Spatial Control of Inositol Phosphate Biosynthesis in Arabidopsis

拟南芥中磷酸肌醇生物合成的空间控制

• 批准号: 0446835
• 负责人: Margaret Johnson
• 金额: --
• 依托单位: University of Alabama Tuscaloosa
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0446835&HistoricalAwards=false
• 关键词: Spatial; Control; Inositol; Phosphate; Biosynthesis

Inositol phosphates play essential roles in regulating numerous cellular processes including signal transduction, cell wall biosynthesis, cell membrane formation, stress response, seed germination, hormone transport, nuclear RNA export, synaptic membrane trafficking, cell surface protein anchoring, and receptor-mediated endocytosis. Little is known of cellular mechanisms that regulate the complex metabolic flux of inositol. Inositol phosphate is synthesized via the internal cyclization of glucose 6-phosphate. Only one enzyme, 1L-myo-inositol1-phosphate synthase (MIPS), is known to catalyze this reaction. The activity of MIPS in Arabidopsis and in other organisms is highly regulated by inositol. In contrast to existing paradigms of inositol phosphate biosynthesis, the evidence obtained in this project suggests that the biosynthesis of inositol phosphate is not restricted to one cellular compartment, the cytosol. In addition to the cytosol, MIPS was localized in membrane bound cellular compartments and extracellularly in plants grown in the absence of inositol. To address mechanisms by which the enzyme is targeted to or through membranes, MIPS genes were analyzed for sorting signals within primary structures and upstream open reading frames that were discovered through sequence analyses. Comprehensive bioinformatics analyses revealed putative transit peptides that are predicted to target MIPS to cellular compartments found to express the enzyme. This project will test the hypothesis that differential gene expression mechanisms selectively target this pivotal biosynthetic enzyme to or through membranes. The specific objectives of this project are: (1) to localize MIPS-GFP fusion proteins in protoplasts of Arabidopsis, (2) to isolate and functionally test tagged cDNAs encoding putative isoforms of MIPS, and (3) to determine the topology of MIPS in microsomes. This research will: (1) define some of the mechanisms controlling inositol phosphate biosynthesis in organelles, (2) determine the orientation of MIPS in membranes, and (3) provide the foundation needed to functionally dissect the complex metabolic regulation of inositol.Broader Impact Resulting from Proposed ActivityThese studies will be carried out utilizing three different Arabidopsis MIPS genes. A minority graduate student and a high school teacher from an underrepresented group will study one of the three genes. Other graduate students and minority undergraduate researchers will analyze the two remaining genes. In addition to acquiring valuable research skills to enhance their teaching abilities, the graduate students and high school teacher will also have the opportunity to develop professional work ethics while learning to think critically.

肌醇磷酸在调节许多细胞过程中起重要作用，包括信号转导、细胞壁生物合成、细胞膜形成、应激反应、种子萌发、激素转运、核RNA输出、突触膜运输、细胞表面蛋白锚定和受体介导的内吞作用。很少有人知道的细胞机制，调节复杂的代谢流肌醇。肌醇磷酸是由葡萄糖6-磷酸经内环化反应合成的。只有一种酶，1 L-肌醇1-磷酸合酶（MIPS），已知催化这一反应。拟南芥和其他生物中MIPS的活性受到肌醇的高度调节。与磷酸肌醇生物合成的现有范例相反，在该项目中获得的证据表明，磷酸肌醇的生物合成不限于一个细胞隔室，即细胞质。除了胞质溶胶，MIPS定位于膜结合的细胞室和细胞外在植物中生长的肌醇的情况下。为了解决酶靶向或穿过膜的机制，分析了MIPS基因的一级结构和上游开放阅读框内的分选信号，这些信号是通过序列分析发现的。全面的生物信息学分析揭示了推定的转运肽，预测其将MIPS靶向至发现表达该酶的细胞区室。这个项目将测试的假设，差异基因表达机制选择性地针对这一关键的生物合成酶或通过膜。本项目的具体目标是：（1）定位MIPS-GFP融合蛋白在拟南芥原生质体中的位置，（2）分离并功能性检测编码MIPS亚型的cDNA，（3）确定MIPS在微粒体中的拓扑结构。这项研究将：（1）确定在细胞器中控制肌醇磷酸生物合成的一些机制，（2）确定MIPS在膜中的方向，和（3）为功能性地剖析肌醇复杂的代谢调节提供所需的基础。一名少数民族研究生和一名来自代表性不足群体的高中教师将研究这三个基因中的一个。其他研究生和少数民族本科生研究人员将分析剩下的两个基因。除了获得宝贵的研究技能，以提高他们的教学能力，研究生和高中教师也将有机会发展专业的职业道德，同时学习批判性思考。