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Laboratory | Structural biology


LPSM

Molecular mechanism of membrane transport processes

Published on 27 November 2017
In our lab, we are interested in the molecular mechanism which underlies the transport of various molecules across membranes, and particularly in the transport of cations and lipids catalyzed by the P-type ATPase family. These transmembrane proteins play key roles in various cellular processes. We develop methods for the heterologous expression of membrane proteins in the yeast S. cerevisiae as well as for purification of these proteins (or protein complexes). We then combine biochemical, spectroscopy, and structural approaches for the functional characterization of target proteins.
Team Leaders
Guillaume Lenoir
01 69 08 75 89
guillaume.lenoir@cea.fr

SERCA1a (Marc le Maire, Philippe Champeil, Christine Jaxel, Cédric Montigny)

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 :

  • The crystallization of WT rabbit SERCA1a after its yeast expression and purification thanks to a biotin acceptor domain has opened the way to the crystallization of mutants. We have purified a number of mutants which appear to be particularly interesting based on their former characterization, revealing various functional deficits. Several of these mutants were studied in the team of JP Andersen in Aarhus, Denmark and in collaboration with this team and the neighbouring teams of P. Nissen and JV. Møller, we have now crystallized three of these mutants. Their 3D structure allows to better understand part of the calcium transport process.
  • More recently we have started the study of sarcolipin, a small transmembrane peptide which regulates the Ca2+-ATPase activity.
 
Figure : structure of the E309Q mutant of the Ca2+-transporting ATPase SERCA1a in the sarcoplasmic reticulum membrane. ATP hydrolysis is taken place in the cytosolic region and provides energy for the transport of the two Ca2+ ions (cyan spheres). One of the steps in the transport requires autophosphorylation of an aspartate located between the red and blue domains, about 50A away from the membranous sites of the Ca2+ ion. Some of the important transmembrane helices for Ca2+ transport are labelled M1 to M4. Glutamate 309 is located on M4. The study of its mutation to glutamine helps to better understand the crucial dialog between the two active site regions, rather far away from each other. From Clausen JD, Bublitz M, Arnou B, Montigny C, Jaxel C, Møller JV, Nissen P, Andersen JP, and le Maire M. (2013). SERCA mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation. EMBO J., 32, 3231-3243.
 

Transporters of Plasmodium falciparum, the parasite responsible for Malaria  (Christine Jaxel, José Luis Vázquez-Ibar, Manuel Garrigos, Guillaume Lenoir)

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.

Molecular mechanism of P4-ATPase-dependent lipid transport (Guillaume Lenoir, Hassina Azouaoui, Cédric Montigny, Philippe Champeil)

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.

 

Figure : répartition asymétrique des phospholipides dans les membranes de la voie sécrétoire tardive et rôle des ATPases P4 dans le maintien de cette asymétrie. MP : membrane plasmique ; TGN : trans-Golgi. PC : phosphatidylcholine ; PS : phosphatidylsérine ; PE : phosphatidyléthanolamine
Guillaume Lenoir/Université Paris-Sud
 
 

Principales collaborations

  • Eva Pebay-Peyroula, Institut de Biologie Structurale, Grenoble
  • Bruno Miroux, Institut de Biologie Physico-chimique, Paris
  • Joost Holthuis, Université d’Osnabrück, Allemagne
  • Poul Nissen et Jesper Vuust Møller, PUMPKIN, Université d’Aarhus, Danemark
  • Rosa López-Marqués, PUMPKIN, Université de Copenhague, Danemark
  • Isabelle Florent, Muséum d’Histoire Naturelle, Paris
  • Andréas Barth, Université de Stockholm, Suède