Genetics of secretion in yeast

酵母分泌的遗传学

• 批准号: 8401131
• 负责人: PETER Jay NOVICK
• 金额: $ 40.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8401131
• 关键词: Accounting; Actins; Affect; Alleles; Animals; Bacillus (bacterium); Bacillus anthracis; Back; Binding; Binding Sites; Biological Assay; Bypass; Catalysis; Cell membrane; Cell physiology; Cell surface; Cells; Clathrin; Clathrin Adaptors; Complex; Cytokinesis; Data; Defect; Drosophila genus; Endoplasmic Reticulum; Epithelium; Family; Genes; Genetic; Golgi Apparatus; Growth; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; In Vitro; Indium; Larva; Lead; Link; Malignant Neoplasms; Mammalian Cell; Maps; Membrane; Modeling; Molecular; Mutagenesis; Mutation; Myosin Type V; Organism; Pathway interactions; Play; Preparation; Protein Secretion; Proteins; Recruitment Activity; Recycling; Resistance; Role; SNAP receptor; Secretory Vesicles; Series; Signal Transduction; Site; Surface; System; TFAP2A gene; Targeted Toxins; Temperature; Testing; Time; Tissues; Toxin; Transport Vesicles; Travel; Tumor Suppressor Proteins; Vesicle; Work; Yeasts; anthrax toxin; cell cortex; cell motility; cilium biogenesis; falls; genetic analysis; human disease; in vivo; intercellular communication; loss of function mutation; member; mutant; overexpression; rab GTP-Binding Proteins; retinal rods; target SNARE proteins; vesicular SNARE proteins; yeast genetics

DESCRIPTION (provided by applicant): In virtually all tissues of every eukaryotic organism, polarized export of cell surface components is critical for a range of important cellular functions including protein secretion, assembly of the plasma membrane, cell signaling, cell polarization, cell migration, ciliogenesis, and cytokinesis. Defects in polarized export underlie or exacerbate a number of human diseases including cancer. We pioneered the genetic analysis of polarized export in yeast, identifying and characterizing a system of components that are all structurally and functionally conserved in higher eukaryotic organisms. Sec4p, the founding member of the rab GTPase family, acts as a master regulator by promoting the function of three different effectors: Myo2p a type V myosin that actively transports vesicles along polarized actin cables, the yeast homolog of the Lgl tumor suppressor Sro7p that physically links Sec4-GTP to the t-SNARE Sec9p, and the exocyst a complex of eight different gene products that interacts with components on the vesicle surface as well as polarity determinants on the cell cortex and thus tethers incoming secretory vesicles to these specialized sites. This proposal seeks to probe in greater detail the mechanism by which the exocyst acts to promote polarized export. 1. We will test the hypothesis that the exocyst is recruited to the vesicle surface by recognizing two coincident signals, Sec4-GTP and the v-SNARE Snc2p. 2. We will determine if the exocyst can bind the vSNARE Snc2p and the t-SNARE Sec9p at the same time, forming a Snc2p-exocyst-Sec9p ternary complex. Mutagenesis of the interaction sites will establish their function in vivo. 3. SNAREs will self-assemble in vitro, but at rates far too slow to account for their function in vivo. We will determine if the exocyst, either alone or in combination with its binding partners, can catalyze assembly of the exocytic SNARE complex. 4. The exocyst subunit Exo70p can localize to exocytic sites independent of the actin used for delivery of secretory vesicles, implying an interaction with a polarity determinant at the cell cortex. We will determine the identity of this polarity determinant. 5. Bacillus anthraxis secretes two toxins that impair the function of the exocyst in both drosophila and mammalian cells. We will determine if they also act on the yeast exocyst. This would allow us to exploit the facile genetics of yeast to identify the direct targets of these toxins and define their mechanism of action.

描述（由申请人提供）：在每种真核生物的几乎所有组织中，细胞表面组分的极化输出对于一系列重要的细胞功能至关重要，包括蛋白质分泌、质膜组装、细胞信号传导、细胞极化、细胞迁移、纤毛发生和胞质分裂。两极化输出的缺陷是包括癌症在内的一些人类疾病的基础或加剧。我们开创了酵母中极化输出的遗传分析，识别和表征了在高等真核生物中结构和功能保守的组件系统。Sec 4p是rab GTdR家族的创始成员，通过促进三种不同效应物的功能来充当主调节剂：Myo 2 p是一种V型肌球蛋白，其沿着沿着极化肌动蛋白电缆主动运输囊泡，是Lgl肿瘤抑制因子Sro 7 p的酵母同系物，其将Sec 4-GTP物理连接到t-SNARE Sec 9 p，外囊是由八种不同的基因产物组成的复合体，它与囊泡表面的成分以及细胞皮层上的极性决定簇相互作用，从而将进入的分泌囊泡束缚在这些专门的位点上。这个提议试图更详细地探讨外囊促进极化输出的机制。1.我们将通过识别两个一致的信号，Sec 4-GTP和v-SNARE Snc 2 p来测试外囊被募集到囊泡表面的假设。2.我们将确定外囊是否可以同时结合vSNARE Snc 2 p和t-SNARE Sec 9 p，形成Snc 2 p-exocyst-Sec 9 p三元复合物。相互作用位点的诱变将在体内建立其功能。3. SNARE可以在体外自组装，但速度太慢，无法解释它们在体内的功能。我们将确定外泌囊，无论是单独或与其结合伙伴组合，可以催化组装的外泌陷阱复合物。4.外泌囊亚基Exo 70 p可以定位于外泌位点，独立于用于递送分泌囊泡的肌动蛋白，这意味着与细胞皮质处的极性决定簇的相互作用。我们将确定这个极性决定簇的身份。5.炭疽杆菌分泌两种毒素，损害果蝇和哺乳动物细胞外囊的功能。我们将确定它们是否也作用于酵母外囊。这将使我们能够利用酵母的简单遗传学来识别这些毒素的直接目标并确定其作用机制。