Characterization of a mammalian mutant cell line defective in glycerolipid biosynthesis.

摘要

Glycerolipids are structural components of membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One mutant isolated from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine and triglycerides, but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity, which is encoded by the lipin gene family. Expression cloning revealed a point mutation, Glycine 189 to Glutamate, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans and mice. GPI activity was reduced by 87% in GroDl. GroDl displayed no evidence of reduced glycolysis, presenting normal level of ATP, fructose-6-phosphate and glycerol-3-phosphate. Expression of wild-type GPI restored GPI activity, glycerolipid biosynthesis and PAP1 activity. cDNA from isoforms of lipin 1 (alpha and beta) were isolated from the CHO cell lines and no mutations were detected in GroDl. Over-expression of either isoform resulted in elevated PAP1 activity, in vitro, but this did not correct the glycerolipid biosynthesis deficiency. The glycerolipid biosynthesis deficiency phenotype showed a high correlation with glucose concentration in the growth media and could be reversed by lowering glucose concentrations. Pronounced glucose-6-phosphate accumulation was observed in these cells. Fluorescence microscopy showed that in GroDl cells lipin 1-alpha/GFP fusion protein did not localized to the nucleus, unlike the parent strain. This could be corrected by expression of wild-type GPI. Lipin 1-alpha localization is known to be controlled by the mammalian target of rapamycin (mTOR) protein complex. Western blot analysis showed an increase in Akt phosphorylation, independent of phosphoinositide-dependent kinase-1 (PDK 1) activation, suggesting mTOR activation. Altogether, the data suggest that the loss of GPI activity has a pleiotropic effect on lipin 1 due to accumulation of glucose-6phosphate. These results may also help explain the neuromuscular symptoms and the large variability in the severity of the anemia associated with inherited GPI deficiency.