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.

Lipid somersaults: Uncovering the mechanisms of protein-mediated lipid flipping

“…This led to the proposal that the interplay between transmembrane domains and non-bilayer favoring lipids might be sufficient to allow fast flip-flop in the ER [21–23]. In line with these ideas, recent studies demonstrated that low-complexity synthetic transmembrane peptides are able to increase the rate of flipping of a variety of phospholipids in synthetic vesicles [24, 25]. However, a large number of peptide molecules were needed per vesicle to see this effect.…”

“…However, a large number of peptide molecules were needed per vesicle to see this effect. For example, reconstitution of 100 peptides/vesicle corresponding to the transmembrane protein EDEM1 could improve the rate of NBD-PC flipping to 20 lipids per second [25]. However, this contrasts with the rapid flipping seen when vesicles contain only one or a few copies of an authentic scramblase (see below).…”

Outperforming nature: synthetic enzyme built from DNA flips lipids of biological membranes at record rates

“…In summary, we have shown that our synthetic DNA nanostructure can reproduce the biological function of a scramblase protein by inducing mixing of lipids that reside on opposite leaflets of a biological membrane in vitro and in human cells. Our synthetic DNA scramblase mixes lipids much more rapidly, outperforming both biological and reported artificial scramblases by at least three orders of magnitude 34,35 . Equipped with an activation mechanism and ability to target plasma membranes of specific cell types, our DNA scramblase can be made suitable for biomedical applications with the scrambling activity being controlled by the geometry of the toroidal lipid pore.…”

Microtubule-dependent and independent roles of spastin in lipid droplet dispersion and biogenesis

“…A direct role of spastin-M1 in limiting pre-LD formation is suggested by a study that tested the ability of a peptide comprising spastin-M1 membrane-spanning sequence to promote phospholipid transbilayer movement (flip-flop). Whereas the wild-type peptide increased the flip rate of lipids in artificial membranes, an R65A mutation abolished this ability (Nakao et al, 2016). It is conceivable that cells lacking spastin may have an excess of phospholipids at the cytoplasmic leaflet of the ER membrane, which could favor LD emergence (Chorlay et al, 2019).…”