Disruption of inositol biosynthesis through targeted mutagenesis in Dictyostelium discoideum: generation and characterization of inositol-auxotrophic mutants

Abstract

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 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 mould 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 medium, 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-BPG (2,3-bisphosphoglycerate), evidence could be found that the internal disturbances of inositol homoeostasis trigger the accumulation. In a first attempt to characterize 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-BPG is presented.

Genetic Control of Lithium Sensitivity and Regulation of Inositol Biosynthetic Genes

“…Expression of the Dictyostelium ino1 gene [9], [26] was elevated both in dpoA null cells and cells treated with PO inhibitor (Figure 4A,B). Furthermore both conditions gave a 60-100% increase in expression of the genes encoding IMPase ( impA1 ), IPP ( ippA and ippB ) and Dd5P3, a phosphatase that converts I(1,4,5)P 3 to I(1,4)P 2 (see Table S1).…”

Functional nanofibers in sound absorption, electromagnetic wave attenuation and bioreactor application

“…The specifi city involves discrimination between substrates (substrate specifi city), similar parts of molecules and optical isomers (Fischbach et al ., 2006;Ju and Parales, 2006;Synowiecki and Woosowska, 2006). The mildness and specifi city of enzymes endow them with a high effi ciency for applications in fi ne-chemical/pharmaceutical synthesis, food processing, biosensor fabrication, biodegradation, biofuel cells, bioremediation and protein digestion in proteomic analysis (Polat et al ., 1997;Akgol and Dinckaya, 1999;Choudhary et al ., 2003;Hasan et al ., 2006).…”

Dephosphorylation of 2,3-bisphosphoglycerate by MIPP expands the regulatory capacity of the Rapoport–Luebering glycolytic shunt

“…Nevertheless, in vertebrates and lower eukaryotes, 2,3-BPG fulfills an essential role in glycolysis by priming the phosphoglycerate mutase reaction that converts 3-PG to 2-PG (4). More recently, experiments in Dictyostelium have suggested that 2,3-BPG, through an unknown mechanism, provides a molecular link between the turnover of phosphorylated inositol derivatives and glycolytic flux (5). Nonetheless, the most well studied function for 2,3-BPG is its regulation of whole-body oxygen homeostasis; 2,3-BPG executes this role because it is the main allosteric effector of hemoglobin in the majority of mammals.…”

“…We considered this question to be intriguing because cellular levels of 2,3-BPG are only 1% of the total cellular content of InsP 6 (5,18), one of DdMipp1's alternate canonical substrates (15). The levels of 2,3-BPG in Dictyostelium have been estimated at Ϸ6 M (5), which is too low for our MDD-HPLC system to accurately resolve from other organic phosphates.…”

“…Overall, our data show that the Rapoport-Leubering shunt not only includes an additional catalytic reaction but also should be considered an important regulatory system with several roles in cell physiology. ular link between glycolytic flux and the turnover of phosphorylated inositol derivatives (5). To investigate this idea, we used a bioinformatic approach to examine the enzymes involved in inositol phosphate metabolism for candidate overlapping functions associated with glycolysis.…”