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, SDS͞PAGE separation, and tandem mass spectrometry analysis. To process the large amount of MS͞MS 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 ␣-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. P lastids, and especially chloroplasts, conduct vital biosynthetic functions, and many reactions are located exclusively within these unique organelles. A two-membrane system, the envelope, surrounds all plastid types and separates the plastid stroma from the cytosol. As a consequence, the envelope is involved in the controlled exchange of a variety of ions and metabolites between these two subcellular compartments (1).Chloroplasts import cytoplasmically synthesized precursor proteins from the cytosol. Translocation of precursor proteins across the envelope is achieved by the joint action of Toc and Tic translocons located at the outer and inner envelope membranes, respectively, of the chloroplast envelope (2, 3). Chloroplasts also take up intermediates of various metabolic pathways such as dicarboxylic acids, acetate, and phosphoenolpyruvate. Chloroplasts also have been demonstrated to import inorganic ions like K ϩ , Na (4, 5). As the sole site of biosynthesis of most amino acids (with the exception of sulfur-containing amino acids; refs. 6 and 7), chloroplasts must export these compounds for protein synthesis in the cytosolic and mitochondrial compartments. Finally, because of metabolism compartmentation, several other organic or inorganic comp...
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