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

Abstract

Membrane lipids diffuse rapidly in the plane of the membrane but their ability to flip spontaneously across a membrane bilayer is hampered by a significant energy barrier. Thus spontaneous flip-flop of polar lipids across membranes is very slow, even though it must occur rapidly to support diverse aspects of cellular life. Here we discuss the mechanisms by which rapid flip-flop occurs, and what role lipid flipping plays in membrane homeostasis and cell growth. We focus on conceptual aspects, highlighting mechanistic insights from biochemical and in silico experiments, and the recent, ground-breaking identification of a number of lipid scramblases.

Keywords: Flippase; Floppase; Membrane asymmetry; Phosphatidylserine; Photoreceptor; Scramblase.

Figure 1. Phospholipid motions in a membrane

Phospholipid bilayers are two-dimensional fluids. Individual lipid molecules have a cross-sectional area of ~0.7 nm². In each monolayer of the membrane bilayer they can rotate very rapidly around their head-to-tail axis with a characteristic time of 10⁻⁹ seconds, and diffuse laterally within the plane of a membrane leaflet with a translational diffusion coefficient of ~10⁻⁸ cm² seconds⁻¹, i.e. the time taken for a phospholipid to move ~1 nm to replace a neighboring phospholipid is ~100 nanoseconds. In contrast, spontaneous exchange of phospholipids between leaflets (flip-flop) is slow, taking typically ~100 hours. The energy barrier that must be overcome in order to move the phospholipid headgroup through the hydrophobic interior of the membrane is >20 kcal mol⁻¹. Adapted from Mouritsen ‘Life - As a Matter of Fat’ [5].

Figure 2. Shape change in GUVs on expanding the outer monolayer of the membrane

A, Lysophosphatidylcholine (16:0) was added to a prolate GUV and the sample was observed by differential interference contrast microscopy. A time-lapse sequence is shown, starting at the left and ending at the right. As the phospholipid does not exchange between the leaflets of the bilayer on the time-scale of this experiment (~6 minutes), and because the two leaflets of the membrane are coupled, the GUV undergoes a predicted shape change to minimize bilayer stress caused by the excess lipid in one leaflet [3]. B, The same experiment as in panel A, except that C6 ceramide (d18:1/6:0) was added to a prolate GUV. The shape change induced by excess ceramide in the outer leaflet is evident. However, as C6 ceramide flip-flops rapidly, the number of lipids in the two leaflets of the GUV membrane eventually normalizes to restore the original shape of the GUV. Images courtesy of Patricia Pipaluk Mia Mathiassen.

Figure 3. Lipid transporters and membrane lipid asymmetry

The endoplasmic reticulum (ER) harbors constitutive scramblases that facilitate rapid flip-flop of lipids and allow them to equilibrate between the two membrane leaflets independently of ATP. This system is unable to accumulate a given lipid in one leaflet. Thus, retentive mechanisms are required to trap lipids (e.g. PS) on the luminal side of the ER; also, for example, consumption of glycolipid biosynthetic intermediates such as DLOs on the luminal side of the ER drives scrambling from the cytoplasmic to the luminal leaflet. In the plasma membrane (PM) of eukaryotic cells, flip-flop of phospholipids is constrained by the absence/silencing of constitutive scramblases. Thus, ATP-dependent flippases (P4-ATPase family members) and floppases (ABC transporters) can maintain an asymmetric phospholipid distribution by moving specific lipids towards or away from the cytosolic leaflet. Cellular activation triggered by cytosolic calcium, caspases or other stimuli can collapse the lipid asymmetry by the transient activity of ATP-independent scramblases. Note that the term “flippase” is sometimes used to designate an enzyme that catalyses lipid flip-flop in both directions [33]. PC, phosphatidylcholine; PS, phosphatidylserine; DLOs, lipid-linked oligosaccharides.

Figure 4. MprF-mediated bacterial CAMP resistance

MprF is a bifunctional protein. Its synthase domain (S) transfers lysine from lysyl-tRNA to phosphatidylglycerol (PG) to synthesize lysyl-PG, whereas its flippase domain (F) transfers lysyl-PG across the inner membrane to the exoplasmic/periplasmic side. Negatively charged PG attracts cationic anti-microbial peptides (CAMPs), whereas lysyl-PG being neutral does not.

