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

LSST: from Science Drivers to Reference Design and Anticipated Data Products

(Abridged) We describe here the most ambitious survey currently planned in the optical, the Large Synoptic Survey Telescope (LSST). A vast array of science will be enabled by a single wide-deep-fast sky survey, and LSST will have unique survey capability in the faint time domain. The LSST design is driven by four main science themes: probing dark energy and dark matter, taking an inventory of the Solar System, exploring the transient optical sky, and mapping the Milky Way. LSST will be a wide-field ground-based system sited at Cerro Pach\'{o}n in northern Chile. The telescope will have an 8.4 m (6.5 m effective) primary mirror, a 9.6 deg$^2$ field of view, and a 3.2 Gigapixel camera. The standard observing sequence will consist of pairs of 15-second exposures in a given field, with two such visits in each pointing in a given night. With these repeats, the LSST system is capable of imaging about 10,000 square degrees of sky in a single filter in three nights. The typical 5$\sigma$ point-source depth in a single visit in $r$ will be $\sim 24.5$ (AB). The project is in the construction phase and will begin regular survey operations by 2022. The survey area will be contained within 30,000 deg$^2$ with $\delta<+34.5^\circ$, and will be imaged multiple times in six bands, $ugrizy$, covering the wavelength range 320--1050 nm. About 90\% of the observing time will be devoted to a deep-wide-fast survey mode which will uniformly observe a 18,000 deg$^2$ region about 800 times (summed over all six bands) during the anticipated 10 years of operations, and yield a coadded map to $r\sim27.5$. The remaining 10\% of the observing time will be allocated to projects such as a Very Deep and Fast time domain survey. The goal is to make LSST data products, including a relational database of about 32 trillion observations of 40 billion objects, available to the public and scientists around the world.Comment: 57 pages, 32 color figures, version with high-resolution figures available from https://www.lsst.org/overvie

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

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

The chloroplast is an organelle specific of the plant cell. It is surrounded by a double membrane or envelope which carries numerous transport systems for ions and metabolites, essential for the function of the chloroplast and the plant cell. At this point, only a few transporters associated with chloroplastic envelope had been identified: An iron transporter, a magnesium transporter and two copper P1B ATPases: HMA1 and PAA1. PAA1 is known to be the main pathway of copper import in the chloroplast, metal mainly used for photosynthesis. HMA1 is thought to be an additional pathway for copper import, essential to cope with an oxidative stress witch appears when the plant is grown under high light condition. In order to better understand the respective roles of these two ATPases in chloroplastic copper's homeostasis, two complementary approaches had been developed: - An in planta approach had been initiated to obtain some new lines affected in the expression of one (paa1 mutant overexpressing HMA1) or both ATPases (hma1/paa1 double knock out), then to identify conditions (light stress, saline stress, metal excess) revealing the essential role of these ATPases or inducing a different transcriptional response for genes encoding for theses ATPases or other actors of copper's homeostasis... Results obtained show that the function of HMA1, known to be essential during a light stress, is also required when plants have to cope with a salt stress. These results are new evidences that HMA1 has a role in copper's delivery to the chloroplastic copper/zinc superoxyde dismutase. Moreover, we have shown that in photoautotrophic culture condition, copper can partially restore the photosensibility of a hma1 mutant thus validating that HMA1 in linked with copper homeostasis. We also have demonstrated that HMA1 and PAA1 function are not redundant. The copper imported by these two ATPases is very likely delivered to target proteins by distinct pathways. At last, we have shown that a third copper import pathway in the chloroplast exists, pathway still not characterized. - An in vitro approach meant to produce HMA1 and PAA1 ATPases in the Lactococcus lactis heterologous expression system had been developed to determine their ionic specificities and biochemical characteristics. The prokaryotic expression system had been developed in the laboratory is perfectly suited for membrane protein production including HMA1 and PAA1. Yields obtained are appropriate for further biochemical analyses. We have determined the conditions of solubilisation and purification of these two proteins. So far, we had not been able to measure ATPase activity associated with these proteins but our data indicates that both of the proteins can bind one of their substrate: ATP. We also demonstrated that PAA1 can bind copper under its 1+ and 2+ form. Finally, these data suggests that the control of chloroplastic copper homeostasis requires several independent transport systems and a tight regulation of these copper import pathways in order to fill up the needs linked to photosynthesis and resistance mechanism to oxidative stress.Le chloroplaste est un organite spécifique de la cellule végétale. Il est délimité par une double membrane ou enveloppe qui renferme de nombreux systèmes de transports d'ions et de métabolites essentiels au fonctionnement du chloroplaste et de la cellule végétale. A ce jour, seuls quelques transporteurs de métaux associés à l'enveloppe des plastes ont été identifiés : un transporteur de fer, un transporteur de magnésium ainsi que deux ATPases à cuivre de type P1B : HMA1 et PAA1. PAA1 représenterait la voie principale d'import du cuivre dans le chloroplaste, notamment pour alimenter la photosynthèse. HMA1 constituerait une voie additionnelle d'import du cuivre, voie essentielle pour répondre à un stress oxydatif qui apparaît en particulier lorsque la plante est cultivée en lumière forte. Afin de mieux comprendre les rôles respectifs de ces deux ATPases dans la régulation de l'homéostasie du cuivre chloroplastique, deux approches complémentaires ont été développées : - une approche in planta visant à produire de nouvelles lignées affectées dans l'expression de l'une (mutant paa1 surexprimant HMA1) ou de ces deux ATPases (double mutant hma1/paa1), puis à identifier des conditions (stress lumineux, stress salin, excès de métaux) révélant le rôle essentiel de ces ATPases ou induisant une réponse transcriptionnelle différente des gènes codant ces ATPases ou d'autres acteurs liés à l'homéostasie du cuivre... Les résultats obtenus montrent que la fonction de HMA1, connue pour être essentielle lors d'un stress lumineux, est aussi requise lorsque la plante subit un stress salin. Ces résultats confortent le rôle de HMA1 dans la délivrance du cuivre à la superoxyde dismutase (cuivre/zinc) du chloroplaste. D'autre part, nous avons montré qu'en condition de culture photoautotrophe, le cuivre permet de supprimer partiellement la photosensibilité du mutant hma1, validant ainsi l'implication de HMA1 dans l'homéostasie du cuivre. Nous avons aussi démontré que les fonctions de HMA1 et PAA1 ne sont pas redondantes. Le cuivre importé par ces deux ATPases est probablement délivré à des protéines cibles par des voies différentes. Enfin, nous avons montré 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 ATPases HMA1 et PAA1 dans le système hétérologue Lactococcus lactis afin de déterminer leurs spécificités ioniques et leurs caractéristiques biochimiques. Le système d'expression procaryote L. lactis a été mis en place au laboratoire et s'avère parfaitement adapté à la production de protéines membranaires de plantes. Ce système a permis de produire plusieurs protéines membranaires d'Arabidopsis, dont HMA1 et PAA1, en quantités compatibles avec des analyses biochimiques. Nous avons déterminé les conditions de solubilisation et de purification de ces deux protéines. Nous n'avons pas pu mesurer d'activité ATPase associée à ces protéines. En revanche, nos données indiquent que ces deux protéines recombinantes peuvent lier l'un de leur substrat ; l'ATP. Nous avons aussi pu démontrer que PAA1 peut lier du cuivre sous forme 1+ et sous forme 2+. Au bilan, ces données suggèrent que le contrôle de l'homéostasie du cuivre chloroplastique requiert plusieurs systèmes de transport indépendants et une régulation fine de ces voies d'import de cuivre afin d'alimenter les besoins liés à la photosynthèse et les besoins liés aux mécanismes de résistance aux stress oxydatifs

