Towards an understanding of the mechanism of transport of membrane lipids

Flippases are membrane proteins which help to maintain lipid asymmetry between the two layers of the cell membrane. They are essential for processes as different as insulin secretion in animals and plant responses to thermal stress. In man, mutations of these proteins are responsible for rare genetic diseases. Research carried out by scientists from the Institut de biologie intégrative de la cellule (Integrated Cell Biology Institute) (Paris-Sud University/CEA/CNRS), Aarhus University (Denmark) and the Max Planck Institute in Frankfurt (Germany) has for the first time revealed structures of one of these proteins at high resolution. The study, published 26 June 2019 in Nature throws light on the mechanism of regulation of these membrane proteins and suggests a pathway whereby lipids can cross the membranes. This work represents a first important stage towards a better understanding of the molecular basis of flippase-associated diseases.

Possible method of transport of lipid across the lipid bilayer. The transmembrane helix 4 of Drs2p, which borders the cavity is represented in yellow. The rest of the transmembrane domain of Drs2p is represented in light brown. The cytosolic domains A, N and P of Drs2p are respectively coloured yellow, red and light blue. The sub-unit Cdc50p is shown in pink. © Guillaume Lenoir / I2BC

The plasma membrane consists of two lipid leaflets in which proteins are partially or completely embedded. The lipid composition of the two leaflets is asymmetrical in order to control many biological functions (traffic across the membrane, cell signalling, etc.). This lipid asymmetry is a product of a number of mechanisms, with the main contributors to these being lipid-transporting transmembrane proteins. The floppases catalyse the transport of lipids from the inner membrane leaflet (cytosolic) to the outer one (exoplasmic), while the flippases work in the reverse direction. In man, mutations of several flippase protein homologues are implicated in rare forms of intrahepatic cholestasis and neurological conditions (cerebellar atxia associated with intellectual deficit). Work in rodents has shown their importance in insulin secretion, the maintenance of red cell shape and survival of retinal photoreceptors. In plants, flippases are essential for the response to thermal stress. They are also thought to contribute to the virulence of certain pathogens. Considerable efforts have been devoted to determining the structure of these proteins so as to elucidate lipid transport mechanisms. In this respect the flippases are intriguing. They are the only sub-type (P4) of the type P family of ATPases that transport lipids. The other sub-types, P1 to P3, only transport ions, molecules some ten times smaller than lipids (sub-type P5’s substrate is not known). How is it that, while P4-type ATPases have the same overall architecture (1) as the other sub-types, they have evolved away from them to create within their structure a sufficiently broad “path” to allow a “giant” substrate to pass?

Teams from the Institut de biologie intégrative de la cellule (Paris-Sud University/CEA/CNRS), Aarhus University and the Max Planck Institute in Frankfurt worked together to determine the structure of the Drs2 molecule complexed with its associated sub-unit Cdc50, a yeast flippase. More specifically, this flippase structure has been studied in three different conformations: one auto-inhibited, another active and a third intermediate (2) one. Achieving this landmark for the flippase family was made possible by electronic cryo-microscopy (an imaging technique for which the Nobel prize in chemistry was awarded to three scientists in 2017). The three structures, obtained at resolutions of 2.8 to 3.7 Å, reveal the mechanism whereby the C-terminal end of Drs2 inhibits the action of the complex and the first steps in the lifting of this auto-inhibition by PI4P2. Comparison of the structures also reveals the  existence of a cavity into which the polar end of the lipid substrate (principally phosphatidylserine) is taken when being ferried from one leaflet to the other of the cell membrane.

Protein Flippases in the plasma membrane. © Guillaume Lenoir / I2BC

This publication describes for the first time the molecular architecture of a flippase but other structures of type P4 ATPases in the presence of their lipid substrate and/or partner regulators will have to be obtained to fully elucidate the mechanism of lipid “flipping”..

1 The presence of a number of residues which are conserved in the various sub-types is in favour of a common catalytic mechanism. In addition, high resolution structures that are already known for members of sub-types P1, P2 and P3 indicate that the architecture might be conserved throughout the family.

2 The protein contains a section at its C-terminal end which inhibits its action. In the presence of phosphatidylinositol-4-phosphate (PI4P), this auto-inhibition is partially lifted (intermediate conformation). To obtain an active conformation of the protein, a form of the enzyme truncated at its C-terminal end was used.

References :
Structure and autoregulation of a P4-ATPase lipid flippase
Milena Timcenko, Joseph A. Lyons, Dovile Januliene, Jakob Ulstrup, Thibaud Dieudonné, Cédric Montigny, Miriam Rose Ash, Jesper Lykkegaard Karlsen, Thomas Boesen, Werner Kühlbrandt, Guillaume Lenoir, Arne Möller, Poul Nissen, Nature  DOI : 10.1038/s41586-019-1344-7