PhD defense - Pauline Funke (eq. Lenoir - B3S dpt)

Speaker: Pauline Funke 

Title: In vitro reconstitution of lipid transport mediated by the lipid flippase Drs2-Cdc50

Composition du jury :
-Sophie DUPRE-CROCHET - Professeure des Universités, Laboratoire de Génétique et Biologie Cellulaire (LGBC), Université Paris Saclay
-Maike BUBLITZ-MEIER - Associate Professor, Institute of Translational Medicine, University in the Principality of Liechtenstein (UFL)
-Cédric ORELLE - Directeur de recherche, Molecular Microbiology and Structural Biochemistry (MMSB), CNRS UMR 5086, Université de Lyon
-Anne Marie WEHENKEL - Directrice de recherche, Bacterial Cell Cycle Mechanisms Unit, Institut Pasteur, Université Paris Cité
-Guillaume DRIN - Directeur de recherche, Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Université Côte d’Azur

Abstract:
In eukaryotic membranes of the late secretory and endocytic pathways, anionic phospholipids like phosphatidylserine (PS) or phosphatidylinositol (PI) are restricted to the cytosolic leaflet, which is essential for cellular processes like vesicular trafficking or signalling. P4-ATPases (flippases) maintain this asymmetry by actively transporting lipids from the exoplasmic to the cytosolic leaflet and their dysfunction is associated with pathophysiological conditions. For example, mutations in the human P4-ATPase ATP8B1 are directly linked to a severe liver disease. Despite recent advances in the structural characterisation of P4-ATPases, how their dysfunction leads to diseases remains unclear, primarily due to the difficulties in accurately characterising their substrate specificity and regulatory mechanisms.

We study the yeast flippase Drs2, a homolog of human ATP8B1, as a model system. Drs2 is mainly located in the trans-Golgi network (TGN), has been shown to transport PS and tightly

associates with a Cdc50 subunit. The Drs2-Cdc50 complex requires phosphatidylinositol-4-phosphate (PI4P) for activation and is autoinhibited by its N- and C-terminal extensions. Drs2 interacts with the small GTPase Arl1 and the Arf guanine-nucleotide-exchange factor Gea2, and both proteins are involved in membrane trafficking at the TGN. Deletion of Arl1 or Gea2 impairs PS transport in isolated TGN membranes. However, their exact interaction mechanisms with Drs2 and involvement in lipid transport remain to be elucidated. We hypothesize that Arl1 and Gea2 binding relieves Drs2 autoinhibition in vivo.

The objectives of this thesis are threefold. First, I set out to reconstitute Drs2 in proteoliposomes to demonstrate lipid transport in vitro. Second, using this reconstitution system, we aim to revisit the substrate specificity of Drs2. Although PS has long been considered the primary substrate, previous work suggested that PS is not strictly required for Drs2 function, raising the possibility that other phospholipids may also be transported. We found that ATPase activity of detergent-solubilized Drs2 is stimulated not only by PS but also by the anionic phospholipids PI, phosphatidylglycerol (PG), and phosphatidic acid (PA). In contrast, the zwitterionic phospholipids phosphatidylethanolamine (PE) and phosphatidylcholine (PC) did not stimulate ATPase activity of Drs2-Cdc50.

To directly monitor lipid translocation, we reconstituted Drs2-Cdc50 with PI4P and fluorescently labelled phospholipids into PC-liposomes. Using a fluorescence-based lipid transport assay, we

demonstrate Drs2-mediated translocation of NBD-PS, -PI, -PG, and -PA. Cryo-EM structures of Drs2-Cdc50 further revealed PS, PG, and PI occlusion in the substrate binding pocket.

These findings provide strong evidence that Drs2-Cdc50 exhibits a broader substrate specificity than initially suggested, especially for anionic phospholipids such as PI, uncovering a previously unrecognised and critical role of P4-ATPases in the turnover of signalling lipids.

The third objective of this thesis is to reconstitute full-length Drs2-Cdc50 together with its potential regulators Arl1 and Gea2 in proteoliposomes to investigate their roles in Drs2 activation and to further explore the mechanisms governing substrate selectivity and lipid transport regulation. To this end, I characterize interactions between purified Drs2-Cdc50, Arl1 and Gea2 in vitro using various biophysical methods. This work lays the foundation for reconstituting the complete tripartite complex in artificial membranes, enabling future functional and structural studies.