Laser Texturing to Increase the Wear Resistance of an Electrophoretic Graphene Coating on Copper Substrates.

In the present paper, different surface preparations are investigated with the aim of increasing the wear behaviour of an electrophoretic graphene coating on a copper plate. The study was divided into two steps: In the first step (pre-tests), to detect the most promising pretreatment technology, five different surface preparations were investigated (electropolishing, sandblasting, degreasing and pickling, laser cleaning and laser dots).In the second step, on the basis of the results of the first step, a 32 full factorial plan was developed and tested; three treatment types (pickled and degreased, laser-cleaned, and laser dots) and three different voltages (30, 45 and 60 V) were adopted. Analysis of variance (ANOVA) was used to evaluate their influence on wear resistance; in particular, the maximum depth and width of the wear tracks and the coating break distance were investigated. The results of this study show that, in optimal conditions, laser treatment (particularly laser dots) canlead to as high as a four-fold increase in wear resistance.

Calmodulin is involved in the dual subcellular location of two chloroplast proteins.

Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.

Unraveling Hidden Components of the Chloroplast Envelope Proteome: Opportunities and Limits of Better MS Sensitivity.

The chloroplast is a major plant cell organelle that fulfills essential metabolic and biosynthetic functions. Located at the interface between the chloroplast and other cell compartments, the chloroplast envelope system is a strategic barrier controlling the exchange of ions, metabolites and proteins, thus regulating essential metabolic functions (synthesis of hormones precursors, amino acids, pigments, sugars, vitamins, lipids, nucleotides etc.) of the plant cell. However, unraveling the contents of the chloroplast envelope proteome remains a difficult challenge; many proteins constituting this functional double membrane system remain to be identified. Indeed, the envelope contains only 1% of the chloroplast proteins (i.e. 0.4% of the whole cell proteome). In other words, most envelope proteins are so rare at the cell, chloroplast, or even envelope level, that they remained undetectable using targeted MS studies. Cross-contamination of chloroplast subcompartments by each other and by other cell compartments during cell fractionation, impedes accurate localization of many envelope proteins. The aim of the present study was to take advantage of technologically improved MS sensitivity to better define the proteome of the chloroplast envelope (differentiate genuine envelope proteins from contaminants). This MS-based analysis relied on an enrichment factor that was calculated for each protein identified in purified envelope fractions as compared with the value obtained for the same protein in crude cell extracts. Using this approach, a total of 1269 proteins were detected in purified envelope fractions, of which, 462 could be assigned an envelope localization by combining MS-based spectral count analyses with manual annotation using data from the literature and prediction tools. Many of such proteins being previously unknown envelope components, these data constitute a new resource of significant value to the broader plant science community aiming to define principles and molecular mechanisms controlling fundamental aspects of plastid biogenesis and functions.

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics.

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

The Main Functions of Plastids.

Plastids are semiautonomous organelles like mitochondria, and derive from a cyanobacterial ancestor that was engulfed by a host cell. During evolution, they have recruited proteins originating from the nuclear genome, and only parts of their ancestral metabolic properties were conserved and optimized to limit functional redundancy with other cell compartments. Furthermore, large disparities in metabolic functions exist among various types of plastids, and the characterization of their various metabolic properties is far from being accomplished. In this review, we provide an overview of the main functions, known to be achieved by plastids or shared by plastids and other compartments of the cell. In short, plastids appear at the heart of all main plant functions.

AT_CHLORO: The First Step When Looking for Information About Subplastidial Localization of Proteins.

Plastids contain several key subcompartments. The two limiting envelope membranes (inner and outer membrane of the plastid envelope with an intermembrane space between), an aqueous phase (stroma), and an internal membrane system terms (thylakoids) formed of flat compressed vesicles (grana) and more light structures (lamellae). The thylakoid vesicles delimit another discrete soluble compartment, the thylakoid lumen. AT_CHLORO ( http://at-chloro.prabi.fr/at_chloro/ ) is a unique database supplying information about the subplastidial localization of proteins. It was created from simultaneous proteomic analyses targeted to the main subcompartments of the chloroplast from Arabidopsis thaliana (i.e., envelope, stroma, thylakoid) and to the two subdomains of thylakoid membranes (i.e., grana and stroma lamellae). AT_CHLORO assembles several complementary information (MS-based experimental data, curated functional annotations and subplastidial localization, links to other public databases and references) which give a comprehensive overview of the current knowledge about the subplastidial localization and the function of chloroplast proteins, with a specific attention given to chloroplast envelope proteins.

Preparation of Membrane Fractions (Envelope, Thylakoids, Grana, and Stroma Lamellae) from Arabidopsis Chloroplasts for Quantitative Proteomic Investigations and Other Studies.

