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

Abstract

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.

Fig. 1.

Analysis of chloroplast fractions: overview of complete strategy. Chloroplasts from Arabidopsis leaves were purified on Percoll density gradients. The chloroplast envelope and other plastid subfractions (thylakoids and stroma) were purified on a sucrose gradient. To get a better view of the chloroplast envelope proteome, we complemented proteomics studies performed on envelope fractions extracted from WT plants with an additional proteomics analysis performed on chloroplast envelope fractions from the iep18 mutant (gray area). Red arrows, analyses corresponding to the proteins included in the initial set used for statistical measurements. Insol., insoluble.

Fig. 2.

Processing pipeline used to generate AMT tag database AT_CHLORO.

Fig. 3.

Cumulative frequencies of reverse and forward database hits as a function of peptide length (A) are Mascot score (B).

Fig. 4.

Subplastidial localization according to spectral count data (819 proteins). A, distribution of protein subplastidial localization as determined using spectral count (see supplemental Data 9, column “Loc_SC”). B, correlation between spectral count-based localizations and PPDB and/or TAIR annotations. Chloroplast location, the localization according to PPDB and/or TAIR (see supplemental Data 9, column “Annotated as plastidial”): black box, proteins annotated as plastidial; gray box, proteins with no localization annotation; white box, proteins annotated as localized in other subcellular compartments. Subplastidial location, for proteins annotated as plastidial, correlation between the annotation at the subplastidial level (PPDB and/or TAIR) and localization based on spectral count (see supplemental Data 9, column “Correlation loc. SC and annotation at the subplastidial level”): black box, proteins whose subplastidial annotation (PPDB and/or TAIR) matches spectral count-based subplastidial localization; gray box, proteins with no subplastidial localization annotation; white box, proteins annotated as localized in another subplastidial compartment compared with spectral count-based subplastidial localization.

Fig. 5.

Spectral count-based subplastidial localization of proteins retrieved from envelope fractions. In good agreement with the levels of cross-contaminations measured using Western blot (WB) experiments (see supplemental Data 1), envelope membranes appear to contain up to 10% stroma proteins and 6% thylakoid membranes proteins. Contamination thresholds were selected accordingly to evaluate subplastidial localization of the proteins identified within the purified envelope fraction (see supplemental Data 10). Nuc, nucleus; Cyto, cytoplasm; Mito, mitochondrion; Tono, tonoplast; PM, plasma membrane; Perox, peroxisome; S+E, stroma and envelope; S, stroma; Th+E, thylakoid and envelope; Th, thylakoid; E?, envelope?, OM, outer membrane; IM, inner membrane.

Fig. 6.

Evaluation of coverage of chloroplast envelope proteome when combining present data with earlier analyses targeted to same membrane system. A, Venn diagram indicating the weight of protein identified during this work when compared with previous data obtained by Ferro et al. (26, 59) or Froehlich et al. (58). Numbers in parentheses are the numbers of proteins identified throughout the diverse studies. For the present study, only 644 of the 700 proteins identified in the purified envelope fraction were considered (proteins classified as “contaminants” and suspected to be derived from non-plastid subcompartments were excluded; see supplemental Data 10). When combining all proteomics studies performed on the chloroplast envelope, a total of 762 proteins was identified. Note that 360 proteins (47%) were only identified during this work. B, overlap of the studies targeted to the chloroplast envelope with the recent and extensive study (more than 1300 identified proteins) performed at the whole chloroplast level (59). In this case, numbers in parentheses are the numbers of proteins identified during this work that were also identified in previous studies. According to these data, of the 360 new envelope proteins identified during this work, 224 proteins were previously detected in the chloroplast (Zybailov et al. (57)), and 136 proteins were only identified during this work. Note that targeting the envelope membrane system to perform the proteomics analyses allowed identification of proteins that could not be detected in more complex chloroplast subfractions. IEM, inner envelope membrane; OEM, outer envelope membrane; E?, envelope?; S/E, stroma/envelope; T/E, thylakoid/envelope.

Fig. 7.

Envelope composition: curated annotation and functional categories. Functional annotations were retrieved from supplemental Data 10. Two sets of proteins were analyzed: the “whole envelope proteome” (460 proteins) corresponding to proteins with abundance in purified envelope fraction above measured cross-contamination levels (first 460 proteins in supplemental Data 10) (A) and the more specific envelope proteome (298 proteins), which only contains proteins displaying increased abundance in envelope fractions compared with other plastid compartments (first 298 proteins in supplemental Data 10) (B). In both cases, non-plastid proteins were excluded.

Fig. 8.

Relative spectral counts and subplastidial localization of proteases. Subplastidial localization of the different classes of proteases as defined by MapManBin (42) is shown. The size of the spots is proportional to the number of proteins having the same relative spectral count distribution between envelope (ENV), stroma (STR), and thylakoids localization.

Fig. 9.

KEA1 and KEA2 were detected in chloroplast envelope proteome. These two proteins are of similar size when compared with their homologues in bacteria, algae, and some plants. However, some predicted rice proteins contain a huge additional N terminus. The N terminus of KEA2 (AT4G00630.1) was identified as the HP57 protein (AT4G00640.1) that was also detected in the envelope fraction during this work (supplemental Data 14). The N terminus of KEA1 (AT1G01790.1) was identified as an HP57-like protein (no AGI accession number; Q9LQ77_ARATH) (supplemental Data 14) for which peptides were also detected during this work. PCR amplification demonstrated that these predicted proteins are two parts of the same large envelope protein. The N terminus of one of the two HP57 proteins is predicted to contain a classical plastid targeting peptide in good agreement with the genuine localization of this protein within the inner membrane of the chloroplast envelope. Chr, chromosome; Ac Nb, accession number.

Fig. 10.

Relative spectral counts and subplastidial localization for selected functional classes. The size of the spots is proportional to the number of proteins having the same relative spectral count distribution between envelope, stroma, and thylakoids fractions. A, how to read the diagrams. x axis, percentage of spectral count corresponding to envelope fractions; y axis, percentage of spectral count corresponding to stroma fractions; yellow box (ENV), proteins that are mainly localized in the envelope; orange box (STR), proteins that are mainly localized in the stroma; green box (THY), proteins that are mainly localized in the thylakoids. B–H, diagrams showing the subplastidial localization, based on spectral count, for some functional classes as defined by MapManBin (42) (see supplemental Data 9, column “MapManBin”). The functional classes considered are transport (B), amino acid metabolism (C), photosystems (D), not assigned (E), protein degradation (F), protein synthesis (G), and Calvin cycle (H).