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Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) associates a chloroplast- and a nucleus-encoded subunit (LSU and SSU). It constitutes the major entry point of inorganic carbon into the biosphere as it catalyzes photosynthetic CO2 fixation. Its abundance and richness in sulfur-containing amino acids make it a prime source of N and S during nutrient starvation, when photosynthesis is downregulated and a high RuBisCO level is no longer needed. Here we show that translational attenuation of ClpP1 in the green alga Chlamydomonas reinhardtii results in retarded degradation of RuBisCO during S- and N-starvation, suggesting that the Clp protease is a major effector of RubisCO degradation in these conditions. Furthermore, we show that ClpP cannot be attenuated in the context of rbcL point mutations that prevent LSU folding. The mutant LSU remains in interaction with the chloroplast chaperonin complex. We propose that degradation of the mutant LSU by the Clp protease is necessary to prevent poisoning of the chaperonin. In the total absence of LSU, attenuation of ClpP leads to a dramatic stabilization of unassembled SSU, indicating that Clp is responsible for its degradation. In contrast, attenuation of ClpP in the absence of SSU does not lead to overaccumulation of LSU, whose translation is controlled by assembly. Altogether, these results point to RuBisCO degradation as one of the major house-keeping functions of the essential Clp protease. In addition, we show that non-assembled subunits of the ATP synthase are also stabilized when ClpP is attenuated. In the case of the atpA-FUD16 mutation, this can even allow the assembly of a small amount of CF1, which partially restores phototrophy.

The Plant Proteomics Database (PPDB; http://ppdb.tc.cornell.edu), launched in 2004, provides an integrated resource for experimentally identified proteins in Arabidopsis and maize (Zea mays). Internal BLAST alignments link maize and Arabidopsis information. Experimental identification is based on in-house mass spectrometry (MS) of cell type-specific proteomes (maize), or specific subcellular proteomes (e.g. chloroplasts, thylakoids, nucleoids) and total leaf proteome samples (maize and Arabidopsis). So far more than 5000 accessions both in maize and Arabidopsis have been identified. In addition, more than 80 published Arabidopsis proteome datasets from subcellular compartments or organs are stored in PPDB and linked to each locus. Using MS-derived information and literature, more than 1500 Arabidopsis proteins have a manually assigned subcellular location, with a strong emphasis on plastid proteins. Additional new features of PPDB include searchable posttranslational modifications and searchable experimental proteotypic peptides and spectral count information for each identified accession based on in-house experiments. Various search methods are provided to extract more than 40 data types for each accession and to extract accessions for different functional categories or curated subcellular localizations. Protein report pages for each accession provide comprehensive overviews, including predicted protein properties, with hyperlinks to the most relevant databases.

Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize (Zea mays) leaves are specialized to accommodate C4 photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression, and homeostasis functions are quantitatively distributed across BS and M chloroplasts. This yielded new insights into cellular specialization. The experimental analysis was based on high-accuracy mass spectrometry, protein quantification by spectral counting, and the first maize genome assembly. A bioinformatics workflow was developed to deal with gene models, protein families, and gene duplications related to the polyploidy of maize; this avoided overidentification of proteins and resulted in more accurate protein quantification. A total of 1,105 proteins were assigned as potential chloroplast proteins, annotated for function, and quantified. Nearly complete coverage of primary carbon, starch, and tetrapyrole metabolism, as well as excellent coverage for fatty acid synthesis, isoprenoid, sulfur, nitrogen, and amino acid metabolism, was obtained. This showed, for example, quantitative and qualitative cell type-specific specialization in starch biosynthesis, arginine synthesis, nitrogen assimilation, and initial steps in sulfur assimilation. An extensive overview of BS and M chloroplast protein expression and homeostasis machineries (more than 200 proteins) demonstrated qualitative and quantitative differences between M and BS chloroplasts and BS-enhanced levels of the specialized chaperones ClpB3 and HSP90 that suggest active remodeling of the BS proteome. The reconstructed pathways are presented as detailed flow diagrams including annotation, relative protein abundance, and cell-specific expression pattern. Protein annotation and identification data, and projection of matched peptides on the protein models, are available online through the Plant Proteome Database.