Molecular Mechanisms that Regulate Lysosomal Protein Transport

描述（由申请人提供）：本提案的长期目标是定义调节整体膜蛋白运输到溶酶体降解的分子机制。最近的证据表明，泛素对跨膜蛋白的修饰足以使蛋白质进入这一途径。ESCRT机制是一组保守的内体蛋白复合物，被认为直接结合泛素化的膜蛋白，并控制它们进入特化的多泡内体（MVEs）的囊泡。这一过程对于激素受体的下调以防止构成性信号传导尤为重要，这可能导致发育异常和疾病。ESCRT机制如何协调泛素化底物到MVEs的有效捕获和运输将在本提案中讨论。秀丽隐杆线虫种系和早期胚胎是研究完整发育动物膜动力学的有力模型系统。利用RNA干扰可以有效地从卵母细胞中去除特定的蛋白质。此外，卵母细胞成熟和受精可重复触发多种跨膜蛋白的内化和降解，为研究溶酶体蛋白转运提供了理想的生理相关系统。秀丽隐杆线虫是高度易受基因操作的，可以被设计成稳定地表达荧光标记的蛋白质，包括可以通过活细胞显微镜监测的细胞表面受体。利用这一独特的属性组合，本提案的具体目标是：1)确定ESCRT机制识别底物的机制，2)定义PTH-2（一种新发现的ESCRT-0结合蛋白）的作用，以及3)定义调节货物进入ESCRT途径的机制。我们的初步遗传和生化研究已经发现了与ESCRT机制相关的溶酶体转运途径的新成分。这些相互作用的意义将使用基于荧光显微镜的功能分析、生物物理测量和体外重构实验的组合来测试。这些研究将为未来研究哺乳动物细胞中高度相关的通路提供一个框架。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to define molecular mechanisms that regulate the trafficking of integral membrane proteins to the lysosome for degradation. Recent evidence indicates that modification of transmembrane proteins by ubiquitin is sufficient for protein sorting into this pathway. The ESCRT machinery, a set of conserved endosomal protein complexes, is proposed to directly bind to ubiquitinylated membrane proteins and govern their entry into vesicles that bud into the lumen of specialized multivesicular endosomes (MVEs). This process is particularly important for the downregulation of hormone receptors to prevent constitutive signaling, which can lead to developmental abnormalities and disease. How the ESCRT machinery coordinates the efficient capture and transport of ubiquitinylated substrates to MVEs will be addressed in this proposal. The C. elegans germline and early embryo are powerful model systems to study membrane dynamics in an intact, developing animal. Specific proteins can be efficiently depleted from oocytes using RNA interference. Additionally, oocyte maturation and fertilization reproducibly trigger the internalization and degradation of multiple transmembrane proteins, providing an ideal, physiologically relevant system for studying lysosomal protein transport. C. elegans is highly amenable to genetic manipulation and can be engineered to stably express fluorescently tagged proteins, including cell surface receptors that can be monitored by live cell microscopy. Taking advantage of this unique combination of attributes, the specific aims of this proposal are: 1) to determine mechanisms by which the ESCRT machinery recognizes substrates, 2) to define the role of PTH-2, a newly discovered ESCRT-0 binding protein, and 3) to define mechanisms that regulate cargo entry into the ESCRT pathway. Our preliminary genetic and biochemical studies have uncovered new components of the lysosomal transport pathway that associate with the ESCRT machinery. The significance of these interactions will be tested using a combination of fluorescence microscopy-based functional assays, biophysical measurements, and in vitro reconstitution experiments. These studies will provide a framework for future investigation into highly related pathways in mammalian cells.