3. P4-ATPases (Flippases) are unique to eukaryotes, where they are present in the plasma membrane
and membranes of the secretory pathway. And is essential for maintaining membrane lipid
asymmetry, signaling, vesicle formation, and regulation of membrane protein activity.
The term ‘flippase’ refer to the bidirectional lipid transporters.
Perez et al., 2015
doi:10.1038/nature14953
4. So how does Flippase work?
The flippase homodimer has three main structural features: a nucleotide binding site, a positively
charged pocket within the membrane-spanning helices, and an external helix.
Perez et al., 2015
doi:10.1038/nature14953
5. The P4-ATPases subfamily comprises five members in yeast (Drs2,
Dnf1, Dnf2, Dnf3 and Neo1)
• The active unit of flippases is a heterodimeric αβ complex: while the P4-ATPase
constitutes the catalytic α-subunit, the β-subunit exhibits chaperone-like features
and belongs to the Cdc50 family.
• The P4-ATPases present in Saccharomyces cerevisiae are well characterized, and
are involved in phospholipid translocation and vesiculating.
• In total, S. cerevisiae harbours five members of this family, namely Neo1p
(Neomycin resistant 1), Drs2p (Deficient for Ribosomal Subunit 2), Dnf1p
(Drs2p/Neo1p family), Dnf2p and Dnf3p.
• Neo1p is the only essential member of the family while the four remaining
members can be knocked out in all combinations, except for all four at once,
without leading to lethality.
6. Substrate specificities and biological roles of flippases.
Radhakrishnan Panatala et al. J Cell Sci 2015;128:2021-2032
7. Flippases participate in key biological processes:
Vesicular trafficking
Radhakrishnan Panatala et al. J Cell Sci 2015;128:2021-2032
8. Potential participation of components of the endosomal sorting complex required for transport
(ESCRT) machinery, GRASP and Flippases in the biogenesis of fungal extracellular vesicles
Oliveira et al., 2013, 14(5), 9581-9603; doi:10.3390/ijms14059581
11. The transport cycle of flippases and Na+/K+-pumps.
Flippases evolved from a family of cation pumps
Radhakrishnan Panatala et al. J Cell Sci 2015;128:2021-2032
12. Experimental procedures
• Fungal strains, media, and culture conditions
• Constructs for gene deletion and complementation mutants
• Construction of GFP and RFP fusion cassettes
• Yeast two-hybrid analyses
• Split-ubiquitin yeast two-hybrid (Y2H) assay
• Microscopic examinations of hyphal and conidial morphology
• Growth, conidiation, and stress-sensitivity tests
• Virulence assay
• Western blot analysis
• Affinity capture–mass spectrometry analysis
• Co-immunoprecipitation assay
Editor's Notes
The apo-conformation of the flippase homodimer (indicated in orange and grey above, left) forms a large cavity towards the cytoplasmic side (indicated in green). The other conformation that is ADP-bound (right) adopts a completely different structure, and forms a larger cavity that opens out to the periplasmic space. This particular flippase also contains an external helix (boxed) that also forms a small hydrophobic groove. This region may recognize the lipid tail of the lipid-linked oligosaccharide (LLO), which is shown in the center for size reference.
a) The flippase homodimer has three main structural features: a nucleotide binding site, a positively charged pocket within the membrane-spanning helices, and an external helix. b) Once the lipid tail of the LLO binds the external helix of the flippase dimer, it promotes ADP/ATP exchange and generates an outward-open state of the dimer. c) The pyrophosphate-oligosaccharide head of the LLO enters the positively-charged cavity of the dimer. d) Upon ATP hydrolysis, the dimer returns to the outward-occluded conformation that squeezes the LLO head region to the outward side of the membrane. e) The LLO tail disassociates from the flippase and extends to the cytoplasmic (i.e. opposite) side of the membrane.
Role of flippases in vesicle biogenesis. (A) Membrane curvature during vesicle budding requires a selective increase in surface area of the cytoplasmic leaflet. In ER and cis-Golgi membranes, phospholipids can readily cross the bilayer in both directions owing to loose lipid packing (low sterol content). In these flexible membranes, assembly of a protein coat would be sufficient to deform the bilayer into a bud. However, in the TGN and plasma membrane, free flip-flop of phospholipids is constrained owing to high sterol levels. Here, coat assembly might no longer be sufficient to drive vesicle budding, and this process would require assistance of a phospholipid pump whose activity helps to expand the cytoplasmic leaflet at the expense of the luminal one. Selectivity of this unidirectional flippase would prevent destabilization of the bilayer, generate transbilayer phospholipid asymmetry (marked by the light and dark gray membrane leaflets), and help establish a phospholipid environment favourable for coat recruitment. (B) The TGN-resident yeast P4-ATPase Drs2p is activated by phosphatidylinositol-4-phosphate (PI4P) and the guanine nucleotide exchange factor Arf-GEF. Both activators bind to an auto-inhibitory domain in the C-terminal tail of Drs2p, suggesting a coincidence detection system to control flippase activity in coordination with cargo loading and vesicle biogenesis. Note that Arf-GEF triggers membrane association of the ADP ribosylation factor Arf, a key regulator of clathrin coat assembly. AP, adaptor protein.
