MECHANISMS OF ENDOSOME TO LYSOSOME TRANSPORT IN YEAST

这项修订后的提案旨在定义 内分泌和胞内的间室蛋白转运 通向溶酶体的途径。蛋白质转运的正确调控 分泌和内吞途径对急性淋巴细胞白血病的正常功能是必不可少的 真核细胞。有缺陷的细胞内蛋白质流量在这种情况下发生 各种人类疾病，如乳腺癌和阿尔茨海默氏症。这个 溶酶体是一种对蛋白质表现出特殊倾向的细胞器。 交通错误，溶酶体储存存在近40种紊乱， 溶酶体中的蛋白质周转是治疗某些疾病的中心 神经退行性疾病。了解分子细节是如何 真核细胞特异性地将蛋白质运输到溶酶体将具有 与基础细胞生物学有着广泛的相关性。这些基本问题是 最好的答案是多样化的实验设计。在这项研究中， 酵母，酿酒酵母，将被用作真核生物的模式 因为分子、遗传和生物化学方法是 易于整合以研究蛋白质向其溶酶体的转运- 就像细胞器，液泡。把遗传学和生物化学结合起来 导致对细胞内蛋白质运输的更彻底的阐明 早期分泌途径的机制。然而，这项工作， 特别关注新发现的蛋白质的运动 溶酶体/空泡的前腺泡内小体间隔，这是很差的 在哺乳动物和酵母中都能理解。最不确定的领域是 这一过程关系到运输商是否会对穿梭货物产生囊泡 在这些细胞器之间或晚些时候的内体“成熟”成溶酶体。至 证明或反驳这些模型，实验旨在建立 特定载体囊泡运输蛋白质的假说 从泡前室到溶酶体/液泡。使用 结合体外重组试验和分子遗传学， 在小泡形成、小泡运输或小泡中起作用的蛋白质 将对消费进行识别和研究。具体地说，(1)胞质 蛋白质将被提纯，并用 通透性细胞间蛋白质转运分析；(2) 开发新的体外分析方法，使用亚细胞组分来测量阶段- 从前泡间室到大脑皮层的特定事件 液泡；(3)运输运输的载体小泡的身份 从泡前间隔到液泡的蛋白质货物将是 已建立的；以及(4)所选择的空泡蛋白子集的作用 分选(VPS)缺陷酵母突变株具有内体到溶酶体蛋白 流量将被确定。

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.