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

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

Recherche de nouveaux systèmes de transport à travers l'enveloppe du chloroplaste. Caractérisation de nouvelles protéines hydrophobes.

The chloroplast is an organelle totally integrated in the metabolism of the plant cell. It contains its own metabolic pathways like photosynthesis, aminoacid synthesis. The chloroplast is limited by the envelope composed of two membranes and an intermembrane space. Envelope membranes are the site of transport of metabolites, ions, proteins and information between the plastid and the cytosol. Then, they contain many transport systems, but only some of them have been characterised. Hydrophobicity and low representation are the main limitations for the study of these proteins. In order to characterised new transport systems, we have developed an approach that allowed us to identify new proteins.This approach is based of the hydrophobicity of translocators that allows solubilisation of these proteins in organic solvents. Hydrophobic proteins were differentially extracted in various mixture of chloroform/methanol according to their hydrophobicity. Then, proteins were separated by SDS-PAGE and analysed by microsequencing. Several new proteins were identified including IE16 and IE18, localized in the inner membrane of the chloroplast envelope.Functional characterisation of these proteins was continued by analysing sequences homologies with proteins of known function. We have also obtained mutants of cyanobacteria and Arabidopsis thaliana in which genes coding for IE16 and IE18 are disrupted. The analysis of their phenotypes provide informations on the function of these proteins. In particular, IE18 could be involved in the transport of K+ and/or H+. In addition, these proteins have been expressed in heterologous systems. They are produced in the system baculovirus/insect cells. Their biochemical and electrophysiological characterisation are currently in progress.One of the proteins extracted in organic solvent was demonstrated to correspond to a 35 kDa annexin. The binding of annexin to chloroplasts and its function were studied. This annexin copurifies with chloroplasts and envelope membranes in the presence of calcium. The sulfolipid, a chloroplast specific lipid, is a high affinity site for interaction with annexin. This protein does not form an ionic channel when integrated in lipidic planar bilayers. This annexin does not possess a peroxydase activity. The signification of the interaction of annexin with the chloroplast envelope remains to be determined.We have developed an approach that can be used systematically for studying transport in various membranes systems.Le chloroplaste est un organite totalement intégré dans le métabolisme de la cellule végétale. Il possède des voies métaboliques qui lui sont propres comme la photosynthèse, la synthèse d'acides gras, la synthèse d'acides aminés. Le chloroplaste est limité par l'enveloppe, constituée de deux membranes distinctes et d'un espace intermembranaire. Par sa localisation, l'enveloppe du chloroplaste constitue un site privilégié de transport de métabolites, d'ions, de protéines et d'informations entre le stroma du chloroplaste et le cytosol de la cellule végétale. Ainsi, l'enveloppe doit renfermer de nombreux systèmes de transport permettant ces échanges. Malgré l'importance de ces transporteurs, très peu d'entre eux ont été totalement caractérisés. La limitation principale provient de la très faible représentation de ces protéines et de leur hydrophobicité. Afin de caractériser de nouveaux systèmes de transport, nous avons développé une nouvelle approche qui nous a permis d'identifier de nouvelles protéines. Nous avons ensuite poursuivi la caractérisation fonctionnelle de ces protéines.L'approche que nous avons développée repose sur le caractère hydrophobe des transporteurs qui permet de les solubiliser dans des mélanges de solvants organiques, comme le chloroforme/méthanol. Cette approche permet l'extraction de protéines hydrophobes et mineures de l'enveloppe, elle est spécifique de la localisation subcellulaire. D'autre part, suivant leur hydrophobicité, les protéines peuvent être extraites de façon différentielle en fonction des rapports de solvants organiques. Les protéines extraites sont séparées par électrophorèse en conditions dénaturantes, et analysées par microséquençage. Plusieurs protéines ont été identifiées dont les protéines IE16 et IE18.Nous avons poursuivi la caractérisation fonctionnelle de IE16 et IE18 en analysant d'une part les homologies de séquences avec des protéines de fonction connue. D'autre part, nous avons obtenu des mutants de cyanobactéries et d'Arabidopsis thaliana dont les gènes correspondant aux protéines IE16 et IE18 ont été interrompus. L'analyse des phénotypes peut fournir des informations sur la fonction des protéines mutées. Notamment, la protéine IE18 pourrait être impliquée dans le transport des ions K+ et/ou H+. Afin d'effectuer des analyses biochimiques sur ces protéines, elles ont été exprimées dans des systèmes hétérologues. Ces protéines sont produites dans le système d'expression cellules d'insecte/baculovirus alors qu'aucune expression n'est détectée dans le système E. coli. Ces protéines forment des homodimères. Les analyses fonctionnelles par électrophysiologie sont en cours actuellement.Lors des premiers séquençages des protéines extraites dans les solvants organiques, une séquence correspondant à une annexine a été obtenue. Nous avons poursuivi l'étude de cette protéine. Le sulfolipide, lipide spécifique du plaste, est un site à haute affinité permettant l'interaction de l'annexine avec l'enveloppe. L'annexine copurifie avec les chloroplastes et des vésicules d'enveloppe en présence de calcium. Cette protéine ne forme pas de canal ionique lorsqu'elle est insérée dans des bicouches lipidiques planes. Elle ne présente pas non plus d'activité péroxydase. La signification de son interaction avec l'enveloppe du chloroplaste reste à être déterminée.L'approche que nous avons développée peut être utilisée de façon systématique pour l'étude des transports dans différents systèmes membranaires