Chloroplasts are semiautonomous organelles found in plants and protists. They are surrounded by a double membrane system, or envelope. These envelope membranes contain machineries to import nuclear-encoded proteins, and transporters for ions or metabolites, but are also essential for a range of plastid-specific metabolisms. The inner membrane surrounds a stroma, which is the site of the carbon chemistry of photosynthesis. Chloroplasts also contain an internal membrane system, or thylakoids, where the light phase of photosynthesis occurs. The thylakoid membranes themselves have a bipartite structure, consisting of grana stacks interconnected by stroma lamellae. These thylakoid membranes however form a continuous network that encloses a single lumenal space. Chloroplast-encoded or targeted proteins are thus addressed to various sub-compartments that turn out to be flexible systems and whose main functions can be modulated by alterations in the relative levels of their components. This article describes procedures developed to recover highly purified chloroplast membrane fractions (i.e., envelope, crude thylakoid membranes, as well as the two main thylakoid subdomains, grana and stroma lamellae), starting from Percoll-purified Arabidopsis chloroplasts. Immunological markers are also listed that can be used to assess the purity of these fractions and reveal specific contaminations by other plastid membrane compartments. The methods described here are compatible with chloroplast proteome dynamic studies relying on targeted quantitative proteomic investigations.

Identification of Two Conserved Residues Involved in Copper Release from Chloroplast PIB-1-ATPases.

Copper is an essential transition metal for living organisms. In the plant model Arabidopsis thaliana, half of the copper content is localized in the chloroplast, and as a cofactor of plastocyanin, copper is essential for photosynthesis. Within the chloroplast, copper delivery to plastocyanin involves two transporters of the PIB-1-ATPases subfamily: HMA6 at the chloroplast envelope and HMA8 in the thylakoid membranes. Both proteins are high affinity copper transporters but share distinct enzymatic properties. In the present work, the comparison of 140 sequences of PIB-1-ATPases revealed a conserved region unusually rich in histidine and cysteine residues in the TMA-L1 region of eukaryotic chloroplast copper ATPases. To evaluate the role of these residues, we mutated them in HMA6 and HMA8. Mutants of interest were selected from phenotypic tests in yeast and produced in Lactococcus lactis for further biochemical characterizations using phosphorylation assays from ATP and Pi Combining functional and structural data, we highlight the importance of the cysteine and the first histidine of the CX3HX2H motif in the process of copper release from HMA6 and HMA8 and propose a copper pathway through the membrane domain of these transporters. Finally, our work suggests a more general role of the histidine residue in the transport of copper by PIB-1-ATPases.

Functional expression of plant membrane proteins in Lactococcus lactis.

The study of most membrane proteins remains challenging due to their hydrophobicity and their low natural abundance in cells. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations and is nowadays widely used in biotechnology for large-scale production of heterologous proteins. This system has been successfully used for the production of prokaryotic and eukaryotic membrane proteins. The purpose of this chapter is to provide detailed protocols for (1) the expression of plant peripheral or intrinsic membrane proteins and then for (2) their solubilization, from Lactococcus membranes, for further purification steps and biochemical characterization.

Deciphering thylakoid sub-compartments using a mass spectrometry-based approach.

Photosynthesis has shaped atmospheric and ocean chemistries and probably changed the climate as well, as oxygen is released from water as part of the photosynthetic process. In photosynthetic eukaryotes, this process occurs in the chloroplast, an organelle containing the most abundant biological membrane, the thylakoids. The thylakoids of plants and some green algae are structurally inhomogeneous, consisting of two main domains: the grana, which are piles of membranes gathered by stacking forces, and the stroma-lamellae, which are unstacked thylakoids connecting the grana. The major photosynthetic complexes are unevenly distributed within these compartments because of steric and electrostatic constraints. Although proteomic analysis of thylakoids has been instrumental to define its protein components, no extensive proteomic study of subthylakoid localization of proteins in the BBY (grana) and the stroma-lamellae fractions has been achieved so far. To fill this gap, we performed a complete survey of the protein composition of these thylakoid subcompartments using thylakoid membrane fractionations. We employed semiquantitative proteomics coupled with a data analysis pipeline and manual annotation to differentiate genuine BBY and stroma-lamellae proteins from possible contaminants. About 300 thylakoid (or potentially thylakoid) proteins were shown to be enriched in either the BBY or the stroma-lamellae fractions. Overall, present findings corroborate previous observations obtained for photosynthetic proteins that used nonproteomic approaches. The originality of the present proteomic relies in the identification of photosynthetic proteins whose differential distribution in the thylakoid subcompartments might explain already observed phenomenon such as LHCII docking. Besides, from the present localization results we can suggest new molecular actors for photosynthesis-linked activities. For instance, most PsbP-like subunits being differently localized in stroma-lamellae, these proteins could be linked to the PSI-NDH complex in the context of cyclic electron flow around PSI. In addition, we could identify about a hundred new likely minor thylakoid (or chloroplast) proteins, some of them being potential regulators of the chloroplast physiology.

Complementary biochemical approaches applied to the identification of plastidial calmodulin-binding proteins.

