Disruption of inositol biosynthesis through targeted mutagenesis in Dictyostelium discoideum: generation and characterization of inositol-auxotrophic mutants
Résumé
myo-Inositol and its downstream metabolites participate in diverse physiological processes. Nevertheless, considering their variety, it is likely that additional roles are yet to be uncovered. Biosynthesis of myo-inositol takes place via an evolutionarily conserved metabolic pathway and is strictly dependent on D-myo-inositol 3-phosphate synthase (EC 5.5.1.4). Genetic manipulation of this enzyme will disrupt the cellular inositol supply. Two methods, based on gene deletion and antisense strategy, were used to generate mutants of the cellular slime mold Dictyostelium discoideum. These mutants are inositol-auxotrophic and show phenotypic changes under inositol starvation. One remarkable attribute is their inability to live by phagocytosis of bacteria, which is the exclusive nutrient source in their natural environment. Cultivated on fluid media, the mutants lose their viability when deprived of inositol for longer than 24 h. Here we report a study of the alterations in the first 24 h in cellular inositol, inositol phosphate and phosphoinositide concentrations, whereby a rapidly accumulating phosphorylated compound was detected. After its identification as 2,3-bisphosphoglycerate, evidence could be found that the internal disturbances of inositol homeostasis triggers the accumulation. In a first attempt to characterise this as a physiologically relevant response, the efficient in vitro inhibition of a D. discoideum inositol polyphosphate 5-phosphatase (EC 3.1.3.56) by 2,3-bisphosphoglycerate is presented.
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