Regulation of exocyst-mediated post-Golgi vesicle tethering

外囊介导的高尔基体后囊泡束缚的调节

• 批准号: 7329112
• 负责人: Kelly Anne Orlando
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7329112
• 关键词: Apical; Binding; Biochemical Genetics; Biological; Biological Process; Cell Cycle Stage; Cell Polarity; Cell membrane; Cells; Complex; Defect; Diabetes Mellitus; Disease; Epithelial; Event; Exocytosis; Family; Generations; Genes; Golgi Apparatus; Hormones; Kinetics; Lead; Localized; Location; Malignant Neoplasms; Mammals; Mediating; Membrane; Membrane Fusion; Methods; Molecular; Monomeric GTP-Binding Proteins; Mutagenesis; Mutation; Neurotransmitters; Pattern; Phospholipids; Play; Polycystic Kidney Diseases; Proteins; Public Health; Range; Regulation; Role; Saccharomyces cerevisiae; Saccharomycetales; Secretory Vesicles; Staging; Time; Travel; Vesicle; Yeasts; base; basolateral membrane; cell growth; controlled release; daughter cell; migration; mutant; neurotransmitter release; novel; release factor; research study; rho

DESCRIPTION (provided by applicant): Polarized exocytosis is fundamental to a wide range of biological processes such as the release of neurotransmitters and the generation of epithelial basolateral asymmetry. Exocytosis occurs when post-Golgi secretory vesicles are tethered to and fuse with the plasma membrane (PM). Vesicle tethering is mediated by the exocyst, an evolutionarily conserved octameric protein complex. The exocyst component Exo70 physically interacts with phospholipids and is localized to the bud tip in the budding yeast Saccharomyces cerevisiae. Exo70 may act as a "landmark" protein to target vesicles to the PM. The spatial and temporal regulation of vesicle tethering controls the location and the kinetics of exocytosis; therefore, studying the regulation of Exo70 should be important for the understanding of the molecular basis of polarized exocytosis and cell growth. Aim 1: Determine the role of Gic1 and Gic2 on Exo70 function at the PM. The small GTPase Cdc42 is a regulator of polarized exocytosis. Cdc42 controls localization of Exo70 at the bud tip, but not through direct binding, suggesting that it controls Exo70 through downstream effector(s). Gic1 and Gic2, effectors of Cdc42, are localized to the bud tip during early budding stages in yeast and are important for the establishment of cell polarity. The interactions between Gic1/2 with the exocyst components (specifically Exo70), and their role in secretion and Exo70 polarization will be examined through genetic, biochemical, and cell biological methods. Aim 2: Identify and characterize novel regulators of Exo70 function. Recent studies indicate that Exo70 functions in parallel with another exocyst component, Sec3, in vesicle tethering. While mutations in either Sec3 or Exo70 have little effect on secretion and cell growth, mutations in both genes result in cell lethality. In order to identify additional regulators of exocytosis, yeast cells expressing mutant Sec3 will undergo random mutagenesis. If the mutation occurs in Exo70 or an Exo70 regulator, its combination with the Sec3 mutation should lead to cell growth defect or inviability. Experiments will be performed to identify these proteins and characterize their roles in polarized exocytosis. Relevance to Public Health: Exocytosis is a basic biological process conserved from yeast to mammals; it controls release of factors such as hormones and neurotransmitters, and incorporation of membrane for cell growth and migration. Defects in the timing and/or location of exocytosis may play a role in diseases such as cancer, diabetes and polycystic kidney disease. This study will focus on potential regulators of exocytosis in order to better understand the spatial and temporal control of exocytosis.

描述（由申请人提供）：两极分化的胞吐作用是广泛的生物学过程，例如神经递质的释放和上皮基底外侧不对称的产生。当高尔基后分泌后囊泡与质膜（PM）融合并融合时，会发生胞吐作用。囊泡的束缚是由外囊肿（一种进化保守的八聚体蛋白复合物介导的。外囊肿成分Exo70与磷脂物理相互作用，并位于酿酒酵母的发芽酵母菌中的芽尖。 EXO70可以用作将囊泡靶向PM的“地标”蛋白。囊泡束缚的空间和时间调节控制了胞吞作用的位置和动力学。因此，研究EXO70的调节对于理解两极分化胞吐和细胞生长的分子基础应该很重要。 AIM 1：确定GIC1和GIC2在PM上exo70功能的作用。小GTPase CDC42是极化胞吐作用的调节剂。 CDC42控制着芽尖处的Exo70的定位，但不能通过直接结合来控制它通过下游效应子（S）控制EXO70。 Cdc42的效应子GIC1和GIC2在酵母的早期萌芽阶段位于芽尖，对于建立细胞极性很重要。 GIC1/2与外囊肿成分（特别是EXO70）之间的相互作用以及它们在分泌和EXO70极化中的作用将通过遗传，生化和细胞生物学方法来检查。 AIM 2：识别和表征EXO70功能的新型调节剂。最近的研究表明，EXO70与囊泡束缚中的另一个外囊肿成分Sec3并行起作用。尽管SEC3或EXO70中的突变对分泌和细胞生长几乎没有影响，但两种基因的突变都会导致细胞致死性。为了鉴定胞吐作用的其他调节剂，表达突变体Sec3的酵母细胞将经历随机的诱变。如果该突变发生在EXO70或EXO70调节剂中，则其与SEC3突变的组合应导致细胞生长缺陷或不可侵袭性。将进行实验以鉴定这些蛋白质并表征它们在极化胞吐作用中的作用。 与公共卫生的相关性：胞吐作用是从酵母到哺乳动物保守的基本生物学过程；它控制了诸如激素和神经递质等因素的释放，以及膜的掺入细胞生长和迁移。胞吐作用的时间和/或位置的缺陷可能在癌症，糖尿病和多囊性肾脏疾病等疾病中起作用。这项研究将集中于胞吐作用的潜在调节剂，以便更好地了解胞吐作用的空间和时间控制。