Proteomics of the Chloroplast: Systematic Identification and Targeting Analysis of Lumenal and Peripheral Thylakoid Proteins

Abstract

The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.

A survey of the plant mitochondrial proteome in relation to development

“…1a), are very abundant proteins in the mitochondrial space since they represent around 3.4% and 1.8% of the total soluble proteins respectively. Like their chloroplastic counterparts [19], they are present in multiforms. The 2-D map in Fig.…”

“…The recent development of proteomics [14][15][16] opens the path toward a deeper exploration of mitochondrial functions through their protein complement. Previous work dealing with organelle proteome analysis has proven the usefulness of the approach, for instance in the case of human mitochondria [17], macrophage phagosome [18], thylakoid proteins of chloroplast [19] and chloroplast envelope membranes [20].…”

Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry

“…As an alternative approach, high-throughput MALDI based peptide mass fingerprinting combined with searching against EST databases has recently been employed in characterizing the expressed proteins in maize leaf, albeit at a relatively modest success rate [9]. Despite this slow start, there has been a noticeable increase in the number of plant proteomics publications in recent years, ranging from analysis of whole tissues and tissue fractions such as leaves [10], roots [11], and seed endosperm [12] to targeted studies involving characterization of organelles such as chloroplasts [13], golgi [14], mitochondria [15] and endoplasmic reticulum [14].…”

Analytical and biological variances associated with proteomic studies of Medicago truncatula by two-dimensional polyacrylamide gel electrophoresis

“…The resolving power of this separation technology has found great utility in proteomic studies of plants [6][7][8][9][10][11], animals [12][13][14] and microorganisms [15,16]. Comparative analyses of 2-DE images yield quantitative information concerning changes in protein expression levels that can be used to understand gene function or to correlate cellular responses to biotic or abiotic stimuli [17,18].…”