Lactococcus lactis, an Alternative System for Functional Expression of Peripheral and Intrinsic Arabidopsis Membrane Proteins

International audienceBACKGROUND: Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system. CONCLUSIONS/SIGNIFICANCE: Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins

Oligomeric Status and Nucleotide Binding Properties of the Plastid ATP/ADP Transporter 1: Toward a Molecular Understanding of the Transport Mechanism

Background: Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters. Methodology/Principal Findings: In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana. By biochemical and biophysical analyses, we identified the best detergent for solubilization and purification of functional proteins, LAPAO. Purified NTT1 was found to accumulate as two independent pools of well folded, stable monomers and dimers. ATP and ADP binding properties were determined, and Pi, a co-substrate of ADP, was confirmed to be essential for nucleotide steady-state transport. Nucleotide binding studies and analysis of NTT1 mutants lead us to suggest the existence of two distinct and probably inter-dependent binding sites. Finally, fusion and deletion experiments demonstrated that the C-terminus of NTT1 is not essential for multimerization, but probably plays a regulatory role, controlling the nucleotide exchange rate. Conclusions/Significance: Taken together, these data provide a comprehensive molecular characterization of a chloroplas

Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

Couplage singulet-triplet du glyoxal : etude statistique par anticroisement

SIGLECNRS T 60143 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

ETUDE DES RELATIONS STRUCTURE - FONCTION DES FACTEURS CYTOSOLIQUES DU COMPLEXE DE LA NADPH OXYDASE

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Etude des interactions entre les facteurs cytosoliques p40phox et p47phox du complexe de la NADPH oxydase

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Editorial overview: Membranes.

International audienc

Etudes structurales du transporteur mitochondrial d'ADP et d'ATP

Le transporteur mitochondrial d'ADP et d'A TP (AAC) permet l'échange des nucléotides adényliques au niveau de la membrane interne des mitochondries. Cette protéine essentielle, qui comporte six hélices transmembranaires, est le représentant le plus abondant et le mieux connu d'une large famille de transporteurs mitochondriaux. Afin de comprendre le mécanisme de ce transport, nous avons entrepris une étude structurale avec un double objectif: d'une part déterminer l'organisation oligomérique de l'AAC, et d'autre part en obtenir une nouvelle structure à l'échelle atomique. Nos expériences de diffraction de neutrons, d'ultracentrifugation analytique, couplées aux résultats de cristallographie permettent de remettre en cause sur des bases solides le dogme affirmant que l'AAC est dimérique en solution. En présence de Lapao, comme probablement la plupart du temps en présence de détergent, la protéine est un monomère. Nos résultats sont néanmoins compatibles avec une organisation dense dans les membranes. Nous avons mis en évidence l'importance structurale de trois cardiolipides qui entourent la protéine et participent à la stabilité de son repliement. Nous avons tenté de purifier des complexes entre l'AAC et deux de ses partenaires protéiques connus: la cyclophiline D, une protéine de la matrice, et Vpr, un peptide viral du Vlli. Nous montrons par co-purification et résonance plasmonique de surface que, dans nos conditions expérimentales, l'interaction entre ces protéines est inexistante.ADP-ATP carriers (AACs) are major and essential constituents of the inner mitochondrial membrane. They drive the import of ADP and the export of neo-synthesized ATP. AAC belongs to the mitochondrial carrier family (MCF). There are more than 45 MCF members in human that handle a tremendous diversity of substrates implicated in the mitochondrial metabolic cycles over the inner membrane. ln order to get a precise insight into the molecular mechanism of transport, we aimed to (i) characterize the controversial oligomeric state of the protein (ii) solve its atomic structure in a new conformation. Our neutron scattering and analytical ultracentrifugation experiments -coupled to crystallography- give a rational basis to de fend a monomeric protein in the presence of Lapao. This is in contradiction with the dogma that the protein is dimeric in solution. Rowever our results are compatible with the existence of dense patches. We evidenced the structural importance of three cardiolipin molecules bound to the protein that enhance the folding's stability. We tried to purify complexes of AAC with known partner protein: cyclophilin D which is a soluble matrix protein and Vpr which a RN peptide. We showed, using co purification and surface plasmon resonance, that no interaction exists in our experimental conditions.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Etudes cristallographiques aux rayons X et aux neutrons du cytochrome P450cam et de la D-xylose isomerase

Les protons ont un rôle essentiel dans les mécanismes enzymatiques. Notre compréhension de ces processus peut donc grandement bénéficier de la localisation des hydrogènes aux sites actifs. Cette information peut-être obtenue par la cristallographie aux neutrons, aux résolutions modestes de 1.5 - 2.0 A. Cependant la cristallographie aux neutrons implique de nombreux challenges. De larges cristaux sont nécessaires pour palier aux faibles flux de neutrons disponibles. De plus, l'importante diffusion incohérente de l'hydrogène réduit considérablement le signal/bruit. Cela peut être contourné en deutérant l'échantillon, partiellement par trempage en solution deutérée ou complètement en produisant une protéine deutérée. La perdeutération offre un gain important en améliorant le signal/bruit d'un ordre de grandeur (permettant l'utilisation de cristaux plus petits) et en renforçant la visibilité des cartes neutroniques. Les cytochromes P450 sont impliqués dans de nombreux processus vitaux. Ils catalysent l'addition d'oxygène à des composés non-activés. La compréhension de leur mécanisme nécessite la connaissance de la position des hydrogènes au site actif. Pour bénéficier du gain offert par la perdeutération, le cytochrome P450cam perdeutéré a été produit. Son intégrité a été évaluée par FTIR et cristallographie aux rayons X. La D-xylose isomerase est une enzyme utilisée industriellement pour convertir la glucose en fructose. La structure neutronique de la D-xylose isomerase partiellement deutérée a été déterminée pour définir des détails critiques de son mécanisme enzymatique.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF