Regulation of ER to Golgi Transport by Luminal Calcium

描述(由申请人提供)：正确的细胞功能和避免疾病需要亚细胞靶向和运输囊泡与受体膜的融合。囊泡外衣、拉布、绳索和圈套构成了膜内膜系统囊泡运输机械的核心。尽管这些蛋白质家族的主要功能点已经确定，但在意想不到的步骤中，新的功能角色和新的调节相互作用不断出现，连接和整合它们的功能。在哺乳动物中，内质网到高尔基体的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，已经在体内得到了广泛的表征，并在体外进行了重组。总的来说，内质网到高尔基体的运输包括：1)由COPII外壳介导的货物分拣和囊泡萌发；2)由Rab1、p115和TRAPP1介导的同型COPII囊泡拴系，以及由ER/高尔基圈套介导的融合形成称为囊状小管簇(VTCs)的前高尔基体细胞器；以及3)VTC介导的货物分拣和沿微管运输导致与高尔基体融合。这个项目将使用活细胞钙测量，在完整的哺乳动物细胞系中ER/高尔基体运输的动力学分析，以及从亚细胞组分的体外运输现象的重建，以剖析腔钙，钙传感器ALG-2和COPII外壳在这一途径的特定阶段中的作用。这些研究的动机是广泛的指导性假设，即从高尔基体前分泌细胞器逃逸的鲁米诺钙与运输机制相互作用并对其进行调节，显著影响货物的运输和/或分拣。拟议的实验有三个特定的目的：1)测试内质网和中间隔室之间的腔内钙浓度急剧下降的假设。2)验证ALG-2和sec31a信号转导腔内钙信号调节VTC形成和功能的多个步骤的假说。3)确定COPII囊泡上的钙靶标。

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.