Reference : UMR9198-GUILEN-003
Workplace : SACLAY
Date of publication : Friday, May 6, 2022
Scientific Responsible name : Guillaume Lenoir
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 1 October 2022
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly
Description of the thesis topic
This PhD project aims to understand the molecular mechanism of lipid transport by flippases, which are transmembrane proteins involved in maintaining lipid asymmetry in eukaryotic cell membranes. Flippases are active transporters that couple the transport of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to ATP hydrolysis, thus maintaining lipid asymmetry in cell membranes. The enrichment of PS on the cytosolic leaflet of membranes, through the activity of flippases such as the Drs2-Cdc50 complex, allows the recruitment of regulatory proteins involved in key cellular processes such as membrane trafficking or cell signaling. In humans, mutation of certain flippase residues can lead to various pathologies, for example intrahepatic cholestasis. This pathology, which results from mutations in the ATP8B1 gene, is characterized by a defect in the secretion of bile acids by liver cells and may become fatal in severe cases. It is therefore essential to understand the molecular details of lipid transport by flippases.
We recently observed that Drs2-Cdc50 was autoinhibited by its C-terminal tail and activated by a phoshatidylinositol-4-phosphate (PI4P), a phosphoinositide lipid. Furthermore, it was shown that the small G protein Arl1 interacts with the N-terminus of Drs2 and that the Gea2 protein interacts with the C-terminus of Drs2 and that these two proteins stimulate lipid transport in vivo. The objective of this thesis is to identify, at the molecular level, the mechanisms by which the autoinhibition of Drs2-Cdc50 by its C-terminus is overcome and activation of transport by PI4P is achieved, and to reconstitute in vitro the mechanism by which Arl1 and Gea2 activate lipid transport by Drs2-Cdc50. More specifically, the main objectives of this doctoral thesis are as follows:
1. To develop a proteoliposome-based lipid transport assay to reveal the activity of the Drs2-Cdc50 flippase.
2. To identify the portion of the C-terminal sequence of Drs2 that leads to its autoinhibition.
3. To express and purify the Drs2-Cdc50 complex partners, the small G protein Arl1 and the GDP-GTP exchange factor for Arf1, Gea2.
4. To assemble purified Arl1 and Gea2 with reconstituted Drs2-Cdc50 into proteoliposomes and monitor the influence of complex formation on lipid transport by Drs2-Cdc50.
Lipid transport by Drs2-Cdc50 is intimately linked to the regulation of vesicular trafficking between the TGN and the plasma membrane and between the TGN and endosomes. In addition, the small G protein Arl1 controls vesicular trafficking at the TGN and Gea2 activates Arf1, itself involved in the regulation of vesicular trafficking at the Golgi apparatus. Therefore, we hope through this project to contribute to a better understanding of the mechanisms that control vesicle formation at the TGN. This project will also contribute to a longer-term goal, i.e. obtaining the high-resolution structure of the Drs2-Cdc50-Gea2-Arl1 complex by cryo-electron microscopy. Finally, knowing that the regulatory mechanism of the yeast flippase Drs2-Cdc50 is conserved in certain human flippases, the knowledge acquired during this project will probably prove to be essential for a better understanding of the functioning of human flippases as well as the functional consequences of disease-related ATP8B1 mutations.
This PhD project is funded thanks to a collaborative ANR grant. In this frame, the PhD student will have the opportunity to work in close relationship with project's partner laboratories:
- J. Cherfils: LBPA, ENS Paris-Saclay, France
- C. Bechara: IGF, Université de Montpellier, France
- J. Lyons: Aarhus University, Denmark
Material and financial scientific conditions of the research project:
All the equipment present in the laboratory and necessary for the realization of the thesis will be made available to the PhD student. The thesis is supported by an ANR research contract, which will provide not only for the student's salary but also for the project to be carried out in good material conditions.
Objective of promoting the research work of the doctoral student:
The work resulting from the PhD student's research will be published in international peer-reviewed journals. This work is not subject to any particular confidentiality. The doctoral student will also be encouraged to present his/her work at national and international conferences, either through oral presentations or posters.
Dieudonné T, Herrera SA, Laursen MJ, Lejeune M, Stock C, Slimani K, Jaxel C, Lyons JA, Montigny C, Pomorski TG*, Nissen P*, Lenoir G*, Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders. eLife 11, e75272 (2022).
G. Lenoir*, J.M. D'Ambrosio, T. Dieudonné, A. Čopič*, Transport pathways that contribute to the cellular distribution of phosphatidylserine. Front Cell Dev Biol 9, 737907 (2021).
M. Timcenko, T. Dieudonné, C. Montigny, T. Boesen, J.A. Lyons, G. Lenoir, P. Nissen, Structural basis of substrate-independent phosphorylation in a P4-ATPase lipid flippase. J Mol Biol 433, 167062 (2021).
M. Timcenko, J.A. Lyons, D. Januliene, J.J. Ulstrup, T. Dieudonné, C. Montigny, M.R. Ash, J.L. Karlsen, T. Boesen, W. Kühlbrandt, G. Lenoir*, A. Moeller*, P. Nissen*, Structure and autoregulation of a P4-ATPase lipid flippase. Nature 571, 366 (2019).
H. Azouaoui, C. Montigny, T. Dieudonné, P. Champeil, A. Jacquot, J.L. Vázquez-Ibar, P. Le Maréchal, J. Ulstrup, M.R. Ash, J.A. Lyons, P. Nissen, G. Lenoir, High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions. J Biol Chem 292, 7954 (2017).
H. Azouaoui, C. Montigny, M.R. Ash, F. Fijalkowski, A. Jacquot, C. Grønberg, R.L. López-Marqués, M.G. Palmgren, M. Garrigos, M. le Maire, P. Decottignies, P. Gourdon, P. Nissen, P. Champeil, G. Lenoir, A high-yield co-expression system for the purification of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate. PLoS One 9, e112176 (2014).
P.-C. Tsai, J.-W. Hsu, Y.-W. Liu, K.-Y. Chen, F.-J. S. Lee, Arl1p regulates spatial membrane organization at the trans-Golgi network through interaction with Arf-GEF Gea2p and flippase Drs2p. Proc Natl Acad Sci USA. 110, E668 (2013).
The PhD student will work in the Laboratory of Membrane Proteins and Membrane Systems (LPSM), which is interested in the molecular mechanisms that underly the transport of molecules or ions across membranes, using biochemical, biophysical and in silico approaches. More specifically, we investigate, at a molecular level, the protein machineries that allow maintaining transbilayer lipid asymmetry across membranes, an essential feature of eukaryotic cell membranes. The LPSM is part of the Institute for Integrative Biology of the Cell (I2BC), an interdisciplinary institute at the forefront of biochemistry/structural biology, cell biology, microbiology, genetics and virology. The I2BC is located in Gif-sur-Yvette, 25 km south-west of Paris, at the heart of the world-class Université Paris-Saclay, a wonderful place to live and work. Financial support for the doctoral thesis comes from a grant of the Agence Nationale de la Recherche (ANR).
Constraints and risks
The successful candidate shall be enrolled in a PhD program of the 'Signalisation et Réseaux Intégratifs en Biologie (BIOSIGNE), a doctoral school from the University Paris-Saclay. The PhD student might potentially use radioisotopes in the frame of phosphorylation experiments, in a safe and dedicated place. The LPSM has a long-standing experience in the handling of radioisotopes. The PhD student will receive a specific training beforehand. Short stays in the partner's laboratories may be expected in the course of the project.
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