Regulation of ER to Golgi Transport by Luminal Calcium

腔内钙对 ER 至高尔基体运输的调节

• 批准号: 8496964
• 负责人: JESSE C HAY
• 金额: $ 31.96万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8496964
• 关键词: Area; Binding; Biogenesis; Biological Assay; Biosensor; Calcium; Cell Line; Cell physiology; Cells; Coated vesicle; Disease; Drops; Event; Factor V; Factor VIII; Failure; Familial benign pemphigus; Golgi Apparatus; Hemorrhage; Human; Human poliovirus; In Vitro; Kinetics; Knowledge; Life; Link; Mammalian Cell; Mammals; Measurement; Mediating; Membrane; Membrane Protein Traffic; Microtubules; Neuropathy; Organelles; Pathology; Pathway interactions; Polioviruses; Protein Family; Proteomics; Rattus; Reaction; Regulation; Research Design; Role; Severe Acute Respiratory Syndrome; Signal Transduction; Site; Sorting - Cell Movement; Source; Spatial Distribution; Staging; Subcellular Fractions; System; Testing; Training; Transport Vesicles; Tubular formation; Vesicle; Viral; base; falls; graduate student; human disease; in vivo; kidney cell; novel; particle; public health relevance; reconstitution; research study; secretory protein; sensor; trafficking; undergraduate student

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.

描述（由申请人提供）：适当的细胞功能和疾病的避免需要用受体膜的运输囊泡的亚细胞靶向和融合。囊泡外套，兔子，系tethers和网罗构成了内膜系统的囊泡运输机制的核心。尽管建立了这些蛋白质家族的每个功能的主要功能点，但在意外步骤中的新功能作用，而将其功能联系起来的新调节相互作用仍在继续出现。在哺乳动物中，代表分泌途径的限速步骤，并且与运输相关疾病最相关的速率限制步骤已被广泛地在体内表征并在体外重构。从广义上讲，ER到高尔基体的运输已被证明包括：1）Copii Coat介导的货物分类和囊泡发芽； 2）由Rab1，p115和trapp1介导的同种型复印囊裂，以及由ER/Golgi Snares介导的融合，形成称为囊状管状簇（VTCS）的高尔基体前细胞器； 3）VTC介导的货物分选和沿微管的运输，导致与高尔基体融合。该项目将采用活细胞Ca2+测量，在完整的哺乳动物细胞系中进行ER/Golgi转运的动力学测定以及从亚细胞分数中的转运现象的体外重构，以剖析腔内Ca2+的作用，CA2+传感器ALG-2和该路径的特定阶段的CAPII层。这些研究是由广泛的指导假设激发的，即腔内ca2+从前分泌前细胞器与逃脱的逃脱相互作用并调节贩运机制，从而显着影响货物运输和/或分类。提出的实验属于三个特定目的：1）测试腔Ca2+浓度在ER和中间室之间急剧下降的假设。 2）检验以下假设：ALG-2和SEC31A转导腔Ca2+信号以调节VTC形成和功能的多个步骤。 3）确定Capii囊泡上的Ca2+靶标。