Regulation of ER to Golgi Transport by Luminal Calcium

描述（由申请人提供）：适当的细胞功能和避免疾病需要亚细胞靶向和转运囊泡与受体膜的融合。囊泡外壳、小笼、系索和SNAREs构成了膜系统囊泡运输机制的核心。虽然这些蛋白家族的主要功能点已经确定，但在意想不到的步骤中，新的功能角色和连接和整合其功能的新的调节相互作用不断出现。在哺乳动物中，内质网到高尔基体转运是分泌途径中的限速步骤，也是与转运相关疾病最相关的步骤，已经在体内和体外得到了广泛的研究。从广义上讲，内质网到高尔基体的运输包括：1)由COPII外壳介导的货物分选和囊泡出芽；2)由rab1、p115和TRAPP1介导的同型COPII囊泡系结和ER/高尔基SNAREs介导的融合形成前高尔基细胞器，称为囊泡管簇（vtc）；3) vtc介导的货物沿微管分拣和运输导致与高尔基体融合。该项目将采用活细胞Ca2+测量，完整哺乳动物细胞系中ER/高尔基转运的动力学分析，以及亚细胞部分转运现象的体外重构，以剖析腔内Ca2+， Ca2+传感器ALG-2和COPII涂层在该途径特定阶段的作用。这些研究的动机是广泛的指导假设，即从前高尔基分泌细胞器逃逸的腔内Ca2+与运输机制相互作用并调节运输机制，显著影响货物运输和/或分类。所提出的实验有三个具体目的：1)测试内质网和中间室之间腔内Ca2+浓度急剧下降的假设。2)验证ALG-2和sec31A转导腔内Ca2+信号调控VTC形成和功能的多个步骤的假设。3)识别COPII囊泡上的Ca2+靶点。

DESCRIPTION (provided by applicant): Proper cell function and avoidance of disease requires subcellular targeting and fusion of transport vesicles with acceptor membranes. Vesicle coats, rabs, tethers and SNAREs make up the core of the vesicle trafficking machinery for the endomembrane system. Although the primary point of function of each of these protein families is established, new functional roles at unexpected steps, and new regulatory interactions linking and integrating their functions continue to emerge. In mammals, ER-to-Golgi transport, which represents the rate-limiting step in the secretory pathway and the step most relevant to transport-related diseases, has been extensively characterized in vivo and reconstituted in vitro. In broad terms, ER-to-Golgi transport has been shown to comprise: 1) cargo sorting and vesicle budding mediated by the COPII coat; 2) homotypic COPII vesicle tethering mediated by rab1, p115 and TRAPP1, and fusion mediated by ER/Golgi SNAREs to form pre-Golgi organelles called vesicular tubular clusters (VTCs); and 3) VTC-mediated cargo sorting and transport along microtubules leading to fusion with the Golgi. This project will employ live cell Ca2+ measurements, kinetic assays of ER/Golgi transport in intact mammalian cell lines and in vitro reconstitution of transport phenomena from subcellular fractions to dissect the roles of luminal Ca2+, the Ca2+ sensor ALG-2 and the COPII coat in specific stages of this pathway. The studies are motivated by the broad guiding hypotheses that luminal Ca2+ escaping from pre- Golgi secretory organelles interacts with and regulates the trafficking machinery, significantly impacting cargo transport and/or sorting. The proposed experiments fall under three specific aims: 1) Test the hypothesis that luminal Ca2+ concentrations drop dramatically between the ER and the intermediate compartment. 2) Test the hypothesis that ALG-2 and sec31A transduce the luminal Ca2+ signal to regulate multiple steps in VTC formation and function. 3) Identify Ca2+ targets on COPII vesicles.