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 Activity These 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.