Figure 5. Lipid transporters in photoreceptor discs

ABCA4 is an ABC transporter specific for PE and N-retinylidene-PE (NRPE); ATP8A2 is a P4-ATPase specific for PS and PE – however, it is not clear whether it is active in discs (see text); rhodopsin (Rho) is a scramblase that translocates common phospholipids (PL) in an ATP-independent manner. Arrows show the direction of lipid transport. ABCA4 is unusual amongst mammalian ABC transporters because it is the only one reported thus far that functions as an importer or flippase. Figure redrawn from [51].

Figure 6. Glycolipid scrambling is necessary for protein N-glycosylation in the ER

G3M9-DLO, the oligosaccharide donor for protein N-glycosylation, is synthesized in the ER in a multi-step, topologically split pathway. The first 7 steps convert dolichyl-P (dol-P) to M5-DLO on the cytoplasmic face of the ER. Then, M5-DLO is flipped into the ER lumen and extended in 7 further steps to G3M9-DLO. The sugar donors for these luminal reactions are MPD and GPD that are synthesized on the cytoplasmic face of the ER and must be flipped to the luminal side. In addition to its role in N-glycan biosynthesis, MPD is required in the ER lumen for GPI anchor biosynthesis, O-mannosylation, and C-mannosylation. Oligosaccharyltransferase (OST) transfers the oligosaccharide from G3M9-DLO to Asn residues within glycosylation sequences in translocating proteins as they emerge from the translocon into the ER lumen. The dolichyl-PP product of the OST reaction is recycled. The multistep synthesis and transfer of the oligosaccharide require at least 40 gene products.

Figure 7. Bacterial cell wall (peptidoglycan) assembly

The peptidoglycan building block is assembled on the lipid undecaprenyl phosphate on the cytoplasmic side of the bacterial inner membrane (IM). The enzyme MraY uses UDP-N-acetylmuramic acid-L-Ala-γ-D-Glu-A2pm-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) to synthesize Lipid I. MurG then catalyzes the transfer of GlcNAc from UDP-GlcNAc to Lipid I to generate Lipid II. Lipid II is flipped across the inner membrane (depicted here as a bidirectional process, although this is not fully established) where transglycosylases (TG) polymerize the GlcNAc-MurNac-pentapeptide units into glycan chains attached to undecaprenol by a pyrophosphate linkage. These chains are crosslinked to pre-existing peptidoglycan by transpeptidases while the terminal D-Ala residues in each unit are removed by carboxypeptidases. Figure redrawn from Ref. [62].

Figure 8. In silico analysis of pore-mediated lipid flip-flop

Molecular dynamics simulation showing that the appearance of a water pore facilitates the spontaneous migration of lipids across a phospholipid membrane. (A) 0 picoseconds, (C) 118.9 nanoseconds, (D) 122.4 nanoseconds, (E) 152.7 nanoseconds. Lipids (except for the flip-flopped one) are not shown; water is shown in red and white, acyl chains of the flip-flopped lipid are shown in yellow, and its choline and phosphate groups are shown in orange and green, respectively. Adapted and reprinted by permission from [189], American Chemical Society, copyright 2007.

Figure 9. Model for substrate flipping by TMEM16 and ATP8A2

(A) Credit card swiping model. The magnetic strip on the card (= polar headgroup of the phospholipid being transported) is protected from the lipid environment (by passage through the groove in the card reader) as it transits the hydrophobic interior of the membrane. See text for details and for a discussion. Adapted from a figure drawn by Adam Steinberg and published in Ref.[194]. Panel (B) and (C) show alternate views of the proposed conduit groove in nhTMEM16 (PDBID 4WIS) and homology modeled bovine ATP8a2 (Uniprot ID: C7EXK4, cytoplasmic domains are omitted), respectively. Both proteins are shown in surface representation with dark salmon color. The grooves are highlighted in blue with a hypothetical lipid placed inside shown as green CPK representation. The ATP8A2 homology model was generated based on the sarcoplasmic-endoplasmic reticulum Ca²⁺ ATPase I structure (PDBID: 3B9B) and in vacuo energy minimized using GROMOS96 implementation in Swiss-Pdb Viewer [195]. All models were generated with Pymol (DeLano Scientific, San Carlos, California).