Identification of a 350-kDa ClpP protease complex with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana.

A 350-kDa ClpP protease complex with 10 different subunits was identified in chloroplast of Arabidopsis thaliana, using Blue-Native gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight and nano-electrospray tandem mass spectrometry. The complex was copurified with the thylakoid membranes, and all identified Clp subunits show chloroplast targeting signals, supporting that this complex is indeed localized in the chloroplast. The complex contains chloroplast-encoded pClpP and six nuclear-encoded proteins nCpP1-6, as well as two unassigned Clp homologues (nClpP7, nClpP8). An additional Clp protein was identified in this complex; it does not belong to any of the known Clp genes families and is here assigned ClpS1. Expression and accumulation of several of these Clp proteins have never been shown earlier. Sequence and phylogenetic tree analysis suggests that nClpP5, nClpP2, and nClpP8 are not catalytically active and form a new group of Clp higher plant proteins, orthologous to the cyanobacterial ClpR protein, and are renamed ClpR1, -2, and -3, respectively. We speculate that ClpR1, -2, and -3 are part of the heptameric rings, whereas ClpS1 is a regulatory subunit positioned at the axial opening of the ClpP/R core. Several truncations and errors in intron and exon prediction of the annotated Clp genes were corrected using mass spectrometry data and by matching genomic sequences with cDNA sequences. This strategy will be widely applicable for the much needed verification of protein prediction from genomic sequence. The extreme complexity of the chloroplast Clp complex is discussed.

Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins.

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.

Construction of a directory of tobacco plasma membrane proteins by combined two-dimensional gel electrophoresis and protein sequencing.

The polypeptide pattern of the plasma membrane from tobacco was studied by two-dimensional gel electrophoresis. When using classical carrier ampholyte isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) approximately 400 polypeptide spots were detected after silver staining and computer analysis using the QUEST software. This resolution was sufficient to assess physiological effects such as changes in a phytohormone concentration. By using pH 4-8 immobilized pH gradient (IPG)-IEF and 10%T SDS-PAGE gels, approximately 600 polypeptides, corresponding to ca. 80% of the total population expected, were resolved. This cross-section of the plasma membrane polypeptide population was mainly constituted by low or intermediate molecular mass (25 to 45 kDa) and acidic (5.2 < pI < 6.1) polypeptides. After sample application by in-gel rehydration, large amounts of plasma membrane protein (between 5 mg and 10 mg protein) were analyzed using IPG-IEF, and N-terminal protein sequencing was performed for polypeptides collected from one gel. Internal protein sequences were also obtained. Nearly all protein sequences corresponded to unidentified proteins but several of them matched translated sequences from unidentified plant expressed sequence tags (ESTs). It is concluded that the combined use of IPG-IEF gels and in-gel rehydration allows, in the case of plant membrane protein, both analytical and micropreparative separations with an efficiency comparable to that demonstrated for soluble proteins. Finally, it is suggested that a systematic investigation of plant plasma membrane polypeptides is feasible and would constitute a source of new and plant-specific genes.

Identification of 14 kDa auxin-binding proteins in a tobacco plasma membrane subfraction responsive to auxin.

Auxin binding by tobacco plasma membrane proteins was investigated. After photolabeling with [3H]IAA, 350 polypeptides were resolved on 2D gels and analyzed. Thirteen polypeptides were selected according to physico-chemical criteria. The labeling of three of them was further shown to increase, after treatment of cells with auxin, specifically in that plasma membrane subfraction where the sensitivity to the hormone of the H(+)-ATPase is enhanced by the treatment of cells. These polypeptides were those that exhibited the more specific labeling features according to physico-chemical criteria. They had similar apparent molecular weight (ca 14 kDa) that distinguished them from other auxin-binding proteins described up to now, and exhibited similar amino acid compositions. These 14 kDa polypeptides are proposed to constitute a group of new auxin-binding proteins, potentially involved, within specialized plasma membrane domains, in the stimulation of the proton pump by the hormone.

Auxin-induced differential sensitivity of the H+-ATPase in plasma membrane subfractions from tobacco cells.

Plasma membrane vesicles form suspension cultured tobacco cells were fractionated by counter-current phase partitioning. The subfractions exhibited identical biochemical characteristics and differed mainly by their sidedness. In addition, fluorescence polarization experiments indicated slight fluidity or order differences. These characteristics remained unchanged when cells were treated with auxin, prior to membrane purification. The sensitivity to auxin of the H+-ATPase catalysed proton translocation was investigated in plasma membrane subfractions. Identical sensitivity was measured in all subfractions from control cells. After auxin treatment of cells, a dramatic increase in the sensitivity was observed in one subfraction. Results are discussed in relation to the recent report of an auxin-induced clustering of the putative auxin-receptor (Diekmann et al., Proc. Natl. Acd. Sci USA 92, 3425-3429, 1995).