Phospholipid flippases and Sfk1p, a novel regulator of phospholipid asymmetry, contribute to low permeability of the plasma membrane

Phospholipid flippase (type 4 P-type ATPase) plays a major role in the generation of phospholipid asymmetry in eukaryotic cell membranes. Loss of Lem3p-Dnf1/2p flippases leads to the exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell surface in yeast, resulting in sensitivity to PS- or PE-binding peptides. We isolated Sfk1p, a conserved membrane protein in the TMEM150/FRAG1/DRAM family, as a multicopy suppressor of this sensitivity. Overexpression of SFK1 decreased PS/PE exposure in lem3 Delta mutant cells. Consistent with this, lem3 Delta sfk1 Delta double mutant cells exposed more PS/PE than the lem3 Delta mutant. Sfk1p was previously implicated in the regulation of the phosphatidylinositol- 4 kinase Stt4p, but the effect of Sfk1p on PS/PE exposure in lem3 Delta was independent of Stt4p. Surprisingly, Sfk1p did not facilitate phospholipid flipping but instead repressed it, even under ATP-depleted conditions. We propose that Sfk1p negatively regulates transbilayer movement of phospholipids irrespective of directions. In addition, we showed that the permeability of the plasma membrane was dramatically elevated in the lem3 Delta sfk1 Delta double mutant in comparison with the corresponding single mutants. Interestingly, total ergosterol was decreased in the lem3 Delta sfk1 Delta mutant. Our results suggest that phospholipid asymmetry is required for the maintenance of low plasma membrane permeability.