Membrane trafficking to lysosomes

项目摘要 溶酶体是细胞的主要分解代谢部位，用于降解由内分泌细胞内化的细胞外物质。 胞吞作用和指定用于周转的细胞内组分。这些项目和水解酶， 降解它们通过多种膜运输途径被递送到溶酶体，这些途径由 从酵母到人类进化上保守的细胞蛋白质机制。 这些机制的活动与遗传疾病有关，病原体利用这些贩运途径， 建立感染。许多操作膜运输机器的分子机制是非- 知道的我的实验室解决了关于膜运输到溶酶体的基本问题， 酵母Saccharomyces cerevisiae作为遗传学上易处理的模式生物。我们计划解决的主要问题 未来五年的工作包括以下内容。1)是什么生理机制调节 将细胞表面受体运输到溶酶体的水解内部？这条贩运途径的功能- 在核内体上的作用是将内吞的受体分类到膜封闭的运输囊泡中， 进入溶酶体腔。我实验室的最新进展表明这些囊泡的形成 与其他对细胞生理学至关重要的过程协调，包括泛素蛋白质稳态 和内吞pH调节。我们计划找出连接这些不同细胞系统的机制 并确定它们如何控制介导囊泡反式形成的蛋白质机制， 进入溶酶体腔。这些结果将确定受体降解与受体降解之间的协调方式。 细胞生理学2)是什么机制介导了新合成的transferase的传递， 膜蛋白是注定要在溶酶体膜上发挥作用的？利用我们发明的新的基因工具 为了发现和诊断，我们最近确定了两种特定的细胞机制和几种额外的细胞机制。 在这一贩运途径中发挥作用的候选机制。我们计划确定机制， 这些机制在运输中发挥作用，这将建立生物起源的基本原则。 的溶酶体。我们的工作得到了持续的富有成效的合作的支持， 技术，包括高分辨率显微镜，生物物理分析的蛋白质组装重建， 体外、蛋白质组学和遗传筛选。展望未来，我们的总体愿景是继续开发酵母， 发现和机械地理解膜运输到溶酶体，同时也解决了 这些机制在人类细胞中的保守程度。从这项工作中获得的信息将 提供了一个了解如何膜运输途径，溶酶体下正常的生理运作， 逻辑条件以及他们在疾病状态下如何脆弱。

PROJECT SUMMARY Lysosomes are the primary catabolic site of cells, serving to degrade extracellular material internalized by en- docytosis and intracellular components earmarked for turnover. These items and the hydrolytic enzymes that degrade them are delivered to lysosomes by multiple membrane trafficking pathways, which are operated by cellular protein machineries that are evolutionarily conserved from yeast to man. Mutations that disrupt the ac- tivities of these machineries are linked to genetic diseases, and pathogens exploit these trafficking pathways to establish infection. Many of the molecular mechanisms that operate membrane trafficking machineries are un- known. My lab addresses fundamental questions about membrane trafficking to lysosomes using the budding yeast Saccharomyces cerevisiae as a genetically tractable model organism. The major questions we plan to address over the next five years include the following. 1) What are the physiological mechanisms that regulate the trafficking of cell-surface receptors to the hydrolytic interior of the lysosome? This trafficking pathway func- tions at endosomes to sort endocytosed receptors into membrane-enclosed transport vesicles that are deliv- ered into the lysosome lumen. Recent progress from my lab has revealed that the formation of these vesicles is coordinated with other processes that are vital to cellular physiology, including ubiquitin protein homeostasis and endocytic pH regulation. We plan to identify the machineries that interface these different cellular systems and determine how they exert control over the protein machinery that mediates the formation of vesicles trans- ported to the lysosome lumen. These results will define ways in which receptor degradation is coordinated with cellular physiology. 2) What are the mechanisms that mediate the delivery of newly synthesized transmem- brane proteins that are destined to function at the lysosomal membrane? Using novel genetic tools we created for discovery and diagnostics, we recently identified two specific cellular machineries and several additional candidate machineries that function in this trafficking pathway. We plan to define the mechanisms by which these machineries function in transport, which will establish fundamental principles that underly the biogenesis of lysosomes. Our work is bolstered by ongoing productive collaborations that employ diverse interdisciplinary techniques, including high-resolution microscopy, biophysical analyses of protein assemblies reconstituted in vitro, proteomics, and genetic screening. Moving forward, our overall vision is to continue exploiting yeast for discovering and mechanistically understanding membrane trafficking to lysosomes while also addressing the extent to which these mechanisms are conserved in human cells. The information gained from this work will provide an understanding of how membrane trafficking pathways to lysosomes operate under normal physio- logical conditions and how they are vulnerable in disease states.