Differential targeting of VDAC3 mRNA isoforms influences mitochondria morphology.

Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites. Four nucleus-encoded VDACs have been identified in Arabidopsis thaliana. Alternative cleavage and polyadenylation generate two VDAC3 mRNA isoforms differing by their 3' UTR. By using quantitative RT-PCR and in vivo mRNA visualization approaches, the two mRNA variants were shown differentially associated with mitochondria. The longest mRNA presents a 3' extension named alternative UTR (aUTR) that is necessary and sufficient to target VDAC3 mRNA to the mitochondrial surface. Moreover, aUTR is sufficient for the mitochondrial targeting of a reporter transcript, and can be used as a tool to target an unrelated mRNA to the mitochondrial surface. Finally, VDAC3-aUTR mRNA variant impacts mitochondria morphology and size, demonstrating the role of mRNA targeting in mitochondria biogenesis.

Voltage-dependent-anion-channels (VDACs) in Arabidopsis have a dual localization in the cell but show a distinct role in mitochondria.

In mammals, the Voltage-dependent anion channels (VDACs) are predominant proteins of the outer mitochondrial membrane (OMM) where they contribute to the exchange of small metabolites essential for respiration. They were shown to be as well associated with the plasma membrane (PM) and act as redox enzyme or are involved in ATP release for example. In Arabidopsis, we show that four out of six genomic sequences encode AtVDAC proteins. All four AtVDACs are ubiquitously expressed in the plant but each of them displays a specific expression pattern in root cell types. Using two complementary approaches, we demonstrate conclusively that the four expressed AtVDACs are targeted to both mitochondria and plasma membrane but in differential abundance, AtVDAC3 being the most abundant in PM, and conversely, AtVDAC4 almost exclusively associated with mitochondria. These are the first plant proteins to be shown to reside in both these two membranes. To investigate a putative function of AtVDACs, we analyzed T-DNA insertion lines in each of the corresponding genes. Knock-out mutants for AtVDAC1, AtVDAC2 and AtVDAC4 present slow growth, reduced fertility and yellow spots in leaves when atvdac3 does not show any visible difference compared to wildtype plants. Analyses of atvdac1 and atvdac4 reveal that yellow areas correspond to necrosis and the mitochondria are swollen in these two mutants. All these results suggest that, in spite of a localization in plasma membrane for three of them, AtVDAC1, AtVDAC2 and AtVDAC4 have a main function in mitochondria.

Plant organelle proteomics: collaborating for optimal cell function.

Organelle proteomics describes the study of proteins present in organelle at a particular instance during the whole period of their life cycle in a cell. Organelles are specialized membrane bound structures within a cell that function by interacting with cytosolic and luminal soluble proteins making the protein composition of each organelle dynamic. Depending on organism, the total number of organelles within a cell varies, indicating their evolution with respect to protein number and function. For example, one of the striking differences between plant and animal cells is the plastids in plants. Organelles have their own proteins, and few organelles like mitochondria and chloroplast have their own genome to synthesize proteins for specific function and also require nuclear-encoded proteins. Enormous work has been performed on animal organelle proteomics. However, plant organelle proteomics has seen limited work mainly due to: (i) inter-plant and inter-tissue complexity, (ii) difficulties in isolation of subcellular compartments, and (iii) their enrichment and purity. Despite these concerns, the field of organelle proteomics is growing in plants, such as Arabidopsis, rice and maize. The available data are beginning to help better understand organelles and their distinct and/or overlapping functions in different plant tissues, organs or cell types, and more importantly, how protein components of organelles behave during development and with surrounding environments. Studies on organelles have provided a few good reviews, but none of them are comprehensive. Here, we present a comprehensive review on plant organelle proteomics starting from the significance of organelle in cells, to organelle isolation, to protein identification and to biology and beyond. To put together such a systematic, in-depth review and to translate acquired knowledge in a proper and adequate form, we join minds to provide discussion and viewpoints on the collaborative nature of organelles in cell, their proper function and evolution.

The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance.

In plant cells, anion channels and transporters are essential for key functions such as nutrition, resistance to biotic or abiotic stresses, and ion homeostasis. In Arabidopsis, members of the chloride channel (CLC) family located in intracellular organelles have been shown to be required for nitrate homeostasis or pH adjustment, and previous results indicated that AtCLCc is involved in nitrate accumulation. We investigated new physiological functions of this CLC member in Arabidopsis. Here we report that AtCLCc is strongly expressed in guard cells and pollen and more weakly in roots. Use of an AtCLCc:GFP fusion revealed localization to the tonoplast. Disruption of the AtCLCc gene by a T-DNA insertion in four independent lines affected physiological responses that are directly related to the movement of chloride across the tonoplast membrane. Opening of clcc stomata was reduced in response to light, and ABA treatment failed to induce their closure, whereas application of KNO3 but not KCl restored stomatal opening. clcc mutant plants were hypersensitive to NaCl treatment when grown on soil, and to NaCl and KCl in vitro, confirming the chloride dependence of the phenotype. These phenotypes were associated with modifications of chloride content in both guard cells and roots. These data demonstrate that AtCLCc is essential for stomatal movement and salt tolerance by regulating chloride homeostasis.

