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Molecular mechanism of lipid transport and its role in vesicle biogenesis – AsymLip

Molecular mechanism of lipid transport and its role in vesicle biogenesis

Phospholipid asymmetry in biological membranes is essential to numerous cell functions. For instance, the regulated dissipation of lipid asymmetry is associated with (patho)physiological conditions such as recognition of apoptotic cells by macrophages. P4-ATPases are membrane transport proteins that are responsible for translocating lipids across membranes, thus establishing phospholipid asymmetry. Our project focuses, at the molecular level, on this class of transporters called ‘flippases’

To understand the molecular mechanism of P4-ATPase-catalyzed lipid transport and to identify the molecular basis for human diseases associated with mutations of these transporters

P4-ATPases are integral membrane transport proteins that catalyze active transport of lipids across membranes. P4-ATPases do not act alone in lipid translocation as they have been shown to physically interact with members of the Cdc50 protein family. The function of Cdc50 proteins in phospholipid transport is not yet clear, but they are required for exit of P4-ATPases from the endoplasmic reticulum. P4-ATPases are part of the P-type ATPase family and so far, P-type ATPases have been shown to translocate cations. How then did P4-ATPases evolve to be able to translocate phospholipids instead of mere ions, to provide a sizeable pathway for lipid transport? To address these issues, we will characterize the molecular mechanism of phospholipid transport of a flippase from Saccharomyces cerevisiae, the Drs2p/Cdc50p complex. <br />Deletion or inactivation of a number of yeast flippases is associated with alteration of vesicular trafficking in the late endocytic/secretory pathways. Specifically, deletion of Drs2p perturbs the formation of endocytic vesicles at the plasma membrane and bidirectional transport between the trans-Golgi network and the endosomes. Another aim within our project is to unravel the link between lipid transport and vesicle biogenesis.<br />Finally, mutations of human ATPases are associated with severe diseases. For instance, mutations of ATP8B1, which interacts with CDC50A or CDC50B subunits at the plasma membrane, are responsible for progressive familial intrahepatic cholestasis (PFIC1), a fatal disease in its severe form. This part of the project aims at exploring the functional consequences of ATP8B1 mutations found in PFIC1 patients.

Dissecting the catalytic mechanism of the yeast Drs2p/Cdc50p flippase involves expression of the complex, as well as solubilization and purification for subsequent reconstitution into liposomes. Coupled with site-directed mutagenesis, this will allow identification of the regions crucial for lipid transport and/or activity of the flippase.
We make use of the ability of P-type ATPases to undergo autophosphorylation to investigate partial reactions of their catalytic cycle using phosphorylation/dephosphorylation assays. Also, we are now able to study the flippase ATPase activity in detail (e.g. its affinity for ATP, its dependence on pH, its dependence on specific lipids). Upon reconstitution in proteoliposomes, we shall then set-up a lipid translocation assay using phospholipids labeled with a fluorescent moiety (NBD).
Gaining insights into the structure of Drs2p/Cdc50p complex involves the large-scale purification of various Drs2p/Cdc50p constructs, crystallization attempts and structure determination. The complex is purified by affinity chromatography on streptavidine beads and further cleaned by adding an extra size-exclusion chromatography (SEC)-FPLC step
Establishing a link between P4-ATPase-mediated lipid transport and vesicle budding involves the electroformation of GUVs from P4-ATPase-containing proteoliposomes and the monitoring of GUVs shape changes by light microscopy. Because electroformation may affect protein activity, we will also consider using a newly developed protocol, namely a procedure that is independent of electroformation. Once protein complexes will have been reconstituted into GUVs, shape changes will be triggered by the addition of ATP.

