A transmembrane Rab GTPase accelerating protein targeted to peroxisomes

项目摘要 了解细胞如何适应压力和修复损伤是细胞生物学和 健康研究。内质网(ER)是脂肪和蛋白质的关键来源，用于构建和 在细胞中维持几个细胞器，并对刺激做出反应，它能够将资源引导到 各式各样的运输路径。信号蛋白Ypt1/Rab1是一种Rab GTP酶(Rab ER在指导资源和执行受损细胞器质量控制方面的重要作用，包括 线粒体和过氧化物体。我们的长期目标是了解脂肪和蛋白质 根据细胞刺激和压力，货物被安排路线和重新安排路线到特定路线，这将 告知制定有针对性的治疗干预措施，以应对疾病。我们发现， GTPase加速蛋白Gyp8，一种进化上保守但知之甚少的负调控蛋白 Ypt1/Rab1信号通路定位于内质网、过氧化物体和线粒体，影响Ypt1信号通路。我们的 中心假设是Gyp8在早期分泌途径中起到调节Ypt1/Rab1信号的作用 (ER)和经常受到损害的非分泌膜(过氧化物体和线粒体) 通过依赖Ypt1/Rab1的选择性自噬进行质量控制。此外，由于Gyp8定位于细胞器 通常在必要的代谢途径中拴/对接交换物质，我们预测Gyp8 调节依赖于RAB的系链相互作用，特别是在专门用于脂质储存和 新陈代谢。检验我们的中心假说并增进对几种膜生物发生的理解 对于起源于内质网的途径，我们将追求以下具体目标：1)识别基因内和基因外因素 控制Gyp8的亚细胞行程和活性；2)确定靶标Rab GTP酶(S)受 Gyp8在分泌途径中的作用；以及3)Gyp8在调节过氧化物体和线粒体中的作用 动力学。拟议的研究在重点领域和技术方法方面都是创新的。而当 Ypt1/Rab1信号控制着多条对细胞健康至关重要的转导通路，了解 而当信号必须被终止以完成其每个角色时，尤其是不完整的。这个 实验计划结合了细胞器和囊泡的金标准生化和成像技术 转运(酶偶联动力学转运分析和三维电子断层成像)与系统生物学 方法(合成基因芯片和亲和捕获质谱仪蛋白质组学)。拟议的研究 具有重要意义，因为定义如何监管Ypt1/Rab1以支持和实施ER的质量控制， 线粒体和过氧化物体是了解心血管、神经退行性疾病的基础 和新陈代谢紊乱。

Project Summary Understanding how cells adapt to stress and repair damage is one of the highest priorities in cell biology and health research. The endoplasmic reticulum (ER) is a key source of lipid and proteins for building and maintaining several organelles in cells, and in response to stimuli it is capable of directing resources into an assortment of transport pathways. The signaling protein Ypt1/Rab1 is a Rab GTPase (Rab) that plays essential roles in how the ER directs resources and executes quality control of damaged organelles including mitochondria and peroxisomes. Our long-term goal is to understand the mechanisms for how lipid and protein cargos are routed and re-routed to specific itineraries in response to cellular stimuli and stresses, which will inform development of targeted therapeutic interventions to address diseases. We have found that the GTPase accelerating protein Gyp8, an evolutionarily conserved but poorly understood negative regulator of Ypt1/Rab1 signaling, localizes to the ER, peroxisomes and mitochondria and impinges on Ypt1 signaling. Our central hypotheses are that Gyp8 functions to modulate Ypt1/Rab1 signaling in the early secretory pathway (ER) and at non-secretory membranes (peroxisomes and mitochondria) that are subject to frequent damage and quality control via Ypt1/Rab1-dependent selective autophagy. Also, since Gyp8 localizes to organelles that commonly tether/dock to exchange materials in essential metabolic pathways, we predict that Gyp8 regulates Rab-dependent tethering interactions, particularly among organelles specialized for lipid storage and metabolism. To test our central hypotheses and advance understanding of several membrane biogenesis pathways that originate at the ER, we will pursue these specific aims: 1) Identify intra- and extra-genic factors that control the subcellular itinerary and activity of Gyp8; 2) Determine target Rab GTPase(s) regulated by Gyp8 in the secretory pathway; and 3) Define the role of Gyp8 in regulating peroxisomal and mitochondrial dynamics. The proposed research is innovative both for area of focus and technical approach. While Ypt1/Rab1 signaling controls multiple transport pathways essential to cellular health, understanding of where and when signal must be terminated to accomplish each of its roles is particularly incomplete. The experimental plan combines gold standard biochemical and imaging techniques in organelle and vesicular transport (enzyme-coupled kinetic transport assays and 3D electron tomography) with systems biology approaches (synthetic gene array and affinity capture mass spectrometry proteomics). The proposed research is significant because defining how Ypt1/Rab1 is regulated to support and exercise quality control of ER, mitochondria and peroxisomes is foundational to understanding aspects of cardiovascular, neurodegenerative and metabolic disorders.