Ca(2+)/Calmodulin (CaM)-dependent signaling pathways play a major role in the modulation of cell responses in eukaryotes. In the chloroplast, few proteins such as the NAD(+) kinase 2 have been previously shown to interact with CaM, but a general picture of the role of Ca(2+)/CaM signaling in this organelle is still lacking. Using CaM-affinity chromatography and mass spectrometry, we identified 210 candidate CaM-binding proteins from different Arabidopsis and spinach chloroplast sub-fractions. A subset of these proteins was validated by an optimized in vitro CaM-binding assay. In addition, we designed two fluorescence anisotropy assays to quantitatively characterize the binding parameters and applied those assays to NAD(+) kinase 2 and selected candidate proteins. On the basis of our results, there might be many more plastidial CaM-binding proteins than previously estimated. In addition, we showed that an array of complementary biochemical techniques is necessary in order to characterize the mode of interaction of candidate proteins with CaM.

AT_CHLORO: A Chloroplast Protein Database Dedicated to Sub-Plastidial Localization.

AT_CHLORO (www.grenoble.prabi.fr/at_chloro) is a database dedicated to sub-plastidial localization of A. thaliana chloroplast proteins. This information was infered from proteomics experiments obtained from a comprehensive study that allowed the identification of proteins from envelope, stroma, and thylakoid sub-compartments Ferro et al., 2010. In addition to current knowledge regarding sub-plastidial localization, AT_CHLORO provides experimental data that allowed curated information regarding subcellular localizations of chloroplast proteins to be given. A specific focus was given to proteins that were identified in envelope fractions and for which expert functional annotation was provided. The present mini review shows the specificities of AT_CHLORO with respect to available information, data export options and recent improvements in data representation.

Preparation of envelope membrane fractions from Arabidopsis chloroplasts for proteomic analysis and other studies.

Plastids are semiautonomous organelles restricted to plants and protists. These plastids are surrounded by a double membrane system, or envelope. These envelope membranes contain machineries to import nuclear-encoded proteins, and transporters for ions or metabolites, but are also essential for a range of plastid-specific metabolisms. Targeted semiquantitative proteomic investigations have revealed specific cross-contaminations by other cell or plastid compartments that may occur during chloroplast envelope purification. This article describes procedures developed to recover highly purified envelope fractions starting from Percoll-purified Arabidopsis chloroplasts, gives an overview of possible cross-contaminations, provides some tricks to limit these cross-contaminations, and lists immunological markers and methods that can be used to assess the purity of the envelope fractions.

Heterologous expression of membrane proteins: choosing the appropriate host.

BACKGROUND: 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.

Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.

BACKGROUND: 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.

AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins.

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.

Chloroplast proteomics highlights the subcellular compartmentation of lipid metabolism.

Recent advances in the proteomic field have allowed high throughput experiments to be conducted on chloroplast samples and the data are available in several databases such as the Plant Protein Database (PPDB), or the SubCellular Proteomic Database (SUBA). However, the accurate localization of many proteins that were identified in different subplastidial compartments often remains hypothetical, thus making quantitative proteomics important for going a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regard to their accurate localization within the chloroplast. Spectral counting, a semi-quantitative proteomic strategy based on accurate mass and time tags (AMT), was used to build up AT_CHLORO, a comprehensive chloroplast proteome database with curated subplastidial localization. In this review, we focus on about a hundred enzymes involved in fatty acid biosynthesis, export and metabolism (desaturation and oxylipin metabolism), in the synthesis of chloroplast-specific glycerolipids either with a eukaryotic or a prokaryotic structure. Two main chloroplast compartments play a major role in lipid biosynthesis: the initial steps of fatty acid biosynthesis take place in the stroma, then the envelope membranes concentrate most of the proteins involved in chloroplast glycerolipid metabolism.

Chloroplast proteomics and the compartmentation of plastidial isoprenoid biosynthetic pathways.

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database (PPDB, Sun et al., 2009) presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. (2009) went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags (AMT) database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways (i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts) validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts (i.e. in envelope membranes, stroma, and/or thylakoids) that remains unclear until now.

Purification of intact chloroplasts from Arabidopsis and spinach leaves by isopycnic centrifugation.

Chloroplasts 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.

Percoll-purified and photosynthetically active chloroplasts from Arabidopsis thaliana leaves.

The availability of the complete genome sequence of Arabidopsis thaliana and of large collections of insertion mutants paved the way for systematic studies of gene functions in this organism, thus requiring adapting biochemical and physiological tools to this model plant. For physiological analysis of photosynthesis, methods combining high level of chloroplast purity and preservation of the photosynthetic activity were missing. Here, we describe a rapid method (less than 1h) to obtain Percoll-purified and photosynthetically active chloroplasts from Arabidopsis leaves retaining almost 90% of the Vmax of photosynthesis measured in the starting leaves from plants grown under a light intensity of 150mumolphotonm(-2)s(-1) and 80% of their initial photosynthetic rate after 3h of storage.