Study on Membrane Function by the Mutants With Requirement for Phosphatidylinositol.

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Phosphatidylinositol (PI) is one of the major membrane lipids in eukaryotic cells. Its phosphorylated or degraded derivatives are now well known to be involved in important signal transduction pathways.The physiological function of phosphatidylinositol involving synthesis and assembly of bio-membrane was examined with mouse FM3A cells, derived from spontaneous mammary carcinoma of C3H/He mouse FM3A cells.Above 10 uM of myo-inositol (Ins) was required for growth of this cell line. The metabolic label experiments and quatitative analysis of cellular phospholipids in mouse FM3A cells grown in the presence of PI showed 1) increase of PI content, 2) stimulation of phosphoinositides synthesis, 3) decrease of phosphatidylglycerol (PG) content, and 4) stimulation of [^3H]Ins incorporation into lipid fractions. [^3H]labeled PI was incorporated into cellular lipid fractions without influence with the concentration of Ins or the kinds of fetal calf serum during pre-culture. Incorporation of [^3H] … More Ins into lipid fraction by these homogenates (PI synthesizing activity) was assayed at about neutral pH (pH 7.4) and the low concentration of Ins (11 uM). CDP-diacylglycerol independent incorporation of [^3H]Ins into lipid fraction (PIE) was 1.5 fold greater than CDP-diacylglycerol dependent one (PIS). With addition of PI, PIS was almost inhibited, and PIE was increased in proportion to the concentration of supplemented PI. In one of the mutants with requirement for PI on the agar plate (6-2-III), incorporation of [^3H]Ins into cells or cellular lipid fraction reduced to 40 %, 60-70% respectively. The mutant revealed the longer generation time (1.5 - 2 fold) and decrease of cell aggregation than the wild type's. In another mutant (17-6-I), even in the medium without Ins, the addition of PI could maintained the higher growth rate and incorporation of [^3H]PI into cellular lipid fraction was increased about 1.4 fold. The results that (1) the manipulation of cellular PI by the medium supplemented with PI, contribution of PI : Ins exchange enzyme activity to the increase of content of cellular PI and alteration of Ins or PI incorporation into cells or lipid fraction in PI auxotrophic mutants suggested the important consequences to clarify the physiological function of PI and Ins in eukaryotic biomembrane. Less