Identifying Gene Functions at Late Endosome and Lysosome 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。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学工具的优势；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。对液泡运输和功能相关基因的两个独立的全基因组筛选的结果将我们引导到1)几个以前未被描述的在内体和空泡界面起作用的基因(ENV基因)，2)一小部分涉及液泡运输和功能的基因(HHY基因)，这些基因在缺失时与潮霉素B高度敏感。此外，所有hhy突变体还表现出药物敏感性，表明TOR激酶信号通路存在缺陷，TOR激酶信号是通过营养和生长因子感应来调节细胞生长和增殖的主要调节因子。在这一竞争性更新中，我们提出了新基因ENV9的多方面特征，该基因与人视黄醇脱氢酶RDH12有显著的同源性，RDH12是一个与早发性视网膜变性有关的基因。我们还建议通过酵母遗传学、分子和显微方法来探索液泡运输、TOR激酶信号转导和潮霉素B超敏反应的界面。这一领域提议的内在目标是继续培养一名少数民族博士后研究人员，并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学生进行富有成效的研究培训。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。TOR激酶信号的过度激活与良、恶性肿瘤的发生以及包括糖尿病和肥胖症在内的代谢性疾病有关。 公共卫生相关性：拟议的研究涉及与青少年和迟发性神经变性相关的公共卫生问题，分别表现为溶酶体储存疾病和阿尔茨海默病。它还与与肿瘤发生和包括糖尿病和肥胖症在内的代谢性疾病有关的公共卫生问题有关。

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 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 genetics tools; large majority of yeast genes identified in lysosomal trafficking have orthologues in humans. Results of two separate genome wide screens for vacuolar trafficking and function related genes have led us to 1)several previously uncharacterized genes that have a role at endosome and vacuole interface (ENV genes), 2) a small set of genes involved in vacuolar trafficking and function that confer severe hypersensitivity to hygromycin B when deleted (HHY genes). Furthermore, all hhy mutants also showed drug hypersensitivities indicative of defective TOR kinase signaling, a master regulator of cell growth and proliferation through nutrient and growth factor sensing. In this competitive renewal, we propose multifaceted characterization of the novel gene ENV9 which has significant identity to human retinol dehydrogenase RDH12 - a gene implicated in early onset retinal degeneration. We also propose to explore the interface of vacuolar trafficking, TOR kinase signaling, and hygromycin B hypersensitivity by yeast genetics, molecular, and microscopic approaches. Inherent in this AREA proposal, is the aim to continue development of a minority post-doctoral researcher and continue productive research training of undergraduate and Master's 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. Hyper activation of TOR kinase signaling is associated with benign and malignant tumorigenesis, as well as with metabolic diseases including diabetes and obesity. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health issues associated with both juvenile and late onset neurodegeneration as manifested in Lysosomal Storage Diseases and Alzheimer's disease, respectively. It is also relevant to public health issues associated with tumorigenesis and metabolic diseases including diabetes and obesity.