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Laboratory | Molecular mechanisms | Structural biology
The plasma membrane of eukaryotic cells is made of two leaflets which display remarkably different lipid compositions, an arrangement referred to as transbilayer lipid asymmetry. Lipid asymmetry, which serves major roles in various (patho)physiological processes, is maintained by integral membrane proteins called lipid flippases. We aim to decipher the molecular mechanism of flippase-mediated transbilayer lipid transport, using biochemical and biophysical approaches.
Eukaryotic cell membranes are comprised of several thousands of different lipid species. Why would cells generate such a repertoire if lipids were only fulfilling a barrier function? As a matter of fact, lipids are essential molecules, e.g. in cell signaling, as signposts for membrane identity, or for the regulation of membrane protein activity and membrane trafficking. Beyond diversity, lipids are characterized by a non random distribution over the two leaflets of cell membranes. This so-called transbilayer lipid distribution is regulated by three kind of lipid transporters (Figure 1), the flippases (transport toward the
inside), the floppases (transport toward the
outside) and the scramblases (transport in both directions).
The critical role of lipid asymmetry in cell physiology is illustrated by the fact that in humans, mutations of flippases (or P4-ATPases) result in severe neurological disorders, as well as a rare liver disease called progressive familial intrahepatic cholestasis. Furthermore, select flippases have been identified as strong contributors of virulence in the pathogen fungus Cryptococcus neoformans.
In this context, we aim to decipher the molecular mechanism of flippase-catalyzed transbilayer lipid transport, and the link between membrane remodeling and vesicle biogenesis. To this end, we devised a procedure for the co-expression and purification of the S. cerevisiae Drs2/Cdc50 flippase complex in a functional state (Jacquot et al, 2012; Azouaoui et al, 2014; Azouaoui et al, 2016) and identified phosphatidylserine (PS) and phosphatidylinositol-4-phosphate (PI4P) as key to the stability of the purified complex (Azouaoui et al, 2014). This allowed us to identify a critical role for the N- and C-terminal extensions of Drs2 in auto-inhibiting the complex and the contribution of PI4P in regulating its activity (Azouaoui et al, 2017). Using this pure and functional Drs2/Cdc50 complex, we recently obtained in collaboration with the laboratory of Poul Nissen (Aarhus University, Denmark) the first high-resolution structure of a lipid flippase (Timcenko et al, 2019) (Figure 2).
Both in the short and the long term, we develop the following projects :
Although we know the complex is auto-inhibited by its terminal extensions, which part of the C-terminus is necessary for actual auto-inhibition remains to be elucidated. Also, what is the contribution of the N-ter in auto-regulation? Similarly, although the binding site for PI4P has been identified (Figure 2), the mechanism by which PI4P activates the flippase complex is unknown.
CEA is a French government-funded technological research organisation in four main areas: low-carbon energies, defense and security, information technologies and health technologies. A prominent player in the European Research Area, it is involved in setting up collaborative projects with many partners around the world.