PRODUCTION AND CHARACTERIZATION OF A MUTANT-CELL LINE DEFECTIVE IN AMINOPHOSPHOLIPID TRANSLOCASE

Phospholipids are normally asymmetrically distributed between leaflets of the plasma membrane, due to the activity of aminophospholipid tran slocase (APT), a putative plasma membrane Mg2+-ATPase which is thought to selectively transport phosphatidylserine (PS) and other aminophosp holipids from outer to inner membrane leaflet. Although several candid ate proteins have been proposed to serve this function, positive ident ification awaits demonstration of their capacity to restore APT activi ty to a cell line that is deficient in this process. This study descri bes a simple and rapid protocol for the production and selection of mu tant cell lines that are defective in APT activity and suitable for ex pression cloning of cDNAs coding for candidate APT enzymes. By flow cy tometry, we demonstrate the time-dependent uptake of NBD-labeled PS, b ut not phosphatidylcholine (PC), by the mouse fibroblast cell line SV- T2. This uptake was inhibited by known inhibitors of APT, including o- vanadate and N-ethylmaleimide, and by ATP-depletion. SV-T2 cells were mutagenized with ethyl methanesulfonate, and APT-deficient cells were isolated by fluorescence activated cell sorting using NBD-PS as substr ate. From a total of 7.2.10(6) cells passed through the flow cytometer , 98 clones exhibited APT activity that was less than 50% of that obse rved for wild-type SV-T2 cells. One clone which exhibited less than or equal to 25% of that observed for wild-type cells, mutant M2711, was further characterized. The defect in mutant M2711 was specific for NBD -PS, and cellular ATP was unchanged, suggesting that the defect in APT activity was not due to a decrease in cellular ATP levels. Mutant M27 11 exhibited a growth pattern indistinguishable from that of wild-type SV-T2 cells, and SV-40 large T antigen, which is needed for efficient episomal replication of plasmids containing the SV40 origin of replic ation, was unchanged. Finally, transfection of M2711 with cDNAs for ma rker membrane proteins consistently resulted in the same high level of protein expression as that observed for identically-transfected wild- type SV-T2. Thus, flow cytometry can be used for rapid identification of mutants with defects in phospholipid transport that are suitable fo r functional reconstitution by transfection with candidate APT cDNAs.