Quelques observations sur le rôle des ATPases à cuivre, HMA1 et PAA1, dans le contrôle de l'homéostasie du cuivre chloroplastique

Le chloroplaste est un organite spécifique de la cellule végétale, délimité par une enveloppe renfermant de nombreux systèmes de transports d'ions et de métabolites et parmi eux, deux ATPases à cuivre de type PlB: HMAI et PAAL PAAl serait la voie principale d'import du cuivre, notamment pour alimenter la photosynthèse. HMAI constituerait une voie additionnelle d'import du cuivre, essentielle lorsque la plante est cultivée en lumière forte. Afin de mieux comprendre les rôles respectifs de ces A TPases, deux approches complémentaires ont été développées: - une approche in Dlanta visant à produire de nouvelles lignées affectées dans l'expression de l'une ou de ces deux A TPases, puis à identifier des conditions révélant le rôle essentiel de ces ATPases. Les résultats obtenus montrent que la fonction de HMAI est aussi requise lorsque la plante subit un stress salin. D'autre part, l'implication de HMAI dans l'homéostasie du cuivre a été validée. Nous avons aussi démontré que les fonctions de HMAI et PAAl ne sont pas redondantes et qu'il existe une troisième voie d'import de cuivre dans le chloroplaste, voie encore non caractérisée. - une approche in vitro visant à produire ces deux A TPases dans le système hétérologue Lactococcus lactis afin de déterminer leurs spécificités ioniques et leurs caractéristiques biochimiques. Ce système d'expression s'avère parfaitement adapté à la production de protéines membranaires d'Arabidopsis, dont HMAI et PAAl qui ont pu être solubilisées et purifiées. Nos données indiquent que ces deux protéines recombinantes peuvent lier l'un de leur substrat; l'A TP et que PAAl peut lier du cuivre monovalent et divalent.The chloroplast is an organelle specific of the plant cell surrounded by a an envelope which carries numerous transport systems for ions and metabolites and among them, two copper PlB ATPases: HMAI and PAAL PAAl is known to be the main pathway of copper import in the chloroplast, metal mainly used for photosynthesis. HMAI is thought to be an additional pathway for copper import, essential when the plant grows under high light condition. ln order to better understand the respective roles of these two A TPases, two complementary approaches had been developed: - An in Dlanta approach had been initiated to obtain sorne new lines affected in the expression of one or both A TPases, then to identify conditions revealing the essential role ofthese ATPases. Results obtained show that the function ofHMAI, is also required when plants have to cope with a salt stress. We have also validated that HMAI is implied in copper homeostasis. We also have demonstrated that HMAI and PAAl function are not redundant. The copper imported by these two ATPases is very likely delivered to target proteins by distinct pathways and that a third copper import pathway in the chloroplast exists, pathway still not characterized. - An in vitro approach meant to produce HMAI and PAAl A TPases in the Lactococcus lactis heterologous expression system had been developed to determine their ionic specificities and biochemical characteristics. This expression system is perfectly suited for membrane protein production including HMAI and PAAl which have been solubilised and purified. Our data indicates that both of the proteins can bind one of their substrate: A TP and that PAAl can bind copper under its 1+ and 2+ form.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Purification of Intact chloroplasts from Arabidopsis and spinach leaves by isopycnic centrifugation

