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

项目摘要 蛋白质通过膜间隔的准确运输依赖于特定的 囊泡相互识别并进行有效的融合。准确性在一定程度上是由分子决定的 系链，这是与囊泡决定簇结合并培育特定囊泡-囊泡的多价化合物 联系人。处理从细胞表面取出的货物需要内切体-溶酶体运输， 矛盾的是，一些分泌器的形成也是如此。涉及到的两个关键系绳 内溶体运输是CORVET(C类核心空泡/内体连接)和啤酒花(同型 融合和蛋白质分选)。两者都是异六聚体，它们在细胞和生物体中都具有重要意义 人类编码CORVET/HOPS亚单位的基因的变异是相连的，这一事实表明了生理学 患有粘多糖症、肾功能障碍、神经退行性变和癌症等疾病。在 在细胞水平上，CORVET和HOP在同型融合中都起作用，即允许两个囊泡具有相同的Rab 相互识别的决定因素。对CORVET/HOPS的作用机制进行了推导 主要通过体外重建实验。然而，当前车型的关键方面仍有待测试 体内，包括重要的机制细节，如复合体是否稳定地与 膜，单个复合体是否经历循环组装/拆卸，以及是否不同 亚基具有明显的循环动力学。此外，小泡上系绳的组装状态未知， 突出的问题是因为系链亚单位在结构上类似于形成大组装的外壳蛋白 在膜上。在纤毛虫的谱系中，包括模式生物嗜热四膜虫，啤酒花 情结消失了。同时，CORVET复合体在这些细胞中繁殖，具有单独的复合体 成为不同的内溶酶体途径的专门化。由于其特定的进化史结合在一起 凭借实验优势，四膜虫提供了一种独特的新系统来分析这些通用系绳。 需要解决的问题包括与囊泡相关的CORVET复合体的拷贝数、 全息复合体和单个亚基的动力学以及特定蛋白质-蛋白质的作用 互动。实验将基于生化分析、相关光和电子的组合。 显微镜跟踪内源水平表达的标记蛋白，并通过细胞融合检测蛋白 动力学。相关的光学和电子显微镜将被用来显示膜的超微结构 由标记蛋白质装饰的隔间，允许对系绳功能进行详细的体内分析。

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.