Proteomics of the Chloroplast Envelope Membranes from Arabidopsis thaliana

Abstract

The development of chloroplasts and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting envelope membranes. To provide the most exhaustive view of the protein repertoire of chloroplast envelope membranes, we analyzed this membrane system using proteomics. To this purpose, we first developed a procedure to prepare highly purified envelope membranes from Arabidopsis chloroplasts. We then extracted envelope proteins using different methods, i.e. chloroform/methanol extraction and alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to least hydrophobic ones. Liquid chromatography tandem mass spectrometry analyses were then performed on each envelope membrane subfraction, leading to the identification of more than 100 proteins. About Plastids are semiautonomous organelles that present a wide structural diversity and contain unique biosynthetic pathways. They are strongly dependent on proteins that are nuclear encoded, translated in the cytoplasm, and imported into this organelle. A pair of membranes called the envelope surrounds all plastids. As the vast majority of plastid proteins are nuclear encoded, the plastid envelope contains a protein import machinery. Translocation at the envelope membranes is directed by a general import machinery composed of the outer-membrane Toc complex and the inner-membrane Tic complex (for reviews, see Refs. 1-4).Located at the interface between the stroma and the cytosol, the envelope is also the site of various transports and exchanges of ions and metabolites required for the integration of the plastid metabolism within the plant cell. Few envelope transporters have been identified and characterized at the molecular level: the triose-phosphate/phosphate translocator, an ADP/ATP translocator, several substrate-specific outer membrane channels, and two dicarboxylate translocators (for a review, see Ref. 5). Recently, a putative hexose transporter was also identified (6). More recently we described a proteomic approach that allowed the identification of several putative transporters of the chloroplast envelope (7).A unique biochemical machinery is also present in envelope membranes. The chloroplast envelope is the site of specific biosynthetic functions i.e. synthesis of plastid membrane components (glycerolipids, pigments, prenylquinones), chlorophyll breakdown, synthesis of lipid-derived signaling molecules (fatty acid hydroperoxydes, growth regulators, or chlorophyll precursors), and participates in the coordination of the expression of nuclear and plastid genes (for a review, see Ref. 8). So far, and as for other plastid envelope components, few proteins catalyzing these biosynthetic functions have been identified and characterized at the molecular level.Subcellular proteomic studies are essential to get access to protein location in relation with their function (for a review, see Ref. 9). Plant proteomics exemplifies perfectly this functional dimensi...

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

“…Among Arabidopsis MIPs, there are 13 plasma membrane intrinsic proteins (PIPs) and 10 tonoplast intrinsic proteins (TIPs). Three Arabidopsis TIPs, namely TIP1;1, TIP1;2 and TIP2;1 have been detected by Ferro et al [28] using the proteomic approach in the chloroplast envelope of Arabidopsis, but their presence has been regarded as a possible vacuolar contamination. In another proteomic study of the Arabidopsis chloroplast [29], several PIPs (PIP1;2, PIP1;3, PIP2;1 and PIP2;2) and TIPs (TIP2;1 and TIP2;2) were identified in the envelope fraction, but their presence was considered insecure since possible contamination with Water transport across chloroplast membranes and its use during photosynthetic reactions.…”

An improved prediction of chloroplast proteins reveals diversities and commonalities in the chloroplast proteomes of Arabidopsis and rice

“…Relatively small sub-proteomes of the chloroplast have been experimentally identified (Kieselbach et al, 1998;Nakabayashi et al, 1999;Peltier et al, 2000;Ferro et al, 2002;Gomez et al, 2002;Peltier et al, 2002;Schubert et al, 2002;Balmer et al, 2003;Ferro et al, 2003;Schleiff et al, 2003), but a saturating organelle-wide proteomic screen has not yet been performed. Thus, large-scale detection of chloroplast proteins can be attempted only in silico.…”

Proteomic Characterization of A Triton-Insoluble Fraction from Chloroplasts Defines A Novel Group of Proteins Associated with Macromolecular Structures

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