title: Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop.
abstract: The mechanism whereby phospholipids rapidly flip-flop in the endoplasmic reticulum (ER) membrane remains unknown. We previously demonstrated that the presence of a hydrophilic residue in the center of the model transmembrane peptide sequence effectively promoted phospholipid flip-flop and that hydrophilic residues composed 4.5% of the central regions of the membrane-spanning sequences of human ER membrane proteins predicted by SOSUI software. We hypothesized that ER proteins with hydrophilic residues might play a critical role in promoting flip-flop. Here, we evaluated the flip rate of fluorescently labeled lipids in vesicles containing each of the 11 synthetic peptides of membrane-spanning sequences, using a dithionite-quenching assay. Although the flippase activities of nine peptides were unexpectedly low, the peptides based on the EDEM1 and SPAST proteins showed enhanced flippase activity with three different fluorescently labeled lipids. The substitution of hydrophobic Ala with His or Arg in the central region of the EDEM1 or SPAST peptides, respectively, attenuated their ability to flip phospholipids. Interestingly, substituting Ala with Arg or His at a location outside of the central region of EDEM1 or SPAST, respectively, also affected the enhancement of flip-flop. These results indicated that both Arg and His are important for the ability of these two peptides to increase the flip rates. The EDEM1 peptide exhibited high activity at significantly low peptide concentrations, suggesting that the same side positioning of Arg and His in α-helix structure is critical for the flip-flop promotion and that the EDEM1 protein is a candidate flippase in the ER.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakao
authors.affiliation: Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ikeda
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
authors.affiliation: Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. Electronic address: mnakano@pha.u-toyama.ac.jp.
authors.forename: Minoru
authors.initials: M
authors.lastname: Nakano
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D012984:Software; D000074181:Spastin; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism
chemical_list: C486654:EDEM1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; D010743:Phospholipids; D053835:Unilamellar Liposomes; D002784:Cholesterol; D000251:Adenosine Triphosphatases; D000074181:Spastin; C400283:SPAST protein, human