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

Membrane protein expression in Lactococcus lactis.

International audienceMembrane proteins play key roles in cellular physiology, and they are important drug targets. Approximately 25% of all genes identified in sequenced genomes are known to encode membrane proteins; however, the majority have no assigned function. Although the resolution of soluble protein structure has entered the high-throughput stage, only 100 high-resolution structures of membrane proteins have been described until now. Lactococcus lactis is a gram-positive lactic bacterium that has been used traditionally in food fermentations, but it is now used widely in biotechnology for large-scale overproduction of heterologously expressed proteins. Various expression vectors based on either constitutive or inducible promoters exist. The nisin-inducible controlled gene expression (NICE) system is the most suitable for recombinant membrane protein expression allowing for fine control of gene expression based on the autoregulation mechanism of the bacteriocin nisin. Recombinant membrane proteins can be produced with affinity tags for efficient detection and purification from crude membrane protein extracts. The purpose of this chapter is to provide a detailed protocol for the expression of membrane proteins and their detection using the Strep-tag II affinity tag in L. lactis

Biochemical Characterization of AtHMA6/PAA1, a Chloroplast Envelope Cu(I)-ATPase.

International audienceCopper is an essential plant micronutrient playing key roles in cellular processes, among them photosynthesis. In Arabidopsis thaliana, copper delivery to chloroplasts, mainly studied by genetic approaches, is thought to involve two P(IB)-type ATPases: AtHMA1 and AtHMA6/PAA1. The lack of biochemical characterization of AtHMA1 and PAA1, and more generally of plant P(IB)-type ATPases, is due to the difficulty of getting high amounts of these membrane proteins in an active form, either from their native environment or after expression in heterologous systems. In this study, we report the first biochemical characterization of PAA1, a plant copper-transporting ATPase. PAA1 produced in Lactococcus lactis is active, forming an aspartyl phosphate intermediate in the presence of ATP and the adequate metal ion. PAA1 can also be phosphorylated using inorganic phosphate in the absence of transition metal. Both phosphorylation types allowed us to demonstrate that PAA1 is activated by monovalent copper ions (and to a lower extent by silver ions) with an apparent affinity in the micromolar range. In agreement with these biochemical data, we also demonstrate that when expressed in yeast, PAA1 induces increased sensitivities to copper and silver. These data provide the first enzymatic characterization of a P(IB-1)-type plant ATPase and clearly identify PAA1 as a high affinity Cu(I) transporter of the chloroplast envelope

Ballistic Electron Magnetic Microscopy: Towards the Nanometer Scale

International audienc

HMA1 and PAA1, two chloroplast envelope PIB-ATPases, play distinct roles in chloroplast copper homeostasis

International audienceCopper is an essential micronutrient but it is also potentially toxic as copper ions can catalyse the production of free radicals, which result in various types of cell damage. Therefore, copper homeostasis in plant and animal cells must be tightly controlled. In the chloroplast, copper import is mediated by a chloroplast-envelope PIB-type ATPase, HMA6/PAA1. Copper may also be imported by HMA1, another chloroplast-envelope PIB-ATPase. To get more insights into the specific functional roles of HMA1 and PAA1 in copper homeostasis, this study analysed the phenotypes of plants affected in the expression of both HMA1 and PAA1 ATPases, as well as of plants overexpressing HMA1 in a paa1 mutant background. The results presented here provide new evidence associating HMA1 with copper homeostasis in the chloroplast. These data suggest that HMA1 and PAA1 behave as distinct pathways for copper import and targeting to the chloroplast. Finally, this work also provides evidence for an alternative route for copper import into the chloroplast mediated by an as-yet unidentified transporter that is neither HMA1 nor PAA