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J Biol Chem. 2010 Jan 8;285(2):866-77. doi: 10.1074/jbc.M109.068213. Epub 2009 Nov 10.

Isolation of novel animal cell lines defective in glycerolipid biosynthesis reveals mutations in glucose-6-phosphate isomerase.

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1
Department of Physiology and Biophysics, Boston University School of Medicine, Massachusetts 02118, USA.

Abstract

Glycerolipids are structural components for 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 isolate 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. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 --> Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency.

PMID:
19903819
PMCID:
PMC2801288
DOI:
10.1074/jbc.M109.068213
[Indexed for MEDLINE]
Free PMC Article
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