Regulation of ER to Golgi Transport by Luminal Calcium

描述（由申请方提供）：正常的细胞功能和避免疾病需要亚细胞靶向以及转运囊泡与受体膜的融合。囊泡外套、rabs、tether和SNARE构成了内膜系统囊泡运输机制的核心。虽然这些蛋白质家族中的每一个的主要功能点已经建立，但在意想不到的步骤中的新的功能作用以及连接和整合它们的功能的新的调节相互作用继续出现。在哺乳动物中，ER-高尔基体转运，这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，已被广泛表征在体内和体外重建。从广义上讲，ER到高尔基体的转运包括：1）由COPII外壳介导的货物分选和囊泡出芽; 2）由rab 1、p115和TRAPP 1介导的同型COPII囊泡束缚，以及由ER/高尔基体SNARE介导的融合，以形成称为囊泡管状簇（VTC）的前高尔基体细胞器;和3）VTC介导的货物分选和沿沿着微管运输，导致与高尔基体融合。该项目将采用活细胞Ca 2+测量，ER/高尔基体运输在完整的哺乳动物细胞系的动力学测定和从亚细胞组分的运输现象的体外重建来剖析腔Ca 2+，Ca 2+传感器ALG-2和COPII涂层在该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，即从前高尔基体分泌细胞器逃逸的腔Ca 2+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/或分选。所提出的实验分为三个具体目标：1）测试的假设，腔Ca 2+浓度急剧下降之间的ER和中间室。2)验证ALG-2和sec 31 A抑制管腔Ca 2+信号以调节VTC形成和功能中的多个步骤的假设。3)识别COPII囊泡上的Ca 2+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.