Within the first 30 months of the project, we devised a robust protocol for the expression and the purification in a functional form of Drs2p, in association with its non-catalytic subunit Cdc50p. We demonstrated that PI4P, a key phosphoinositide involved in the regulation of membrane trafficking, is mandatory for the activity of the purified complex. Based on preliminary experiments suggesting a prominent regulatory role for a domain of Drs2p, we resorted to limited proteolysis to further study this regulation mechanism. Limited proteolysis allows to excise this regulatory domain, and to stimulate the Drs2p/Cdc50p complex by 30-50-fold, demonstrating the inhibitory role of this domain. Remarkably, the stimulated activity of the proteolyzed complex remains highly sensitive to PI4P, suggesting a 2-step model for autoinhibition relief.
We also made a step forward with respect to structural characterization of the Drs2p/Cdc50p complex. The high yield of our purification protocol allowed our collaborators to start first crystallization trials. In parallel, our Danish collaborators are currently developing structural characterization by single particle cryo-electron microscopy. Whether the purified complex is purified in detergent or reconstituted in nanodiscs does not matter much; in both cases, the complex looks homogenous and suitable for further structural studies.
We already cloned a human homolog of Drs2p, ATP8B1 and the associated CDC50A and CDC50B subunits. Constructs with N-ter or C-ter tags as well as co-expression vectors we CDC50A or CDC50B are now available. ATP8B1 and CDC50A can be overexpressed in S. cerevisiae and preliminary purification attempts suggest the ATP8B1 and CDC50A interact in yeast membranes.

Given that we now managed to express the Drs2p/Cdc50p complex, and to purify it in a functional and stable form, we may now proceed to a detailed characterization of its molecular mechanism. We already started such detailed functional characterization, by using a limited proteolysis approach. Our next goal will consist in setting up a fluorescence-based lipid uptake assay, after reconstitution of the purified complex into proteoliposomes. Such a lipid transport assay will increase the panel of functional tests we have at our disposal, and provide an essential step toward reconstitution in giant vesicles. Beyond providing a link between lipid transport by P4-ATPases and membrane shape changes, reconstitution in GUVs should provide an original system for probing P4-ATPase-mediated transport of natural lipids across membranes.
The identification of highly disordered regions in Drs2p, thanks to our proteolysis approach, is also promising in view of future crystallization attempts. Indeed, disordered regions in proteins are often flexible and thus inhibit the formation of crystals. Drs2p devoid of these disordered regions is going to be soon used as a material for future crystallization attempts.
In parallel with our studies on the yeast Drs2p/Cdc50p lipid flippase, we’re now also working on ATP8B1, co-expressed with CDC50A or CDC50B. We will now check whether the overexpressed transporter is functional in yeast membranes, using phosphorylation assays. If ATP8B1 turns out to be active in yeast membranes, we shall start characterizing its function in greater details and examine the functional consequences of ATP8B1 mutations found in PFIC1 patients.

Azouaoui H, Montigny C, Dieudonné T, Champeil P, Jacquot A, Vázquez-Ibar JL, Le Maréchal P, Ulstrup J, Ash MR, Lyons JA, Nissen P, Lenoir G (2017) 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, 292:7954-7970

Montigny C, Dieudonné T, Orlowski S, Vázquez-Ibar JL, Gauron C, Georgin D, Lund S, le Maire M, Møller JV, Champeil P, Lenoir G (2017) Slow phospholipid exchange between a detergent-solubilized membrane protein and lipid-detergent mixed micelles: brominated phospholipids as tools to follow its kinetics. PLoS One. 12:e0170481

Montigny C, Lyons J, Champeil P, Nissen P, Lenoir G. (2016) On the molecular mechanism of flippase- and scramblase-mediated phospholipid transport. Biochim Biophys Acta, 1861:767-83.

