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Mechanism of secretory cargo sorting at the trans-Golgi Network

跨高尔基体网络的分泌货物分类机制

基本信息

批准号：
323442537
负责人：
Professorin Dr. Julia von Blume, Ph.D.
金额：
--
依托单位：
Department of Cell Biology
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/323442537?language=en
关键词：
Mechanism secretory cargo sorting trans

项目摘要

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.

对运输到细胞表面或从细胞分泌的蛋白质进行分选，对于胰岛素、胶原蛋白、抗体、表面受体、分泌的蛋白酶和GPCRs等蛋白质的胞外活性至关重要。细胞内蛋白质分布的一个主要调节因素是跨高尔基体网络(TGN)，它将分泌的蛋白质分选到特定的载体中，将它们运送到最终目的地。通过甘露糖-6-磷酸受体对TGN上的溶酶体水解酶进行分选是非常清楚的。然而，对分泌蛋白的分选机制仍然知之甚少。我们最近揭示了一种新的过程，它将细胞质肌动蛋白细胞骨架与膜锚定的钙离子ATPase SPCA1和腔内钙结合蛋白Cab45连接到TGN的一类分泌蛋白亚集。然而，监管这些组成部分之间相互作用的潜在机制尚不清楚。因此，我们建议采取一种办法来解决这一至关重要的问题。人类细胞中的质谱学和现代基因组编辑技术将被应用于识别这一分类途径的其他组成部分。超分辨率显微镜和基于DNA-Paint的多路复用将被用来绘制这些组件的TGN组织图。此外，活细胞显微镜将确定复合体的时空组织及其与钙离子依赖的分类的联系。最后，我们将在体外对无细胞系统和模型膜中已识别的成分进行重组，以确定对分泌物进行分类所需的最小机械。总之，我们的发现将揭示TGN对蛋白质进行分类并将其定位到各自目的地的机制，这一过程对细胞比较、组织组织和功能至关重要。