Scramblase Activities of Transmembrane Peptides Depend on Relative Position of Hydrophilic Amino Acid Residues and their Depth in the Membrane

Abstract

Phospholipid transbilayer movement (flip-flop) is very rapid in the ER of eucaryotes, where phospholipids in the cell are mainly synthesized only at the cytoplasmic leaflet. However, the mechanism to promote the phospholipid flip-flop in the ER remains unknown. We have previously revealed that a peptide with a membrane-spanning sequence of a human ER protein, EDEM1, promotes the flip-flop, and that both Arg and His residues in the sequence are necessary for the peptide's activity. Interestingly, these hydrophilic residues are located at the same side of an α-helix structure of EDEM1 peptide. Therefore, we hypothesized that relative position of two hydrophilic residues in the helix may be important for the flip-promotion ability of peptides. In this study, we synthesized model transmembrane peptides containing two hydrophilic residues at various positions in the sequences and evaluated their activity. We demonstrated that the activities of the peptides depended on the turn angle between Arg and His in the helices, and significantly increased when two hydrophilic residues are located at the same side of the helix. The peptides containing other high hydrophilic residues at the same side of the helices also had flip-promotion abilities, whereas the substitution of either Arg or His with a lower hydrophilic or hydrophobic residue inhibited the activities. Therefore, the scramblase activities of the peptides were attributed to the hydrophilicity of the residues at the same side. We also found that the peptides with hydrophilic residues at the deeper position in the membrane showed higher flip-promotion ability. These results provided the possibility that the proteins whose transmembrane regions have similar physicochemical properties as those of the peptides used in this study might be involved in the flip-flop promotion in the ER.