Daphné Seigneurin-Berny

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.

Functional significance of histone deacetylase diversity.

Nucleocytoplasmic shuttling of histone deacetylases is emerging as a major step in determining the composition, and hence the activity, of the corresponding nuclear regulatory complexes. This shuttling process is one of the distinctive characteristics of these enzymes, themselves belonging to structurally and functionally different classes. Considering the specific features of each class of deacetylases, it is possible to determine how each member can contribute to particular cellular functions.

HSP27 Is a Ubiquitin-Binding Protein Involved in I-κBα Proteasomal Degradation

ABSTRACT HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-α). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-κB by degrading its main inhibitor, I-κBα. HSP27 overexpression increases NF-κB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, ΤNF-α, and interleukin 1β. HSP27 does not affect I-κBα phosphorylation but enhances the degradation of phosphorylated I-κBα by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-κBα. A protein complex that includes HSP27, phosphorylated I-κBα, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-κBα. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-κB activity.

Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways

ABSTRACT The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

Integral membrane proteins of the chloroplast envelope: Identification and subcellular localization of new transporters

A two-membrane system, or envelope, surrounds plastids. Because of the integration of chloroplast metabolism within the plant cell, the envelope is the site of many specific transport activities. However, only a few proteins involved in the processes of transport across the chloroplast envelope have been identified already at the molecular level. To discover new envelope transporters, we developed a subcellular proteomic approach, which is aimed to identify the most hydrophobic envelope proteins. This strategy combined the use of highly purified and characterized membrane fractions, extraction of the hydrophobic proteins with organic solvents, SDS/PAGE separation, and tandem mass spectrometry analysis. To process the large amount of MS/MS data, a blast-based program was developed for searching in protein, expressed sequence tag, and genomic plant databases. Among the 54 identified proteins, 27 were new envelope proteins, with most of them bearing multiple α-helical transmembrane regions and being very likely envelope transporters. The present proteomic study also allowed us to identify common features among the known and newly identified putative envelope inner membrane transporters. These features were used to mine the complete Arabidopsis genome and allowed us to establish a virtual plastid envelope integral protein database. Altogether, both proteomic and in silico approaches identified more than 50 candidates for the as yet previously uncharacterized plastid envelope transporters. The predictable function of some of these proteins opens up areas of investigation that may lead to a better understanding of the chloroplast metabolism. The present subcellular proteomic approach is amenable to the analysis of the hydrophobic core of other intracellular membrane systems.

HMA1, a new Cu-ATPase of the chloroplast envelope, is essential for growth under adverse light conditions

Although ions play important roles in the cell and chloroplast metabolism, little is known about ion transport across the chloroplast envelope. Using a proteomic approach specifically targeted to the Arabidopsis chloroplast envelope, we have identified HMA1, which belongs to the metal-transporting P1B-type ATPases family. HMA1 is mainly expressed in green tissues, and we validated its chloroplast envelope localization. Yeast expression experiments demonstrated that HMA1 is involved in copper homeostasis and that deletion of its N-terminal His-domain partially affects the metal transport. Characterization of hma1 Arabidopsis mutants revealed a lower chloroplast copper content and a diminution of the total chloroplast superoxide dismutase activity. No effect was observed on the plastocyanin content in these lines. The hma1 insertional mutants grew like WT plants in standard condition but presented a photosensitivity phenotype under high light. Finally, direct biochemical ATPase assays performed on purified chloroplast envelope membranes showed that the ATPase activity of HMA1 is specifically stimulated by copper. Our results demonstrate that HMA1 offers an additional way to the previously characterized chloroplast envelope Cu-ATPase PAA1 to import copper in the chloroplast.

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.

Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins

As a complementary approach to genome projects, proteomic analyses have been set up to identify new gene products. One of the major challenges in proteomics concerns membrane proteins, especially the minor ones. A procedure based on the differential extraction of membrane proteins in chloroform/methanol mixtures, was tested on the two different chloroplast membrane systems: envolope and thylakoid membranes. Combining the use of classical sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and mass spectrometry analyses, this procedure enabled identification of hydrophobic proteins. The propensity of hydrophobic proteins to partition in chloroform/methanol mixtures was directly correlated with the number of amino acid residues/number of putative transmembrane regions (Res/TM ratio). Regardless of the particular case of some lipid‐interacting proteins, chloroform/methanol extractions allowed enrichment of hydrophobic proteins and exclusion of hydrophilic proteins from both membrane systems, thus demonstrating the versatility of the procedure.

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.

A dual strategy to cope with high light in Chlamydomonas reinhardtii

To protect photosynthetic organisms from photodamage, excess energy in photosystem II must be dissipated. In C. reinhardtii, two alternative mechanisms (energy thermal dissipation [qE] and kinase-mediated migration of light harvesting proteins [qT]) synergistically modulate photoprotection via the reversible migration of LHCSR3, a key qE effector in this alga, between photosystems II and I during qT. Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.