Identify Gene Functions at late endosome & lysosome interface in yeast

描述(由申请人提供)：正确的分子分选和囊泡运输到可降解的溶酶体是所有真核细胞的基本特征。晚期内小体是运输到溶酶体的内吞途径和生物合成途径的交汇点。因此，不同的转运事件发生在内体晚期，因为它与三个隔室对接：TGN、内吞内体和内质体。我们的长期目标是确定晚期内小体到溶酶体界面的调控过程。酿酒酵母的晚期内体和液泡在功能上与哺乳动物晚期内体和溶酶体相同，其空泡递送的羧基肽酶Y(CPY)途径与哺乳动物细胞中的甘露糖-6-磷酸途径平行。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绝大多数酵母基因在人类中都有同源基因。利用一种新的免疫检测屏幕，我们分离到了四个在cpy递送和加工的内体到液泡阶段有缺陷的突变体(env突变体)。突变体的特征已经确定在内吞的晚期步骤和空泡结构/功能上都存在缺陷。该集合的第一个基因已经被克隆；env1是VPS35的一个独特的等位基因，它定义了其保守基因产物的第二个角色。在这项建议中，我们的具体目标是完成一种基因组方法，直接从酵母缺失文库中鉴定更多的ENV基因，并克隆和鉴定ENV3。这一领域建议的固有目标是在一个综合性的拉美裔服务学院内继续对本科生和硕士学生进行生产性研究培训。溶酶体蛋白水解酶和CARO的错误定位与溶酶体储存疾病和阿尔茨海默病(AD)有关。在幼年期和老年性神经退行性疾病中，缺陷特别是在运输的内小体到溶酶体的晚期阶段已经成为可能的潜在机制。 公共卫生相关性：拟议的研究与阿尔茨海默病中表现的衰老神经退行性变相关的公共卫生问题。它还涉及与患有溶酶体储存性疾病的青少年患者的进行性神经变性有关的公共卫生问题。

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.