Genetic Control of Lithium Sensitivity and Regulation of Inositol Biosynthetic Genes
A, Mipp1 enzyme prepared from Mipp1 over-expressing cells were incubated with 10 nmoles InsP6 for the indicated times and analyzed by HPLC-MDD. I(1,4)P2 was generated by contaminating IP3 5-phosphatase activity, and demonstrates that the product of Mipp1 is I(1,4,5)P3. B, Mipp1 activity measured as the rate of IP3 formation in extracts from wild type, mipp1 null mutant and Mipp1overexpressingcells. C, Cell tracks of wild type and mipp1 mutant cells in 7 mM LiCl (or control of 7 mM NaCl) during chemotaxis. mipp, mipp:dpoA and Mipp1 over-expressing mutants (Mipp1oe) are Li+ hypersensitive compared to wild type. D = Directionality, S = cell speed (µm/min). D, Vegetative wild type and mipp1 null cells were treated with either 1.2 mM Z-pro-L-prolinal or an equal volume of DMSO carrier as a control. Samples were then removed at the times indicated and IP3 concentration measured. Values plotted as mean ± standard error of 4 independent experiments. * P<0.05, paired T-test. E, Mipp1 protein extracts were incubated with recombinant DpoA with or without the PO inhibitor Z-pro-L-prolinal. Mipp1 activity is measured by the production of IP3 from IP6. Values plotted as mean ± standard error of 3 independent experiments. ** P<0.01, paired T-test. Samples of Mipp1 protein are shown on Western (underneath).