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. Molecular & Cellular Proteomics 9:1063-1084, 2010.Plastids are semiautonomous organelles that are ubiquitously found in plant cells. They are derived from an endosymbiotic event and are thought to have evolved from an ancient photosynthetic prokaryote related to present-day cyanobacteria. Following endosymbiosis, the plastid genome has been reduced to ϳ100 genes, mainly coding for housekeeping functions (translation and transcription of the plastid genome), proteins required for primary photosynthetic reactions, and a few, yet poorly characterized, gene products (1). The most conspicuous plastid type is the chloroplast, found in leaves and carrying out photosynthesis as its main function. Photosynthesis is an integrated biological process involving the coordinated functioning of ...

Assessment of the requirement for aquaporins in the thylakoid membrane of plant chloroplasts to sustain photosynthetic water oxidation

“…Zybailov et al [30] have detected TIP2;1 in the thylakoid fraction, but no other isoforms. In a more recent study, Ferro et al [31] identified again TIP1;1, TIP1;2 and TIP2;1 in chloroplast membranes from Arabidopsis. TIP1;1 was detected only in the chloroplast envelope, and has been considered as a contaminant because in the same fraction, the vacuolar marker V-ATPase has also been detected.…”

Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

“…chloroplasts reported in the AT-CHLORO database (Ferro et al, 2010) and in Zybailov et al (2008). It appears that the percentage of proteins classified according to the MapMan functional classes is very similar in the two proteomes, particularly for the class corresponding to photosynthesis (Supplemental Fig.…”

“…Protein abundance is expressed as a log 2 . The present graph corresponds to Supplemental Table S3 generated by screening the tomato plastid proteome on the base of AT homologs with the AT-CHLORO subplastidial database (Ferro et al, 2010) for stroma, thylakoids, and envelope proteins and in Lundquist et al (2012) for plastoglobule proteins.…”

“…The tomato plastid proteome referenced in Supplemental Table S2 has been screened with the AT_Chloro database (Ferro et al, 2010) to isolate proteins present in the stroma, thylakoids, and envelope membrane (Supplemental Table S3) and with the list of proteins of the plastoglobules established by Lundquist et al (2012). In agreement with the structural remodeling of the internal membrane system (Spurr and Harris, 1968), this study clearly shows that the abundance of thylakoid proteins fell mostly during the transition from B to R stages while the abundance of proteins of the envelope and of the plastoglobules remained essentially unchanged and proteins of the stroma underwent a slight decrease in abundance (Fig.…”

Plastid Proteome Assembly without Toc159: Photosynthetic Protein Import and Accumulation of N-Acetylated Plastid Precursor Proteins

“…To exclude those proteins whose decreased abundance in ppi2 could be attributed to lower transcript levels, we additionally excluded those from the list of Toc159-dependent proteins that were significantly downregulated in ppi2 compared with wtS. As a final criterion, plastid proteins annotated by PPDB and AT_CHLORO to be localized in the outer envelope were removed Ferro et al, 2010). Together, 44 proteins fulfilled these requirements and constitute the set of Toc159-dependent proteins, whose accumulation is affected at the protein and not at the transcript level (see Supplemental Data Set 11 online).…”