镜将提供新的见解， 活细胞中各组分的功能性相互连接。总之，我们的发现将揭示 TGN将蛋白质分类并靶向其各自目的地的机制， 对于细胞区室化、组织结构和功能至关重要。

Project Summary Sorting of soluble proteins for transport to intracellular compartments and secretion from cells is essential for cell and tissue homeostasis. 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 established. However, the sorting mechanism for secreted proteins remains poorly understood. The recent discovery of a Ca2+-based sorting of secretory cargo at the TGN is beginning to uncover the mechanism by which cells sort secretory cargoes from Golgi residents and cargoes destined to the other cellular compartments. This Ca2+ based sorting involves the cytoplasmic actin cytoskeleton, which through membrane-anchored Ca2+ ATPase SPCA1 and the luminal Ca2+ binding protein Cab45 sorts of a subset of secretory proteins at the TGN. The underlying mechanism regulating the interplay between these components, however, is not clear. We take an interdisciplinary and highly innovative approach to address this issue of fundamental importance. Our aims include in vitro reconstitution of these components in model membranes to identify the minimal machinery required for the sorting of secretory cargoes. Modern genome editing techniques in human cells coupled to fluorescence and super-resolution microscopy will provide new insights into the functional interconnection of the components in living cells. 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 compartmentation, tissue organization, and function.