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. 2016 Jun 21;110(12):2689-2697.
doi: 10.1016/j.bpj.2016.05.023.

Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop

Hiroyuki Nakao  1 , Keisuke Ikeda  2 , Yasushi Ishihama  1 , Minoru Nakano  3
Affiliations

Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop

Hiroyuki Nakao et al. Biophys J. .

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.

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Figures

Figure 1
Figure 1
Amino acid sequences of the synthesized peptides. The residues surrounded by the rectangular box represent the central regions of the sequences. Hydrophilic residues in the central region are shown in bold text. ATF6, activating transcription factor 6; EDEM1, ER degradation-enhancing α-mannosidase-like protein 1; FACL4, long-chain fatty acid-CoA ligase 4; FMO2, flavin-containing monooxygenase 2; JP3, junctophilin-3; JP4, junctophilin-4; MMP23, matrix metalloproteinase-23; ORP5, oxysterol-binding protein-related protein 5; P4HTM, transmembrane prolyl 4-hydroxylase; RNF180, ring finger protein 180; SPAST, spastin; TMED7, transmembrane emp24 domain-containing protein 7; UBE2J2, ubiquitin-conjugating enzyme E2 J2.
Figure 2
Figure 2
CD spectra of 13 synthetic peptides in LUVs. The peptide/lipid ratio was 0.2 mol %. Spectra are provided for (A) ATF6 (blue), EDEM1 (red), FACL4 (yellow), FMO2 (green), JP3 (light blue), JP4 (purple), and MMP23 (black), and (B) ORP5 (blue), P4HTM (red), RNF180 (yellow), SPAST (green), TMED7 (light blue), UBE2J2 (purple). Error bars represent the mean ± SD from n = 2 independent experiments. To see this figure in color, go online.
Figure 3
Figure 3
(A) Flip assays for C6NBD-PC, C6NBD-PE, and C6NBD-PS in LUVs without peptides (open circles), containing 0.1 mol % (for C6NBD-PC), or 0.025 mol % (for C6NBD-PE and -PS) EDEM1 (red circles), or containing 0.1 mol % ATF6 (blue circles), FACL4 (yellow circles), JP3 (light blue circles), JP4 (purple circles), ORP5 (blue triangles), P4HTM (red triangles), RNF180 (yellow triangles), SPAST (green triangles), TMED7 (light blue triangles), or UBE2J2 (purple triangles). The lines are fitting curves generated using Eq. 2. (B) Flip rate constants of C6NBD-PC, C6NBD-PE, and C6NBD-PS in LUVs containing 0–0.1 mol % peptides. The symbols are the same as in (A). The error bars represent the mean ± SD from n = 2 experiments. To see this figure in color, go online.
Figure 4
Figure 4
(A) Flip-rate constants of C6NBD-PC, C6NBD-PE, and C6NBD-PS in LUVs containing 0–0.1 mol% SPAST WT (black), R13A (red), or H18A (blue). (B) Flip-rate constants of C6NBD-PC, C6NBD-PE, and C6NBD-PS in LUVs containing 0–0.1 mol% EDEM1 WT (black), R10A (red), H14A (blue), R10E (green), or 0.025 mol% of both EDEM1 R10A and H14A (purple). Error bars represent the mean ± SD from n = 2 experiments. To see this figure in color, go online.
Figure 5
Figure 5
Arrhenius plots of the rate constants of intrinsic flip (open circles) or EDEM1 (0.025 mol %)-mediated flip (solid circles) of C6NBD-PC (blue), C6NBD-PE (green), and C6NBD-PS (red). Error bars represent the mean ± SD from n = 2 experiments. The rate constants of EDEM1-mediated flip were calculated as the difference of the kflip values in the presence and absence of 0.025 mol % EDEM1. To see this figure in color, go online.
Figure 6
Figure 6
Flip-rate constants of C6NBD-PC, C6NBD-PE, and C6NBD-PS in LUVs containing 0–0.1 mol% EDEM1 WT in the absence (black circles) or presence of 5 (light blue triangles) or 20 (orange circles) mol % cholesterol. Error bars represent the mean ± SD from n = 2 experiments. To see this figure in color, go online.
Figure 7
Figure 7
Schematic representation of flip-flop promotion by the EDEM1 peptide. Arg and His residues (orange) in the α-helix of EDEM1 form a hydrophilic milieu in the hydrocarbon region, through which headgroups of phospholipids can enter the membrane with a reduced activation energy and flip-flop. To see this figure in color, go online.
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