Potential participation of components of the endosomal sorting complex required for transport (ESCRT) machinery, GRASP and flippases in the biogenesis of fungal extracellular vesicles (EVs). The similarities between EVs produced by fungi and mammalian exosomes suggest that ESCRT machinery is required for formation of the fungal compartments (green arrows). Maturation of the late endosome (LE) is accompanied by membrane invagination, giving origin to small intraluminal vesicles and multivesicular bodies (MVB). The ESCRT machinery is recycled through the activity of the Vps4 protein complex. MVB may be directed to vacuolar (VC) degradation pathways, but also to fusion with the plasma membrane, releasing exosomes to the extracellular milieu now receiving the name exosomes. GRASP, a regulator of unconventional secretion by mechanisms that are putatively linked to EV release, was first identified as a structural component of the Golgi cisternae. Alternative roles included tethering activity for endosomal or lysosomal compartments and/or regulation of autophagy-related mechanisms (blue arrows). GRASP may also localize to the plasma membrane, mediating the release of exosomes to the extracellular space. Finally, GRASP can also participate in docking or fusion events involving vesicles originating at the Golgi, thus facilitating anterograde transport through the early secretory pathway (brown arrow).
Flippases are involved in vesicle biogenesis through phospholipid translocation across the lipid bilayers. These enzymes can regulate endocytosis at the plasma membrane level (orange arrows) and also drive the formation of exocytic vesicles (light blue arrows). Flippases can also participate in protein trafficking between the trans-Golgi network and endosomal compartment or between the trans-Golgi network and vacuoles (purple arrows). It has been also proposed that flippases may regulate the retrograde transport pathway from the Golgi apparatus to the ER (pink arrow), as well as vesicle budding at the plasma membrane level (yellow arrow). The possibility that cellular pathways regulated by endosomal proteins, GRASPs and flippases are interconnected cannot be ruled out, as previously described for other unconventional secretory pathways [165]. Most of the mechanisms proposed here have been implicated with the physiology of yeast cells, although they also participate in pathways required for molecular degradation and / or export in other eukaryotes. (PL) phospholipid; (EE) early endosome; (LE) late endosome; (MVB) multivesicular bodies; (APS) autophagosome; (ELC) endosomal/lysosomal compartment; (VC) vacuole.
Regulation of the transbilayer lipid distribution in cellular membranes. In early secretory organelles, such as the endoplasmic reticulum (ER), membrane proteins 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, thereby promoting a symmetric lipid distribution across the bilayer. In contrast, flip-flop of phospholipids across the plasma membrane (PM) is constrained owing to high levels of cholesterol and sphingolipids and/or the absence of constitutive bi-directional flippases. Thus, ATP-dependent flippases can maintain an asymmetric lipid distribution by moving specific lipids towards (P-type ATPase family members) or away from the cytosolic leaflet (ABC transporters). Cellular activation triggered by cytosolic calcium can collapse the lipid asymmetry by the transient activity of an ATP-independent scramblase. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SM, sphingomyelin; GL, glycolipids; Chol, cholesterol.
Role of energy-coupled lipid flippases in triggering membrane budding. (A) ATP-driven lipid translocation may be required to generate a lipid imbalance across the bilayer by increasing the proportion of total lipids in one monolayer and thereby driving budding of vesicles. noL, number of lipid molecules in the outer leaflet, niL, number of lipid molecules in the inner leaflet (e.g. at the level of the head groups, the cytoplasmic leaflet of a 60 nm diameter vesicle contains 1.5 times the number of lipid molecules of the lumenal leaflet). (B) ATP-driven lipid translocation may help to create a high local concentration of aminophospholipids in the cytosolic leaflet favorable for recruitment of peripheral proteins, such as ARF, clathrin, amphiphysin and endophilins.
Architecture and transport cycle of flippases and Na+/K+-pumps. (A) Flippases and Na+/K+-pumps share a common architecture. Flippases comprise a P4-ATPase catalytic chain associated with a Cdc50 subunit. Na+/K+-pumps comprise a P2C-ATPase catalytic chain associated with β- and γ-subunits. Both the Cdc50 and β-subunit have a bulky N-glycosylated ectodomain with conserved disulfide bridges (S-S). Na+/K+-pumps utilize an ion-binding pocket in the center of the helical bundle or M domain. Transmembrane segment M4 harbors a conserved glutamate (E) that binds Na+ or K+ ions. In flippases, the residue located at this position is an isoleucine (I). M, transmembrane region; A, actuator domain; N, nucleotide-binding domain; P, phosphorylation domain. The P with a red background shows phosphorylation. (B) Cartoons of a flippase and Na+/K+-pump illustrating domain reorientation and subunit rearrangements during the transport reaction cycle (see also Box 2). In Na+/K+-pumps, the transition from E1P to E2P involves a vertical movement of M4, allowing delivery of Na+ ions bound at the M4 glutamate (E) to the exoplasm and loading of the enzyme with exoplasmic K+ ions. During transition from E2 to E1, M4 moves in the opposite direction to release K+ ions into the cytosol. In flippases, the isoleucine (I), which is present in the place of the M4 glutamate, is crucial for translocating phospholipid to the cytoplasmic leaflet, presumably by functioning as a hydrophobic gate for the polar headgroup (brown circle). Transition from E1P to E2P is accompanied by tighter binding of the ATPase to its subunit, involving a high-affinity interaction with the ectodomain of the subunit. In this way, the subunit might stabilize E2P to help load the ATPase with luminal substrate (K+ ions or phospholipid) or serve as a ‘lid’ to close access to the substrate-binding site from the exoplasm.
