Mechanisms Controlling Organelle Acidification Along the Regulated Secretory Pathway

沿着调节的分泌途径控制细胞器酸化的机制

• 批准号: 9983342
• 负责人: Hsiao-Ping Moore
• 金额: $ 37.4万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9983342&HistoricalAwards=false
• 关键词: Mechanisms; Controlling; Organelle; Acidification; Along

The eukaryotic cell is exquisitely compartmentalized into membrane-enclosed spaces that provide sequestered microenvironments particularly suited for various metabolic functions. In particular, one aspect of these microenvironments within the subcellular compartments is the pH: for example, some compartments, such as lysosomes, are extremely acidic, while others, such as the endoplasmic reticulum, provide a pH environment close to neutral. Some of these compartments are part of the dynamic membrane trafficking system of the inter-related secretory and endocytic pathways, which are involved in the processing and secretion of proteins and other macromolecules synthesized by the cell and the uptake of proteins and hormones from the cell's surface and its environment. A key feature of this inter-related set of trafficking pathways is the differences in pH among the various compartments. Acidification of the exocytic and endocytic pathways controls a variety of cellular processes, including viral entry, protein sorting, and proteolytic processing. A specific example of this generalization, which forms the model system on which this project is based, is the regulated secretory pathway for the peptide hormone ACTH (adrenocorticotropic hormone). Here, the establishment of distinct ionic milieus within individual compartments is essential for proper sorting and processing of the various peptide hormones and their precursor molecules. For example, the trans-Golgi network (TGN) maintains a lumen that is acidic enough for sorting of regulated secretory products, and yet alkaline enough to prevent premature activation of prohormones and lysosomal enzymes. Upon budding from the TGN, secretory granules (SG's) acidify further to allow processing of the packaged prohormones. Thus, molecular mechanisms must be in place to ensure that the TGN and SG each maintains its unique level of acidity. How this is accomplished remains mysterious. A major difficulty has been the lack of a systematic method to study acidification of individual organelles along the regulated secretory path. The goal of this project is to use a novel fluorescence targeting method to study the regulatory mechanisms that control acidification of the secretory pathway. This elegant but technically challenging method, which involves genetic engineering and novel pH-sensitive fluorescent dyes, is expected to allow the systematic introduction of pH-sensitive dyes into specific compartments such that the pH dynamics of each organelle can be measured in living cells. The mouse pituitary cell line AtT-20 will be used as a model system because of its well characterized regulated secretory pathway and the role of organelle acidification in prohormone processing. The fluorescence targeting method will be used to determine the pH values of individual compartments along the regulated secretory pathway. The mechanisms that maintain individual organelles at their respective pH's will be elucidated. In particular, Drs. Moore and Machen will test the hypothesis that progressive acidification along the secretory pathway is generated by iterative retention of proton leaks within the earlier compartments.

真核细胞被精细地划分成膜封闭的空间，提供特别适合各种代谢功能的隔离微环境。特别是，亚细胞区室内这些微环境的一个方面是pH值：例如，一些区室，如溶酶体，是极端酸性的，而其他的，如内质网，提供接近中性的pH值环境。其中一些室室是相互关联的分泌和内吞途径的动态膜运输系统的一部分，参与细胞合成的蛋白质和其他大分子的加工和分泌，以及从细胞表面及其环境中摄取蛋白质和激素。这组相互关联的运输途径的一个关键特征是不同隔间之间pH值的差异。胞外和内吞途径的酸化控制着多种细胞过程，包括病毒进入、蛋白质分选和蛋白质水解加工。这一概括的一个具体例子是肽激素ACTH（促肾上腺皮质激素）的调节分泌途径，它构成了本项目所基于的模型系统。在这里，在单个隔间内建立不同的离子环境对于正确分类和处理各种肽激素及其前体分子是必不可少的。例如，反式高尔基网络（TGN）维持一个足够酸性的管腔，以分类受调节的分泌产物，但又足够碱性，以防止激素原和溶酶体酶的过早激活。从TGN出芽后，分泌颗粒（SG’s）进一步酸化，以允许包装的原激素的处理。因此，分子机制必须到位，以确保TGN和SG各自保持其独特的酸度水平。这是如何实现的仍然是个谜。一个主要的困难是缺乏一种系统的方法来研究沿调节分泌路径的单个细胞器的酸化。本项目的目的是利用一种新的荧光靶向方法来研究控制分泌途径酸化的调节机制。这种优雅但技术上具有挑战性的方法，涉及基因工程和新颖的pH敏感荧光染料，有望允许系统地将pH敏感染料引入特定的隔室，从而可以在活细胞中测量每个细胞器的pH动态。小鼠垂体细胞系at -20将被用作模型系统，因为它具有良好的调节分泌途径和细胞器酸化在激素原加工中的作用。荧光靶向法将用于确定沿调节分泌途径的单个室的pH值。将阐明维持单个细胞器各自pH值的机制。特别是，dr。Moore和Machen将测试一个假设，即分泌途径上的渐进式酸化是由质子泄漏在早期隔间内的反复保留所产生的。