ATP binding to the C terminus of the Arabidopsis thaliana nitrate/proton antiporter, AtCLCa, regulates nitrate transport into plant vacuoles.

Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.

Two anion transporters AtClCa and AtClCe fulfil interconnecting but not redundant roles in nitrate assimilation pathways.

* In plants, the knowledge of the molecular identity and functions of anion channels are still very limited, and are almost restricted to the large ChLoride Channel (CLC) family. In Arabidopsis thaliana, some genetic evidence has suggested a role for certain AtCLC protein members in the control of plant nitrate levels. In this context, AtClCa has been demonstrated to be involved in nitrate transport into the vacuole, thereby participating in cell nitrate homeostasis. * In this study, analyses of T-DNA insertion mutants within the AtClCa and AtClCe genes revealed common phenotypic traits: a lower endogenous nitrate content; a higher nitrite content; a reduced nitrate influx into the root; and a decreased expression of several genes encoding nitrate transporters. * This set of nitrate-related phenotypes, displayed by clca and clce mutant plants, showed interconnecting roles of AtClCa and AtClCe in nitrate homeostasis involving two different endocellular membranes. * In addition, it revealed cross-talk between two nitrate transporter families participating in nitrate assimilation pathways. The contribution to nitrate homeostasis at the cellular level of members of these different families is discussed.

Extent of N-terminal modifications in cytosolic proteins from eukaryotes.

Most proteins in all organisms undergo crucial N-terminal modifications involving N-terminal methionine excision, N-alpha-acetylation or N-myristoylation (N-Myr), or S-palmitoylation. We investigated the occurrence of these poorly annotated but essential modifications in proteomes, focusing on eukaryotes. Experimental data for the N-terminal sequences of animal, fungi, and archaeal proteins, were used to build dedicated predictive modules in a new software. In vitro N-Myr experiments were performed with both plant and animal N-myristoyltransferases, for accurate prediction of the modification. N-terminal modifications from the fully sequenced genome of Arabidopsis thaliana were determined by MS. We identified 105 new modified protein N-termini, which were used to check the accuracy of predictive data. An accuracy of more than 95% was achieved, demonstrating (i) overall conservation of the specificity of the modification machinery in higher eukaryotes and (ii) robustness of the prediction tool. Predictions were made for various proteomes. Proteins that had undergone both N-terminal methionine (Met) cleavage and N-acetylation were found to be strongly overrepresented among the most abundant proteins, in contrast to those retaining their genuine unblocked Met. Here we propose that the nature of the second residue of an ORF is a key marker of the abundance of the mature protein in eukaryotes.

Anion channels and transporters in plant cell membranes.

Anion channels/transporters appear as key players in signaling pathways leading to the adaptation of plant cells to abiotic and biotic environmental stresses, in the control of metabolism and in the maintenance of electrochemical gradients. Focusing on the most recent advances, this review aims at providing a description of the role of these channels in various physiological functions such as control of stomatal movements, plant-pathogen interaction, xylem loading, compartmentalization of metabolites and coupling with proton gradients. These functions have been demonstrated by a combination of electrophysiology, pharmacology and genetics approaches, the key issue being to identify the corresponding proteins and genes.

Purification and fractionation of membranes for proteomic analyses.

Proteomics is a very powerful approach to link the information contained in sequenced genomes, such as Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. However, membrane proteomics remains a challenge. One way to bring into view the complex mixture of proteins present in a membrane is to develop proteomic analyses based on (1) the use of highly purified membrane fractions and (2) fractionation of membrane proteins to retrieve as many proteins as possible (from the most to the less hydrophobic ones). To illustrate such strategies, we choose two types of membranes, the plasma membrane and the chloroplast envelope membranes. Both types of membranes can be prepared in a reasonable degree of purity from different types of tissues: the plasma membrane from cultured cells and the chloroplast envelope membrane from whole plants. This article is restricted to the description of methods for the preparation of highly purified and characterized plant membrane fractions and the subsequent fractionation of these membrane proteins according to simple physicochemical criteria (i.e., chloroform/methanol extraction, alkaline or saline treatments) for further analyses using modern proteomic methodologies.

Plant membrane proteomics.

Plant membrane proteins are involved in many different functions according to their location in the cell. For instance, the chloroplast has two membrane systems, thylakoids and envelope, with specialized membrane proteins for photosynthesis and metabolite and ion transporters, respectively. Although recent advances in sample preparation and analytical techniques have been achieved for the study of membrane proteins, the characterization of these proteins, especially the hydrophobic ones, is still challenging. The present review highlights recent advances in methodologies for identification of plant membrane proteins from purified subcellular structures. The interest of combining several complementary extraction procedures to take into account specific features of membrane proteins is discussed in the light of recent proteomics data, notably for chloroplast envelope, mitochondrial membranes and plasma membrane from Arabidopsis. These examples also illustrate how, on one hand, proteomics can feed bioinformatics for a better definition of prediction tools and, on the other hand, although prediction tools are not 100% reliable, they can give valuable information for biological investigations. In particular, membrane proteomics brings new insights over plant membrane systems, on both the membrane compartment where proteins are working and their putative cellular function.

