Mechanisms of tether function in endolysosomal trafficking - Renewal - Resubmission 01

项目摘要 蛋白质通过膜区室的准确运输取决于特异性的蛋白质转运的能力。 囊泡相互识别并进行有效的融合。精确度部分由分子水平决定 系链，系链是与囊泡决定簇结合并促进特定囊泡-囊泡的多价复合物， 联系人.内体-溶酶体运输是处理从细胞表面摄取的货物所必需的， 而且矛盾的是，它也用于一些分泌细胞器的形成。两个关键的系绳参与 内溶酶体运输是CORVET（C类核心空泡/内体束缚）和HOPS（同型 融合和蛋白分选）。两者都是异源六聚体复合物，其在细胞和生物体中的重要性 编码CORVET/HOPS亚基的基因中的人类变异体与编码CORVET/HOPS亚基的基因中的人类变异体相关联， 粘多糖沉积症、肾功能不全、神经变性和癌症以及其他疾病。在 在细胞水平，CORVET和HOPS都在同型融合中起作用，即，允许具有相同Rab的两个囊泡 决定性的因素是互相认识。推导了CORVET/HOPS的作用机制 主要通过体外重构实验。然而，目前模式的关键方面还有待测试 在体内，包括重要的机制细节，如复合物是否稳定地与 膜，单个复合物是否经历循环组装/拆卸，以及是否不同 亚基具有不同的循环动力学。此外，系链在囊泡上的组装状态是未知的， 这是一个突出的问题，因为系链亚基在结构上类似于形成大组件的外壳蛋白 在膜上。在包括模式生物嗜热四膜虫（Tetrahymena thermophila）在内的纤毛虫谱系中， 复合体丢失了。与此同时，CORVET复合物在这些细胞中繁殖， 变得专门用于不同的内溶酶体途径。由于其特定的进化历史， 凭借实验优势，四膜虫提供了一个独特的新系统来分析这些通用的系绳。 要解决的问题包括与囊泡相关的CORVET复合物的拷贝数， 全复合物和单个亚基的动力学，以及特定蛋白质-蛋白质的作用 交互.实验将基于生物化学分析、相关光和电子的结合 显微镜观察以内源水平表达的标记蛋白，细胞融合以检测蛋白 动力学用相应的光镜和电镜观察细胞膜的超微结构 通过标记的蛋白质装饰的隔室，允许详细的体内分析系链功能。

Project Summary The accurate trafficking of proteins through membrane compartments depends on the ability of specific vesicles to recognize one another and undergo efficient fusion. Accuracy is conferred in part by molecular tethers, which are multivalent complexes that bind to vesicle determinants and foster specific vesicle-vesicle contacts. Endosome-lysosome trafficking is required for the processing of cargo taken up from the cell surface, and also paradoxically for the formation of some secretory organelles. Two key tethers involved in endolysosomal trafficking are CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and protein sorting). Both are hetero-hexameric complexes, whose importance in cell and organismal physiology is shown by the fact that human variants in the genes encoding CORVET/HOPS subunits are linked with mucopolysaccharidosis, renal dysfunction, neurodegeneration and cancer, among other diseases. At the cellular level, CORVET and HOPS both function in homotypic fusion, i.e., allow two vesicles with the same Rab determinants to recognize one another. The mechanisms of action of CORVET/HOPS have been deduced primarily via in vitro reconstitution experiments. However, key aspects of current models have yet to be tested in vivo, including important mechanistic details such as whether the complexes are stably associated with membranes, whether individual complexes undergo cyclical assembly/disassembly, and whether different subunits have distinct cycling dynamics. In addition, the assembly state of tethers on vesicles is unknown, a salient question because the tether subunits are structurally similar to coat proteins that form large assemblies on membranes. In the lineage of ciliates including the model organism Tetrahymena thermophila, the HOPS complex was lost. Concurrently, CORVET complexes multiplied in these cells, with individual complexes becoming specialized for distinct endolysosomal pathways. Due to its specific evolutionary history combined with experimental strengths, Tetrahymena offers a unique new system to analyze these universal tethers. Issues to be addressed include the copy number of CORVET complexes associated with vesicles, the dynamics of both the holo-complexes and individual subunits, and the role of specific protein-protein interactions. Experiments will be based on a combination of biochemical analysis, correlated light and electron microscopy to follow tagged proteins expressed at endogenous levels, and cell fusion to detect protein dynamics. Correlative light and electron microscopy will be used to visualize the ultrastructure of membrane compartments decorated by tagged proteins, allowing for detailed in vivo analysis of tether function.