Abstract
Phosphatidylserine (PS) in mammalian cells is synthesized through the exchange of free L-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine (PE). The serine base exchange in Chinese hamster ovary (CHO) cells is catalyzed by at least two enzymes, PS synthase (PSS) I and II. A PSS I-lacking mutant of CHO-K1 cells, PSA-3, which exhibits approximately 2-fold lower serine base exchange activity than CHO-K1, is defective in the conversion of phosphatidylcholine to PS but has the ability to convert PE to PS. The PSA-3 mutant requires exogenous PS or PE for cell growth. In the present study, from PSA-3 mutant cells, we isolated a mutant, named PSB-2, with a further decrease in the serine base exchange activity. The activity in the homogenate of PSB-2 mutant cells was approximately 10% that of PSA-3 mutant cells and approximately 5% that of CHO-K1 cells. The PSB-2 mutant exhibited an approximately 80% reduction in the PSS II mRNA level relative to that in PSA-3 mutant and CHO-K1 cells. These results showed that the PSB-2 mutant is defective in PSS II. Like the PSA-3 mutant, the PSB-2 mutant grew well in medium supplemented with PS. However, in the medium supplemented with PE, the PSB-2 mutant was incapable of growth, in contrast to the PSA-3 mutant. In the medium with exogenous PE, the PSB-2 mutant was defective in PS biosynthesis, whereas the PSA-3 mutant synthesized a normal amount of PS. A metabolic labeling experiment with exogenous [32P]PE revealed that the PSB-2 mutant was defective in the conversion of exogenous PE to PS. This defect and the growth and PS biosynthetic defects of the PSB-2 mutant cultivated with exogenous PE were complemented by the PSS II cDNA. In addition, the cDNA of the other PS synthase, PSS I, was shown not to complement the defect in the conversion of exogenous PE to PS of the PSB-2 mutant, implying that PSS I negligibly contributes to the conversion of PE to PS in CHO-K1 cells. These results indicated that PSS II is critical for the growth and PS biosynthesis of PSA-3 mutant cells cultivated with exogenous PE and suggested that most of the PS formation from PE in CHO-K1 cells is catalyzed by PSS II.
Highlights
Phosphatidylserine (PS)1 is one of the major phospholipids in mammalian cells, comprising about 10% of the total membrane phospholipids
Regardless of the decreases in the serine and ethanolamine base exchange activities, the PSB-1 mutant showed no remarkable alteration in cell growth, PS biosynthesis, or phospholipid composition compared with the parental strain, PSA-3
PS formation in mammalian cells occurs through the exchange of L-serine with the base moiety of PC or PE (8, 20 –22)
Summary
Cells and Culture Conditions—CHO-K1 cells were obtained from the American Type Culture Collection and routinely maintained in Ham’s F-12 medium (ICN Biomedicals) supplemented with 10% (v/v) newborn calf serum (ICN Biomedicals), 100 units/ml penicillin G, 100 g/ml streptomycin sulfate, and 1.176 g/liter NaHCO3 at 37 °C under a 5% CO2 atmosphere and 100% humidity. These clones were subjected to two more cycles of colony screening and purified by limited dilution This mutant screening was performed to allow the isolation of temperature-sensitive conditional mutants, because the decrease in the serine base exchange activity might have a lethal effect on PSA-3 mutant cells. Preparation of [32P]PE and [32P]PC—J 774.1 cells were seeded at 2 ϫ 107 cells in a 150-mm-diameter dish containing 30 ml of phosphate-free modified Eagle’s medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin G, and 100 g/ml streptomycin sulfate and labeled with 400 Ci of 32Pi (the Japan Atomic Energy Institute, Ibaragi, Japan) per dish at 37 °C for 3 days. The amount of protein was determined as described (18), using bovine serum albumin as a standard
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