Azouaoui H, Montigny C, Jacquot A, Barry R, Champeil P, Lenoir G (2016) Coordinated overexpression in yeast of a P4-ATPase and its associated Cdc50 subunit: the case of the Drs2p/Cdc50p lipid flippase complex. Methods Mol Biol. 1377:37-55

Champeil P, Orlowski S, Babin S, Lund S, le Maire M, Møller JV, Lenoir G, Montigny C (2016) A robust method to screen detergents for membrane protein stabilization, revisited. Anal Biochem. 511:31-5

Membrane lipids fulfill many essential cellular roles. In membranes of the late secretory pathway, they display an asymmetric distribution, with phosphatidylserine (PS) and phosphatidylethanolamine (PE) primarily restricted to the cytosolic leaflet while sphingomyelin (SM) and glycosphingolipids (GSLs) are enriched in the non-cytosolic leaflet. This asymmetric distribution is implicated in fundamental processes. Conversely, dissipation of lipid asymmetry may trigger various cellular responses, ranging from virus entry to recognition of apoptotic cells by macrophages or activation of the blood coagulation cascade.
P-type ATPases from the P4 subfamily (P4-ATPases) are prime candidates for creating and maintaining this lipid asymmetry (at least as judged from assays with fluorescent analogs of lipids), and they also control vesicle formation both in the endocytic and secretory pathways. Additional proteins, called Cdc50 proteins, have been found to associate with P4-ATPases and to be essential for export of P4-ATPases from the ER. Yet, the exact role in lipid transport and membrane trafficking of the two partners remains to be established.
Our proposed project is at the interface between biochemistry, cell biology, and biophysics. Using functional and structural approaches, it aims at characterizing at the molecular level the complexes of P4-ATPase and Cdc50 protein which are responsible for lipid transport, and at elucidating the link between this transport function and membrane trafficking and associated pathological disorders. Because the yeast P4-ATPase Drs2p is the one for which the most convincing data suggesting implication in lipid transport and vesicular trafficking have been obtained, we will first focus on the complex formed by this ATPase and its associated subunit, Cdc50p. We will then also characterize the human ATP8B1/CDC50A complex, of high medical relevance: mutations in that P4-ATPase give rise to intrahepatic cholestasis, a fatal disease in its severe form. We will also extend our studies to flippases in malaria parasites, as they might provide valuable targets for therapy.
These complexes will be overexpressed in the yeast S. cerevisiae. Crude yeast membrane fractions will first be used for preliminary analysis of the enzymatic and transport cycle catalyzed by Drs2p/Cdc50p and ATP8B1/CDC50A (or parasite flippases), and then for identification by site–directed mutagenesis of residues or domains involved in this activity. We will also examine the consequences of those ATP8B1 mutations which have been identified in patients with intrahepatic cholestasis. Active protein complexes will also be purified by affinity chromatography for detailed functional analysis of the transport cycle.
Simultaneously, purified Drs2p/Cdc50p or ATP8B1/CDC50A (or parasite flippases) complexes will be prepared for crystallogenesis, and their structure elucidated by X-ray crystallography in collaboration with Poul Nissen’s laboratory in Aarhus (Denmark). Such elucidation would be a major breakthrough for understanding the molecular mechanism of lipid transport, and ultimately controlling it.
Independently, the purified material will be reconstituted into giant unilamellar vesicles (GUVs), in collaboration with Joost Holthuis (Osnabrück, Germany). Using such a system, we aim at detecting membrane deformation upon translocation of natural lipids from one leaflet of the vesicle to the other: according to classical biophysics rationale, a small increase in surface area of one leaflet of the bilayer and the concomitant decrease in the surface area of the opposing leaflet will induce bilayer curvature and possibly vesicle budding, which should be observable by optical microscopy.

Project coordinator

Monsieur Guillaume LENOIR (Service de Bioénergétique, Biologie Structurale et Mécanismes, Commissariat à l'Energie Atomique et aux Energies Alternatives)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CEA Service de Bioénergétique, Biologie Structurale et Mécanismes, Commissariat à l'Energie Atomique et aux Energies Alternatives

Help of the ANR 263,536 euros
Beginning and duration of the scientific project: September 2014 - 48 Months

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