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以及运输囊泡与受体膜的融合。囊泡外套、拉布、系绳和圈套构成了内膜系统囊泡运输机制的核心。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作用以及连接和整合其功能的新的调控相互作用不断出现。在哺乳动物中，内质网到高尔基体的转运代表了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，已在体内得到了广泛的表征并在体外进行了重建。从广义上讲，内质网到高尔基体的运输已被证明包括：1）由 COPII 外壳介导的货物分类和囊泡出芽； 2) 由 rab1、p115 和 TRAPP1 介导的同型 COPII 囊泡束缚，以及由 ER/高尔基体 SNARE 介导的融合，形成称为囊泡管状簇 (VTC) 的前高尔基体细胞器； 3) VTC 介导的货物沿着微管分类和运输，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/高尔基体转运的动力学测定以及亚细胞部分转运现象的体外重建，以剖析管腔 Ca22、Ca22 传感器 ALG-2 和 COPII 涂层在此途径特定阶段的作用。这些研究的动机是广泛的指导假设，即从前高尔基体分泌细胞器中逸出的管腔 Ca2与运输机制相互作用并对其进行调节，从而显着影响货物运输和/或分类。所提出的实验属于三个具体目标：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) 测试 ALG-2 和 sec31A 转导管腔 Ca2 信号以调节 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.