Autoradiographic detection of animal cell membrane mutants altered in phosphatidylcholine synthesis.

AUTOR(ES)

Esko, J D

RESUMO

We have screened approximately 20,000 colonies of Chinese hamster ovary cells immobilized on filter paper [Esko, J.D. & Raetz, C.R.H. (1978) Proc Natl. Acad. Sci. USA 75, 1190-1193] for strains unable to incorporate [methyl-14C]-choline into trichloroacetic acid-precipitable phospholipid at 40 degrees C. Mutant 58, identified in this way, was specifically defective in choline incorporation, and other isolates were also blocked in thymidine and leucine incorporation into DNA and protein, respectively. Further analysis of mutant 58 revealed that the strain grew almost normally at 33 degrees C, the permissive temperature, but divided only once at 40 degrees C, the restrictive temperature. After a 20-hr incubation at 40 degrees C, the phosphatidyl-choline level dropped from 41% to 20% in the mutant whereas other phospholipids, including sphingomyelin, continued to accumulate. Wild-type cells contained approximately 50% phosphatidylcholine at both temperatures. Anion-exchange chromatography of the water-soluble choline metabolites extracted from mutant 58 revealed that phosphorylcholine accumulation increased from 6 nmol/mg of protein at 33 degrees C to 42 nmol/mg of protein at 40 degrees C whereas CDP-choline decreased from 0.42 nmol to less than 0.07 nmol per mg of protein. Phosphorylcholine also increased in wild-type cells shifted from 33 degrees C to 40 degrees C (from 1.8 nmol to 16 nmol per mg of protein), but the level of CDP-choline was not altered (from 0.52 nmol to 0.58 nmol per mg of protein). Enzymatic assays of extracts prepared from mutant and wild-type cells revealed a reduction of CTP: phosphorylcholine cytidylyltransferase (EC 2.7.7.15) activity (CDP-choline synthetase) in the mutant to 1/40th that in the wild type, and mixing experiments excluded the production of antagonists to CDP-choline synthesis in the mutant. Thus, the inability of the mutant to generate normal amounts of phosphatidylcholine in vivo was correlated with an enzymatic lesion in the biosynthesis of CDP-choline in vitro.

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