Identify Gene Functions at late endosome & lysosome interface in yeast

描述（由申请人提供）：正确分选和向降解溶酶体输送分子是所有真核细胞的基本特征。内核体晚期是内吞途径和向溶酶体运输的生物合成途径的交汇点。因此，不同的运输事件发生在晚期核内体，因为它与三个室室：TGN，内吞核内体和内溶体相连接。我们的长期目标是确定内核体与溶酶体界面的后期调控过程。酿酒酵母的晚期核内体和液泡在功能上与哺乳动物的晚期核内体和溶酶体相当；它的羧基肽酶Y （CPY）空泡传递途径与哺乳动物细胞中的甘露糖-6-磷酸途径相似。酵母提供了传统和分子遗传工具的优势；在溶酶体运输中发现的大部分酵母菌基因在人类中有同源物。利用一种新的免疫检测筛选方法，我们分离出了四个在CPY传递和加工的核内体到液泡阶段有缺陷的突变体（环境突变体）。突变体的特征已经确定了内吞后期步骤和液泡结构/功能的缺陷。第一个基因已经被克隆；env1是VPS35的一个独特等位基因，它定义了其保守基因产物的第二个作用。在这个提议中，我们的具体目标是完成一种基因组方法，从酵母缺失文库中直接鉴定额外的ENV基因，并克隆和分子表征ENV3。在这个地区的提议中，固有的目标是在一个综合性的西班牙裔服务学院内继续对本科生和硕士生进行富有成效的研究培训。溶酶体蛋白酶和货物的错误定位与溶酶体贮积病和阿尔茨海默病（AD）有关。特别是在内核体到溶酶体运输阶段的缺陷已被认为是青少年和老年神经退行性疾病的可能潜在机制。

DESCRIPTION (provided by applicant): Correct sorting and vesicular trafficking of molecules to the degradative lysosome is an essential feature of all eukaryotic cells. The late endosome is the convergence point of the endocytic pathway and the biosynthetic pathway of trafficking to the lysosome. As such, distinct trafficking events occur at the late endosome as it interfaces with three compartments: TGN, endocytic endosomes, and Iysosomes. Our long term goal is to define the regulatory processes at the late endosome to lysosome interface. The late endosome and vacuole of the yeast Saccharomyces cerevisiae are functionally equivalent to the mammalian late endosome and lysosome; its carboxy peptidase Y (CPY)-pathway of vacuolar delivery parallels the mannose-6-phosphate pathway in mammalian cells. Yeast offers the advantage of both conventional and molecular genetic tools; large majority of yeast genes identified in lysosomal trafficking have orthologues in humans. Using a novel immunodetection screen, we have isolated four mutants that are defective at endosome to vacuole stage of CPY delivery and processing (env mutants). Characterizations of the mutants have established defects in both late endocytic steps and vacuolar structure/function. The first gene of the collection has been cloned; env1 is a unique allele of VPS35 that defines a second role for its conserved gene product. In this proposal, our specific aims are to complete a genomic approach to directly identify additional ENV genes from a yeast deletion library and to clone and molecularly characterize ENV3. Inherent in this AREA proposal, is the aim to continue productive research training of undergraduate and masters students within a comprehensive, Hispanic Serving Institute. Mislocalization of lysosomal proteases and cargo is associated with Lysosomal Storage Diseases and Alzheimer's Disease (AD). Defects specifically at the late endosome to lysosome stage of trafficking have emerged as the possible underlying mechanism in both juvenile and aging neurodegeneration diseases. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health issues associated with neurodegeneration of aging as manifested in Alzheimer's Disease. It is also relevant to public health issues associated with the progressive neurodegeneration of juvenile patients with lysosomal storage diseases.