Sorting and trafficking in the endosomal system

描述（由申请人提供）：本研究项目的主要目标是阐明核内体中的蛋白质和脂质被包装成运输载体的机制，这些运输载体从核内体中产生，并将货物运送到高尔基体和其他细胞器。这些运输途径统称为“内体逆行运输”，对细胞生理和体内平衡、后生动物发育至关重要，它们对抗导致溶酶体储存疾病、癌症、阿尔茨海默病和其他疾病的鲜为人知的过程。在之前的资助周期中，我们发现逆行途径用于调节质膜的组成，以响应环境和营养线索，并且我们确定了可溶性蛋白质分选复合物（称为“逆行物”）和磷脂酰肌醇3-激酶（PI3K）的三种效应物（称为分选连接蛋白（SNX）蛋白）的作用。这些成分在核内体膜上组装，在那里它们捕获并将货物包装成萌芽的运输载体，但潜在的机制尚不清楚。随着逆转录酶的主要细胞功能现在已经确立，该领域的主要挑战是阐明其功能的机制。生化方法将被用来剖析逆转录物在核内体膜上与货物组装的机制。这些实验将辅以逆行运输的活细胞成像，将组装反应置于生理背景下。由于认识到后转录分选连接蛋白是一个更大的SNX蛋白家族的成员，除少数外，所有的功能都已被描述，因此将使用无偏倚的蛋白质组级实验来阐明每个分选连接蛋白的功能，并评估它们对细胞生理学的具体贡献。

DESCRIPTION (provided by applicant): The broad goal of this research project is to elucidate the mechanisms by which proteins and lipids in the endosome are packaged into transport carriers that bud from the endosome and ferry cargo to the Golgi apparatus and other organelles. These trafficking pathways, collectively termed 'endosomal retrograde transport,' are vital for cell physiology and homeostasis, metazoan development and they counter poorly understood processes that contribute to lysosomal storage diseases, cancer, Alzheimer's disease, and other diseases. In previous funding cycles we discovered that retrograde pathways are used to modulate the composition of the plasma membrane in response to environmental and nutritional cues, and we established roles for a soluble protein sorting complex called 'retromer' and three effectors of Phosphatidylinositol 3-kinase (PI3K) called sorting nexin (SNX) proteins. These components assemble on the endosome membrane where they capture and package cargo into budding transport carriers but the underlying mechanisms are unknown. With the principle cellular function of retromer now well established, the major challenge for the field is to elucidate the mechanisms by which it functions. Biochemical approaches will be used to dissect the mechanism of retromer assembly with cargo on the endosome membrane. These experiments will complemented with live cell imaging of retrograde transport that will place the assembly reactions in a physiological context. In recognition that the retromer sorting nexins are members of a much larger SNX protein family for which the functions of all but a few have been described, unbiased, proteome scale experiments will be used to elucidate the functions of each sorting nexin and to assess their specific contributions to cell physiology.