Chloroplast Isolation and Enrichment of Low-Abundance Proteins by Affinity Chromatography for Identification in Complex Proteomes.

Comprehensive knowledge of the proteome is a crucial prerequisite to understand dynamic changes in biological systems. Particularly low-abundance proteins are of high relevance in these processes as these are often proteins involved in signal transduction and acclimation responses. Although technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS), the dynamic range in protein abundance is still the most limiting problem for the detection of low-abundance proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of high-abundance proteins. Therefore, specific enrichment strategies are still required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundance proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.

Chloroplast isolation and affinity chromatography for enrichment of low-abundant proteins in complex proteomes.

Detailed knowledge of the proteome is crucial to advance the biological sciences. Low-abundant proteins are of particular interest to many biologists as they include, for example those proteins involved in signal transduction. Recent technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS). However, the dynamic range in protein abundance still forms a fundamental problem that limits the detection of low-abundant proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of other proteins at an abundance several-fold higher in order of magnitude. Therefore, specific enrichment strategies are required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundant proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.

Chloroplast-localized protein kinases: a step forward towards a complete inventory.

In addition to redox regulation, protein phosphorylation has gained increasing importance as a regulatory principle in chloroplasts in recent years. However, only very few chloroplast-localized protein kinases have been identified to date. Protein phosphorylation regulates important chloroplast processes such as photosynthesis or transcription. In order to better understand chloroplast function, it is therefore crucial to obtain a complete picture of the chloroplast kinome, which is currently constrained by two effects: first, recent observations showed that the bioinformatics-based prediction of chloroplast-localized protein kinases from available sequence data is strongly biased; and, secondly, protein kinases are of very low abundance, which makes their identification by proteomics approaches extremely difficult. Therefore, the aim of this study was to obtain a complete list of chloroplast-localized protein kinases from different species. Evaluation of protein kinases which were either highly predicted to be chloroplast localized or have been identified in different chloroplast proteomic studies resulted in the confirmation of only three new kinases. Considering also all reports of experimentally verified chloroplast protein kinases to date, compelling evidence was found for a total set of 15 chloroplast-localized protein kinases in different species. This is in contrast to a much higher number that would be expected based on targeting prediction or on the general abundance of protein kinases in relation to the entire proteome. Moreover, it is shown that unusual protein kinases with differing ATP-binding sites or catalytic centres seem to occur frequently within the chloroplast kinome, thus making their identification by mass spectrometry-based approaches even more difficult due to a different annotation.

Mining the soluble chloroplast proteome by affinity chromatography.

Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO(2), they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions.

Protein N-acylation overrides differing targeting signals.

In a bioinformatics based screen for chloroplast-localized protein kinases we noticed that available protein targeting predictors falsely predicted chloroplast localization. This seems to be due to interference with N-terminal protein acylation, which is of particular importance for protein kinases. Their N-myristoylation was found to be highly overrepresented in the proteome, whereas myristoylation motifs are almost absent in known chloroplast proteins. However, only abolishing their myristoylation was not sufficient to target those kinases to chloroplasts and resulted in nuclear accumulation instead. In contrast, inhibition of N-myristoylation of a calcium-dependent protein kinase was sufficient to alter its localization from the plasma membrane to chloroplasts and chloroplast localization of ferredoxin-NADP+ reductase and Rubisco activase could be efficiently suppressed by artificial introduction of myristoylation and palmitoylation sites.

The Ca(2+) -dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis.

Plants use different signalling pathways to respond to external stimuli. Intracellular signalling via calcium-dependent protein kinases (CDPKs) or mitogen-activated protein kinases (MAPKs) present two major pathways that are widely used to react to a changing environment. Both CDPK and MAPK pathways are known to be involved in the signalling of abiotic and biotic stresses in animal, yeast and plant cells. Here, we show the essential function of the CDPK CPK3 (At4g23650) for salt stress acclimation in Arabidopsis thaliana, and test crosstalk between CPK3 and the major salt-stress activated MAPKs MPK4 and MPK6 in the salt stress response. CPK3 kinase activity was induced by salt and other stresses after transient overexpression in Arabidopsis protoplasts, but endogenous CPK3 appeared to be constitutively active in roots and leaves in a strictly Ca(2+) -dependent manner. cpk3 mutants show a salt-sensitive phenotype comparable with mutants in MAPK pathways. In contrast to animal cells, where crosstalk between Ca(2+) and MAPK signalling is well established, CPK3 seems to act independently of those pathways. Salt-induced transcriptional induction of known salt stress-regulated and MAPK-dependent marker genes was not altered, whereas post-translational protein phosphorylation patterns from roots of wild type and cpk3 plants revealed clear differences. A significant portion of CPK3 was found to be associated with the plasma membrane and the vacuole, both depending on its N-terminal myristoylation. An initial proteomic study led to the identification of 28 potential CPK3 targets, predominantly membrane-associated proteins.