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Laboratory | Structural biology
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For many years we have been focusing on the membranous Ca2+-transporting ATPase located in muscle sarcoplasmic reticulum (SERCA1a). The goal was to understand the various changes of conformations linked to the coupling between ATP hydrolysis and ion transport. Various 3D X-ray structures of SERCA1a have been published, either with or without Ca2+, ATP or analogues, or in the presence of various inhibitors. In our team we are presently focusing on the two following aspects :
Malaria is a parasite disease (630000 deaths per year) due to a protist parasite of genus Plasmodium, transferred by an insect vector of genus Anopheles. The occurence in 2009 of parasites becoming resistant to the more recent antimalarials used (artemisinin and derivatives) is crucial. In the absence of an efficient vaccine, the treatment of malaria is dependent on the use of drugs.
Our project is to study transporters of Plasmodium falciparum considered to be major potential molecular targets such as P4 ATPases, integral membrane proteins from apicoplast, the Ca2+-ATPase PfATP6, the ADP/ATP transporter PfAdT and the membrane transport proteins with roles in drug resistance PfCRT and PfMDR1.
In the case of PfATP6, we have been successful in isolating the protein after heterologous expression in yeast and affinity chromatography in a pure, active and stable detergent-solubilized form that affords the opportunity to test new potential antimalarials by screening for inhibitors against PfATP6.
In parallel, assays for crystallization of these transporters will be performed, in order to obtain the structural characterization of these major potential targets.
An essential feature of eukaryotic cells is the asymmetric distribution of phospholipids over the two leaflets of membranes from the late secretory pathway (for instance plasma membrane and trans-Golgi network). While phosphatidylserine (PS) and phosphatidylethanolamine are mostly restricted to the inner leaflet of the plasma membrane, phosphatidylcholine and sphingomyelin are primarily found in the outer leaflet (Fig. 1). Inside the cell, the specific distribution of PS is important in many aspects of cell physiology, as its negatively charged headgroup is for instance the target of C2 domain-containing proteins involved in key processes like protein phosphorylation or membrane fusion. Moreover, the contribution of PS to the anionic surface charge of membranes, especially that of the inner leaflet of the plasma membrane, affects both recruitment of some soluble proteins via their polybasic motifs and the function of transmembrane proteins. Conversely, appearance of PS outside the cell is an early indicator of apoptosis and a signal to initiate blood clotting.
Thus, PS distribution must be regulated tightly and in this aim, energy-dependent transporters, the so-called flippases, transfer PS (but also other phospholipid species) from the exoplasmic to the cytosolic leaflet of cell membranes. P-type ATPases from the P4 subfamily (P4-ATPases) are prime candidates for creating and maintaining this asymmetry (Fig. 1), 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 their export from the ER. Yet, the exact role in lipid transport and membrane trafficking of the two partners remains to be established.
So far, P-type ATPases of most subfamilies have been shown to translocate cations. Within the whole family, the fair conservation of key residues involved in catalysis and the predicted similar membrane domain organization suggest similar mechanisms for transport. How then did P4-ATPases evolve to provide a sizeable pathway, allowing translocation of lipids instead of ions? What is the mechanism for lipid transport?
To address these issues, we have worked out procedure for high-yield co-expression of yeast lipid flippase complex, namely Drs2p and Cdc50p (Jacquot et al, 2012). This allowed us to monitor the activity of the complex in crude yeast membranes and to identify a specific role for phosphatidylinositol-4-phosphate (PI4P) in regulation of Drs2p/Cdc50p activity (Jacquot et al, 2012). We also currently develop a method for purification of the Drs2p/Cdc50p complex, for further functional and structural studies. Combining site-directed mutagenesis with detailed functional characterization of the purified complex, we aim at deciphering the mechanism for P4-ATPase-dependent lipid transport.
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.