Flip-Flop Promotion by Membrane-Spanning Sequences in the ER Membrane Proteins

Phospholipid flip-flop in the endoplasmic reticulum (ER) is very rapid and non-specific to phospholipid headgroups, which is necessary to maintain lipid homeostasis in the membrane. Much research suggested that the rapid flip-flop in the ER is mediated by membrane proteins. However, “flippases”, which are responsible for the flip-flop, have not been identified yet. We have previously demonstrated that peptide sequences that contain a hydrophilic residue in the transmembrane region are effective to promote the flip-flop and that such sequences can be seen in the transmembrane region of ER proteins predicted by SOSUI. Therefore, we hypothesized that ER proteins with hydrophilic residues in the transmembrane region may promote the flip-flop. In this study, we synthesized peptides with membrane-spanning sequences of several ER proteins and investigated their flip-flop promotion abilities by using fluorescent-labeled phospholipids. We found that the sequences of EDEM1 and SPAST promoted the flip-flop of fluorescent lipids non-selectively with respect to the glycerophospholipid structure. These two peptides contain both positively charged Arg and non-charged His near the center of the sequences. Thus, we next examined how the flip-flop promotion abilities were altered by substitution of hydrophobic Ala for Arg or His. The Arg to Ala mutation inhibited the flip-flop promotion abilities of each peptides completely, while the His to Ala mutation did partly. These results suggested that both Arg and His are responsible for the flip-flop promotion by the peptides and Arg plays a vital role in the activities. Considering the high activity of the EDEM1 peptide observed at significantly low peptide concentrations, EDEM1 protein might act as a “flippase”.