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

Abstract

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...

ArabidopsisSAMT1 Defines a Plastid Transporter Regulating Plastid Biogenesis and Plant Development

“…As would be predicted for an integral protein, SAMT1 was completely solubilized by treatment with 2% Triton X-100 ( Figure 2E). The localization of SAMT1 in the chloroplast envelope membranes has been suggested previously by bioinformatic analysis (Koo and Ohlrogge, 2002) and demonstrated by proteomics (Ferro et al, 2002(Ferro et al, , 2003, supporting the localization data presented in this study. Collectively, these data demonstrate that SAMT1 is an integral membrane protein that is localized in the chloroplast envelope membranes.…”

Differential expression and phylogenetic analysis suggest specialization of plastid-localized members of the PHT4 phosphate transporter family for photosynthetic and heterotrophic tissues

“…Like TPT, both of these Pi transporters are located in the chloroplast inner envelope membrane. 14,18 Also, transcript levels for the genes increased when plants were exposed to light, although the delay in PHT4;4 transcript accumulation may reflect regulation by photosynthates rather than a direct response to light. It is formally possible that PHT4;4 and PHT2;1 also maintain stromal Pi concentrations through export.…”

“…11 Functional analyses in yeast, however, suggest that PHT2 proteins catalyze H + -dependent Pi transport. [11][12][13] GFP translational fusions with PHT2 proteins from Arabidopsis, Medicago truncatula, spinach and potato are targeted to the chloroplast envelope, [12][13][14][15] and localization within the inner envelope membrane is supported by subcellular proteomics and membrane fractionation/immunodetection. 14 In addition to a putative role in Pi import into the chloroplast, the presence of PHT2;1 transcripts within the root stele suggests that the encoded protein also functions in a subset of non-photosynthetic plastids.…”

Protein Profiling of Plastoglobules in Chloroplasts and Chromoplasts. A Surprising Site for Differential Accumulation of Metabolic Enzymes

“…These identified proteins were cross-correlated to large-scale Arabidopsis proteomics data from envelope (Ferro et al, 2002(Ferro et al, , 2003Froehlich et al, 2003) and other Arabidopsis subcellular proteomes (Borderies et al, 2003;Nuhse et al, 2003;Carter et al, 2004;Heazlewood et al, 2004;Marmagne et al, 2004) and to other literature. Most of the proteins were never identified in the stromal or envelope proteome, clearly indicating that PGs contain a specific protein population (Table I).…”