Review. CLC-mediated anion transport in plant cells.

Plants need nitrate for growth and store the major part of it in the central vacuole of cells from root and shoot tissues. Based on few studies on the two model plants Arabidopsis thaliana and rice, members of the large ChLoride Channel (CLC) family have been proposed to encode anion channels/transporters involved in nitrate homeostasis. Proteins from the Arabidopsis CLC family (AtClC, comprising seven members) are present in various membrane compartments including the vacuolar membrane (AtClCa), Golgi vesicles (AtClCd and AtClCf) or chloroplast membranes (AtClCe). Through a combination of electrophysiological and genetic approaches, AtClCa was shown to function as a 2NO3-/1H+ exchanger that is able to accumulate specifically nitrate into the vacuole, in agreement with the main phenotypic trait of knockout mutant plants that accumulate 50 per cent less nitrate than their wild-type counterparts. The set-up of a functional complementation assay relying on transient expression of AtClCa cDNA in the mutant background opens the way for studies on structure-function relationships of the AtClCa nitrate transporter. Such studies will reveal whether important structural determinants identified in bacterial or mammalian CLCs are also crucial for AtClCa transport activity and regulation.

A novel pathway of cytochrome c biogenesis is involved in the assembly of the cytochrome b6f complex in arabidopsis chloroplasts.

We recently characterized a novel heme biogenesis pathway required for heme c(i)' covalent binding to cytochrome b6 in Chlamydomonas named system IV or CCB (cofactor assembly, complex C (b6f), subunit B (PetB)). To find out whether this CCB pathway also operates in higher plants and extend the knowledge of the c-type cytochrome biogenesis, we studied Arabidopsis insertion mutants in the orthologs of the CCB genes. The ccb1, ccb2, and ccb4 mutants show a phenotype characterized by a deficiency in the accumulation of the subunits of the cytochrome b6f complex and lack covalent heme binding to cytochrome b6. These mutants were functionally complemented with the corresponding wild type cDNAs. Using fluorescent protein reporters, we demonstrated that the CCB1, CCB2, CCB3, and CCB4 proteins are targeted to the chloroplast compartment of Arabidopsis. We have extended our study to the YGGT family, to which CCB3 belongs, by studying insertion mutants of two additional members of this family for which no mutants were previously characterized, and we showed that they are not functionally involved in the CCB system. Thus, we demonstrate the ubiquity of the CCB proteins in chloroplast heme c(i)' binding.

A high content in lipid-modified peripheral proteins and integral receptor kinases features in the arabidopsis plasma membrane proteome.

The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered.

Two members of the Arabidopsis CLC (chloride channel) family, AtCLCe and AtCLCf, are associated with thylakoid and Golgi membranes, respectively.

Though numerous pieces of evidence point to major physiological roles for anion channels in plants, progress in the understanding of their biological functions is limited by the small number of genes identified so far. Seven chloride channel (CLC) members could be identified in the Arabidopsis genome, amongst which AtCLCe and AtCLCf are both more closely related to bacterial CLCs than the other plant CLCs. It is shown here that AtCLCe is targeted to the thylakoid membranes in chloroplasts and, in agreement with this subcellular localization, that the clce mutants display a phenotype related to photosynthesis activity. The AtCLCf protein is localized in Golgi membranes and functionally complements the yeast gef1 mutant disrupted in the single CLC gene encoding a Golgi-associated protein.

A versatile method for deciphering plant membrane proteomes.

Proteomics is a very powerful approach to link the information contained in sequenced genomes, like that of Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. This article summarizes the different steps of a versatile strategy that has been developed to decipher plant membrane proteomes. Initiated with envelope membranes from spinach chloroplasts, this strategy has been adapted to thylakoids, and further extended to a series of membranes from the model plant Arabidopsis: chloroplast envelope membranes, plasma membrane, and mitochondrial membranes. The first step is the preparation of highly purified membrane fractions from plant tissues. The second step in the strategy is the fractionation of membrane proteins on the basis of their physico-chemical properties. Chloroform/methanol extraction and washing of membranes with NaOH, NaCl or any other agent led to the simplification of the protein content of the fraction to be analysed. The next step is the genuine proteomic step, i.e. the separation of proteins by 1D-gel electrophoresis followed by in-gel proteolytic digestion of the polypeptides, analysis of the proteolytic peptides using mass spectrometry, and protein identification by searching through databases. The last step is the validation of the procedure by checking the subcellular location. The results obtained by using this strategy demonstrate that a combination of different proteomics approaches, together with bioinformatics, indeed provide a better understanding of the biochemical machinery of the different plant membranes at the molecular level.

Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.