Regulation of Phosphoinositide Metabolism and Early Responses to Osmotic Stress

磷酸肌醇代谢的调节和对渗透应激的早期反应

• 批准号: 9604285
• 负责人: Wendy Boss
• 金额: $ 24.49万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604285&HistoricalAwards=false
• 关键词: Regulation; Phosphoinositide; Metabolism; Early; Responses

9604285 Boss Technical The survival of plants depends on their ability to alter metabolism in response to changes in their environment. Although physiological changes in response to environmental stresses are well documented, the signal transduction pathways between the initial stimulus and the induction of gene expression are not well understood. We have evidence for rapid changes in the distribution of one isoform of translational elongation factor 1 alpha (EF-1alpha) and in inositol phospholipid metabolism as a result of hyperosmotic stress and treatment with the wasp venom peptide, mastoparan. This proposal focuses on the interaction of inositol lipids and EF-1alpha in initiating early responses to stress. EF-1alpha is essential for protein synthesis and can regulate cytoskeletal structure and activate phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase is the first committed enzyme in the synthesis of polyphosphorylated inositol phospholipids. The inositol phospholipids, phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) in turn can affect cytoskeletal structure. Our working hypothesis is that EF-1alpha and PIP and PIP2 play integral roles in the signal transduction pathway as cells respond and acclimate to environmental stress. To test our hypothesis, we will study the coordinated changes in the distribution of ethanolamine-EF-1alpha and PIP synthesis from the initiation of plasmolysis until the cells reach a new steady state. We propose to biochemically characterize the glycerylphosphoethanolamine posttranslational modification of EF-1alpha; to use site-directed mutagenesis to make recombinant EF-1alpha lacking the ethanolamine attachment site and to determine the effects of this posttranslational modification on the function of EF-1alpha in vitro. Finally, we propose to obtain cDNAs encoding PI 4-kinase and make transgenic plants over producing this protein. The response of the transgenics to hyperosmotic conditions will be stu died microscopically and biochemically to determine whether PIP is involved in the initial signaling event or the recovery to a new steady state. We anticipate observing a difference in the rate or magnitude of plasmolysis and recovery of transgenics compared to wild type cells. The proposed work provides an exciting opportunity for studying the integration of signal transduction pathways during a rapid physiological response. At the same time, the work will contribute new information in two areas: 1) The functional significance of the glycerylphosphoethanolamine posttranslational modification of EF-1alpha. 2) The role of PIP in hyperosmotic stress. Finally, this work will result in future applications by identifying key components of regulatory pathways that can be used to genetically alter cellular homeostasis and cellular responses to environmental stimuli. Nontechnical Plants respond rapidly to environmental changes and stress by changing the types and quantities of proteins synthesized that function to assist to adapt the plant to its new circumstances. How the altered environment is perceived and then transmitted within the plants cell to alter protein synthesis and cellular metabolism is a central problem in plant biology. The protein EF-1alpha (elongation factor-1alpha) is an abundant and highly conserved protein that is essential for protein synthesis. The EF-1alpha regulates the structure of the intracellular network of cytoskeleton. The cytoskeleton has a critical role in defining tand maintaining the shape and morphology of plant cells. This project will examine how plant stress induces the modification of EF-1alpha and its role in transmitting the environmental stress signal. This project is important because it addresses one of the fundamental aspects of plant life, how plants respond and adapt to environmental change. ***

9604285老板技术植物的生存取决于它们因应环境变化而改变新陈代谢的能力。虽然对环境胁迫的生理变化有很好的了解，但在最初的刺激和诱导基因表达之间的信号转导途径还不是很清楚。我们有证据表明，由于高渗应激和黄蜂毒多肽Mastoparan的治疗，翻译延长因子1α的一种异构体(EF-1α)的分布和肌醇磷脂代谢的快速变化。这项建议侧重于肌醇脂类和EF-1α在启动对应激的早期反应中的相互作用。EF-1α是蛋白质合成所必需的，可以调节细胞骨架结构，激活磷脂酰肌醇4-激酶。磷脂酰肌醇4-激酶是合成多聚磷酸化肌醇磷脂的第一个关键酶。肌醇磷脂、磷脂酰肌醇-4-单磷酸(PIP)和磷脂酰肌醇-4，5-二磷酸(PIP2)依次影响细胞骨架结构。我们的工作假设是EF-1α、PIP和PIP2在细胞对环境应激的反应和适应的信号转导通路中发挥着不可或缺的作用。为了验证我们的假设，我们将研究乙醇胺-EF-1α的分布和PIP合成从质壁分离开始到细胞达到新的稳定状态的协调变化。我们建议对EF-1α的甘油基磷酸乙醇胺翻译后修饰进行生化表征；利用定点突变使重组EF-1α缺失乙醇胺结合位点，并在体外确定这种翻译后修饰对EF-1α功能的影响。最后，我们建议获得编码PI4-激酶的cDNA，并使转基因植物大量生产这一蛋白。将从显微镜和生化角度研究转基因生物对高渗透条件的反应，以确定PIP是否参与了最初的信号事件或恢复到新的稳定状态。我们预计，与野生型细胞相比，转基因细胞在质壁分离和恢复的速度或幅度上会有所不同。这项工作为研究快速生理反应过程中信号转导通路的整合提供了一个令人兴奋的机会。同时，这项工作将在两个方面提供新的信息：1)EF-1α的甘油基磷乙醇胺翻译后修饰的功能意义。2)PIP在高渗应激中的作用。最后，这项工作将通过识别可用于从基因上改变细胞内稳态和细胞对环境刺激的反应的调控途径的关键组件，从而导致未来的应用。非技术性植物通过改变合成的蛋白质的类型和数量来快速响应环境变化和胁迫，这些蛋白质的功能是帮助植物适应新的环境。改变的环境如何被感知，然后在植物细胞内传递，从而改变蛋白质的合成和细胞的代谢，这是植物生物学的中心问题。蛋白质EF-1α(延伸因子-1α)是一种含量丰富、高度保守的蛋白质，对蛋白质合成是必不可少的。EF-1α调节细胞内细胞骨架网络结构。细胞骨架在定义和维持植物细胞的形状和形态方面起着至关重要的作用。这个项目将研究植物胁迫如何诱导EF-1α的修饰，以及它在传递环境胁迫信号中的作用。这个项目很重要，因为它解决了植物生命的一个基本方面，即植物如何响应和适应环境变化。***