International audienceChloroplasts are plant‐specific organelles. They are the site of photosynthesis but also of many other essential metabolic pathways, such as syntheses of amino acids, vitamins, lipids, and pigments. This unit describes the isolation and purification of chloroplasts from Arabidopsis and spinach leaves. Differential centrifugation is first used to obtain a suspension enriched in chloroplasts (crude chloroplasts extract). In a second step, Percoll density gradient centrifugation is used to recover pure and intact chloroplasts. The Basic Protocol describes the purification of chloroplasts from Arabidopsis leaves. This small flowering plant is now widely used as a model organism in plant biology as it offers important advantages for basic research in genetics and molecular biology. The Alternate Protocol describes the purification of chloroplasts from spinach leaves. Spinach, easily available all through the year, remains a model of choice for the large‐scale preparation of pure chloroplasts with a high degree of intactness. Curr. Protoc. Cell Biol . 40:3.30.1‐3.30.14. © 2008 by John Wiley & Sons, Inc

Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular‐specific proteomic approach to identify rare intrinsic membrane proteins

International audienceSummary Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane α‐helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS‐PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal receptors or to control metabolic processes and vesicle trafficking

Protocol to study the role of a human nuclear m6A RNA reader on chromatin-associated RNA targets

Summary: Here, we present a detailed protocol to study the role of a human nuclear m6A RNA reader, YTHDC1, on chromatin-associated RNA targets. We describe steps for RNA extraction coupled to subnuclear fractionation to identify and study RNA-based regulations that take place in the chromatin-associated fraction. We then detail an RNA immunoprecipitation procedure adapted to identify chromatin-associated RNA targets. This protocol can be adapted to other human or mammalian chromatin-associated RNA binding proteins.For complete details on the use and execution of this protocol, please refer to Timcheva et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Protocol to study the role of a human nuclear m6A RNA reader on chromatin-associated RNA targets

International audienceHere, we present a detailed protocol to study the role of human YTHDC1, a nuclear m6A RNA reader, on chromatin-associated RNA targets. We describe steps for RNA extraction coupled to subnuclear fractionation to identify and study RNA-based regulations that take place in the chromatin-associated fraction. We then detail an RNA immunoprecipitation procedure adapted to identify chromatin-associated RNA targets. This protocol can be adapted to other human or mammalian chromatin-associated RNA binding proteins

Sulfolipid Is a Potential Candidate for Annexin Binding to the Outer Surface of Chloroplast

International audienceUsing a subcellular-specific proteomic approach, we have identified by protein microsequencing, a putative 35-kDa annexin from among the chloroplast envelope polypeptides. To confirm this identification, we demonstrate that (a) a 35-kDa protein, identified as annexin by antibody cross-reactivity, co-purifies with Percoll-purified chloroplasts and their envelope membranes when extracted in the presence of Ca(2+) and (b) the native spinach annexin protein binds to chloroplast-specific lipids in a Ca(2+)-dependent manner. The binding of the spinach annexin to these glycerolipids occurs at similar Ca(2+) concentrations as those, which promote the interaction of annexins to phospholipids in other membranes. Among chloroplast glycerolipids known to be accessible on the cytosolic face (outer leaflet) of the outer envelope membrane, sulfolipid, and probably phosphatidylinositol, would be the sole candidates for a putative Ca(2+)-dependent interaction of annexin with the chloroplast surface