Spatial Control of Inositol Phosphate Biosynthesis in Arabidopsis

肌醇磷酸盐在调节许多细胞过程中发挥重要作用，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、激素运输、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。对调节肌醇复杂代谢通量的细胞机制知之甚少。磷酸肌醇是通过葡萄糖6-磷酸的内部环化合成的。已知只有一种酶，1l -肌醇-磷酸合成酶（MIPS）能催化该反应。MIPS在拟南芥和其他生物体中的活性受到肌醇的高度调节。与现有的磷酸肌醇生物合成模式相反，本项目获得的证据表明，磷酸肌醇的生物合成并不局限于一个细胞室，即细胞质。除了细胞质外，MIPS还定位于无肌醇生长的植物的膜结合细胞室和细胞外。为了解决酶靶向或穿透膜的机制，研究人员分析了MIPS基因，以对通过序列分析发现的初级结构和上游开放阅读框内的信号进行排序。综合生物信息学分析揭示了假定的转运肽，预测将MIPS靶向表达该酶的细胞区室。该项目将测试不同基因表达机制选择性地将这种关键的生物合成酶靶向或穿过膜的假设。该项目的具体目标是：(1)在拟南芥原生质体中定位MIPS- gfp融合蛋白，(2)分离和功能测试编码MIPS假设亚型的标记cdna，以及(3)确定MIPS在微粒体中的拓扑结构。本研究将：(1)确定细胞器中肌醇磷酸生物合成的一些控制机制；(2)确定膜中MIPS的取向；(3)为从功能上剖析肌醇的复杂代谢调节提供所需的基础。这些研究将利用三种不同的拟南芥MIPS基因进行。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种基因中的一种。其他研究生和少数族裔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外，研究生和高中教师也将有机会在学习批判性思维的同时培养专业的职业道德。

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.