ISOLATION AND CHARACTERIZATION OF A MUTANT DEFECTIVE IN PHOSPHATIDYLETHANOLAMINE BIOSYNTHESIS FROM A MAMMALIAN CELL LINE

MARK ALAN MILLER, Purdue University

Abstract

A selection procedure was devised to facilitate the isolation of mutants with temperature-sensitive defects in the incorporation of ethanolamine into phosphatidylethanolamine (PE) from a mammalian cell line. The selection was based on the enrichment of the initial population for such mutants using (('3)H)ethanolamine suicide, followed by replica screening of the surviving population. One mutant (888.3) isolated by this procedure from the CHO-K1 cell line was further characterized. The mutant was temperature-sensitive for growth and had a reduced rate of incorporation of ethanolamine into PE. The incorporation of ethanolamine was not thermolabile in mutant 888.3 but was reduced equally at 34(DEGREES) and 40(DEGREES). The activity of the CTP:phosphoethanolamine cytidylytransferase in mutant 888.3 was reduced 2-fold relative to the parental CHO-K1 cell line, and the decreased incorporation of ethanolamine into PE correlated with a decrease in the rate of labelling of CDP-ethanolamine in mutant 888.3. Despite the reduced activity of the phosphoethanolamine cytidylyltransferase the incorporation of ('32)PO(,4) into PE and the cellular levels of PE in mutant 888.3 were indistinguishable from those of CHO-K1. Further characterization of the PE biosynthesis in these two cell lines demonstrated that the phosphoethanolamine cytidylyltransferase was rate-limiting in the incorporation of ethanolamine into PE by the Kennedy pathway, and that the flux through this pathway was reduced 2-fold in mutant 888.3. The decrease in PE synthesis through the Kennedy pathway was not compensated through increased decarboxylation of phosphatidylserine, or through the uptake of exogenous serum lipids in the culture medium. Pulse labelling studies using radiolabelled precursors suggested that in the absence of added ethanolamine, cellular PE in both CHO-K1 and mutant 888.3 was derived from serine through the decarboxylation of phosphatidylserine. The results suggest that under normal culture conditions the contribution of the Kennedy pathway to PE biosynthesis is small in the CHO-K1 cell line. In the presence of physiological concentrations of ethanolamine, however, the contribution from this pathway may be as much as 50% of total PE synthesis.

Degree

Ph.D.

Subject Area

Biology

COinS