Membrane trafficking to lysosomes

项目总结 溶酶体是细胞的主要分解代谢部位，其作用是降解内毒素内化的胞外物质。 细胞增多症和指定用于周转的细胞内成分。这些物质和水解酶 降解它们通过多个膜转运途径传递到溶酶体，这些途径是由 从酵母到人类进化上保守的细胞蛋白质机制。破坏Ac-1基因的突变 这些机器的活动与遗传疾病有关，病原体利用这些传播途径 确定感染。许多运行膜转运机制的分子机制都不是 为人所知。我的实验室解决了使用萌发的溶酶体膜运输的基本问题 酿酒酵母是一种遗传易驯化的模式生物。我们计划解决的主要问题 未来五年的地址包括以下内容。1)调节的生理机制是什么 细胞表面受体的运输到溶酶体的水解性内部？这条运输途径的功能是- 在内小体上将内吞的受体分类到膜包裹的运输小泡中，这些小泡是递送的。 进入溶酶体腔。我实验室的最新进展显示，这些囊泡的形成 与其他对细胞生理至关重要的过程相协调，包括泛素蛋白动态平衡 和胞内pH调节。我们计划确定连接这些不同蜂窝系统的机器 并确定它们如何控制介导囊泡形成的蛋白质机制，反式- 移植到溶酶体腔内。这些结果将定义受体降解与 细胞生理学。2)新合成的转运蛋白的传递机制是什么？ 膜蛋白注定要在溶酶体膜上发挥作用？使用我们创造的新型基因工具 为了发现和诊断，我们最近确定了两个特定的细胞机器和几个额外的 在这条贩运途径上发挥作用的候选机器。我们计划定义通过哪些机制 这些机器在运输中发挥作用，这将建立生物发生的基本原理 溶酶体。我们的工作得到了持续的富有成效的合作的支持，这些合作采用了不同的跨学科 技术，包括高分辨率显微镜，重组的蛋白质组件的生物物理分析 试管、蛋白质组学和基因筛查。展望未来，我们的总体愿景是继续开发酵母 发现并机械地理解膜向溶酶体的运输，同时还解决 这些机制在人类细胞中被保守的程度。从这项工作中获得的信息将 提供对溶酶体膜转运途径在正常生理条件下如何运作的理解 合乎逻辑的条件以及它们在疾病状态下的脆弱性。

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.