MECHANISMS OF ENDOSOME TO LYSOSOME TRANSPORT IN YEAST

酵母内体到溶酶体的转运机制

• 批准号: 2190985
• 负责人: THOMAS A VIDA
• 金额: $ 17.36万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190985
• 关键词: MECHANISMS; ENDOSOME; LYSOSOME; TRANSPORT; YEAST

This revised proposal is aimed at defining molecular mechanisms of intercompartmental protein transport from both the secretory and endocytic pathways to the lysosome. Correct regulation of protein transport in the secretory and endocytic pathways is essential for normal function in all eukaryotic cells. Defective intracellular protein traffic occurs in such diverse human diseases as breast cancer and Alzheimer's syndrome. The lysosome is one organelle that shows particular dispositions to protein traffic errors as nearly 40 disorders in lysosomal storage exist and protein turnover in lysosomes is central to treating certain neurodegenerative diseases. Understanding the molecular details of how eukaryotic cells specifically transport proteins to lysosomes will have broad relevance to fundamental cell biology. These fundamental issues are best answered with a diverse experimental design. In this research, the yeast, Saccharomyces cerevisiae, will be used as a model eukaryotic organism because molecular, genetic, and biochemical approaches are readily integrated for protein transport investigations to its lysosome- like organelle, the vacuole. Integrating genetics and biochemistry has lead to a more thorough elucidation of intracellular protein transport mechanisms during the early secretory pathway. This work, however, specifically focuses on movement of proteins from the newly-discovered prevacuolar endosomal compartment to the lysosome/vacuole, which is poorly understood in both mammals and yeast. The greatest area of uncertainty in this process concerns whether transport carrier vesicles shuttle cargo between these organelles or late endosomes "mature" into lysosomes. To prove or disprove these models, experiments are designed to establish the hypothesis that specific carrier vesicles function to transport proteins from the prevacuolar compartment to the lysosome/vacuole. Using a combination of in vitro reconstitution assays and molecular genetics, proteins that function in vesicle formation, vesicle transport, or vesicle consumption will be identified and studied. Specifically, (1) cytosolic proteins will be purified and functionally characterized using intercompartmental protein transport assays in permeabilized cells; (2) develop new in vitro assays using subcellular fractions to measure stage- specific events in transport from the prevacuolar compartment to the vacuole; (3) the identity of carrier vesicles that operate to transport protein cargo from the prevacuolar compartment to the vacuole will be established; and (4) the role a selected subset of vacuolar protein sorting (vps) defective yeast mutants has in endosome to lysosome protein traffic will be determined.

这项修订的建议旨在确定分子机制， 从分泌和内吞的蛋白质跨室转运 通向溶酶体的通路蛋白质转运的正确调节 分泌和内吞途径是所有细胞的正常功能所必需的。 真核细胞有缺陷的细胞内蛋白质运输发生在这样的细胞中， 乳腺癌和老年痴呆症等多种人类疾病。的 溶酶体是一种对蛋白质有特殊处理能力的细胞器 由于存在近40种溶酶体贮积症， 溶酶体中的蛋白质周转是治疗某些 神经退行性疾病了解分子的细节 真核细胞特异性地将蛋白质转运到溶酶体， 与基础细胞生物学的广泛相关性。这些基本问题得到 最好用不同的实验设计来回答。本研究 酵母，酿酒酵母，将被用作模型真核生物 生物体，因为分子，遗传和生物化学方法是 易于整合用于蛋白质转运研究至其溶酶体- 就像细胞器，液泡。整合遗传学和生物化学 从而更彻底地阐明了细胞内蛋白质转运 在早期分泌途径的机制。然而，这项工作， 特别关注蛋白质的运动， 前液泡内体区室的溶酶体/液泡，这是很差的 在哺乳动物和酵母中都有。最大的不确定性是 这一过程涉及运输载体囊泡是否穿梭货物 或晚期内体“成熟”成溶酶体。到 证明或反驳这些模型，实验旨在建立 特异性载体囊泡转运蛋白质的假说 从液泡前区室到溶酶体/液泡。使用 体外重建测定和分子遗传学的组合， 在囊泡形成、囊泡运输或囊泡形成中起作用的蛋白质 将对消费进行识别和研究。具体而言，（1）胞质 蛋白质将被纯化，并使用 透化细胞中的隔室间蛋白质转运测定;（2） 开发新的体外试验，使用亚细胞组分来测量阶段- 从前液泡区室运输到 液泡;（3）载体小泡的身份， 蛋白质货物从prevacuolar室到液泡将是 建立;和（4）的作用，一个选定的子集的液泡蛋白 分选（vps）缺陷酵母突变体具有内体至溶酶体蛋白 交通将被确定。