An isoform of 14-3-3 protein regulates transbilayer lipid movement at the plasma membrane

Exogenous phosphatidylserine (PS) is cytotoxic to Chinese hamster ovary (CHO) cells with a PS-resistant CHO cell mutant exhibiting impaired transbilayer movement of a fluorescent PS analog at the plasma membrane. Here, we demonstrate that both mRNA and protein levels of 14-3-3 zeta are markedly reduced in this mutant, while knockdown of 14-3-3 zeta in wild-type CHO cells confers partial resistance to exogenous PS. Consistently, these knockdown cells display defective transbilayer movement of 1-palmitoyl-2-{6-[(7-nitro-1,3-benz-2-oxadiazol-4-yl) amino] hexanoyl}-sn-glycero-3-phosphoserine (C₆-NBD-PS). However, 14-3-3 zeta knockdown does not further enhance PS resistance in ATP11C-deficient cells. These findings indicate that 14-3-3 zeta plays a regulatory role in ATP11C-dependent transbilayer movement of PS at the plasma membrane. Impact statement 14-3-3ζ regulates ATP11C-dependent phosphatidylserine (PS) transbilayer movement at the plasma membrane. Reduced 14-3-3ζ impairs PS flipping and confers resistance to exogenous PS in CHO cells, identifying 14-3-3ζ as a key modulator of phospholipid asymmetry and lipid-induced cytotoxicity.