Approximate calculation and experimental derivation of native isoelectric points of membrane protein complexes of Arabidopsis chloroplasts and mitochondria.

Electric charges are important intrinsic properties of proteins. They directly affect functionality and also mediate interactions with other molecules such as cofactors, substrates and regulators of enzymatic activity, with lipids as well as other proteins. As such, analysis of the electric properties of proteins gives rise to improved understanding of the mechanism by which proteins fulfil their specific functions. This is not only true for singular proteins but also applies for defined assemblies of proteins, protein complexes and supercomplexes. Charges in proteins often are a consequence of the presence of basic and acidic amino acid residues within polypeptide chains. In liquid phase, charge distributions of proteins change in response to the pH of their environment. The interdependence of protein charge and the surrounding pH is best described by the isoelectric point, which is notoriously difficult to obtain for native protein complexes. Here, experimentally derived native isoelectric points (npIs) for a range mitochondrial and plastid protein complexes are provided. In addition, for four complexes, npIs were calculated by a novel approach which yields results largely matching the experimental npIs.

Physiological and proteomic characterization of manganese sensitivity and tolerance in rice (Oryza sativa) in comparison with barley (Hordeum vulgare).

BACKGROUND AND AIMS: Research on manganese (Mn) toxicity and tolerance indicates that Mn toxicity develops apoplastically through increased peroxidase activities mediated by phenolics and Mn, and Mn tolerance could be conferred by sequestration of Mn in inert cell compartments. This comparative study focuses on Mn-sensitive barley (Hordeum vulgare) and Mn-tolerant rice (Oryza sativa) as model organisms to unravel the mechanisms of Mn toxicity and/or tolerance in monocots. METHODS: Bulk leaf Mn concentrations as well as peroxidase activities and protein concentrations were analysed in apoplastic washing fluid (AWF) in both species. In rice, Mn distribution between leaf compartments and the leaf proteome using 2D isoelectric focusing IEF/SDS-PAGE and 2D Blue native BN/SDS-PAGE was studied. KEY RESULTS: The Mn sensitivity of barley was confirmed since the formation of brown spots on older leaves was induced by low bulk leaf and AWF Mn concentrations and exhibited strongly enhanced H2O2-producing and consuming peroxidase activities. In contrast, by a factor of 50, higher Mn concentrations did not produce Mn toxicity symptoms on older leaves in rice. Peroxidase activities, lower by a factor of about 100 in the rice leaf AWF compared with barley, support the view of a central role for these peroxidases in the apoplastic expression of Mn toxicity. The high Mn tolerance of old rice leaves could be related to a high Mn binding capacity of the cell walls. Proteomic studies suggest that the lower Mn tolerance of young rice leaves could be related to Mn excess-induced displacement of Mg and Fe from essential metabolic functions. CONCLUSIONS: The results provide evidence that Mn toxicity in barley involves apoplastic lesions mediated by peroxidases. The high Mn tolerance of old leaves of rice involves a high Mn binding capacity of the cell walls, whereas Mn toxicity in less Mn-tolerant young leaves is related to Mn-induced Mg and Fe deficiencies.

The Mitochondrial Complexome of Medicago truncatula.

Legumes (Fabaceae, Leguminosae) are unique in their ability to carry out an elaborate endosymbiotic nitrogen fixation process with rhizobia proteobacteria. The symbiotic nitrogen fixation enables the host plants to grow almost independently of any other nitrogen source. Establishment of symbiosis requires adaptations of the host cellular metabolism, here foremost of the energy metabolism mainly taking place in mitochondria. Since the early 1990s, the galegoid legume Medicago truncatula Gaertn. is a well-established model for studying legume biology, but little is known about the protein complement of mitochondria from this species. An initial characterization of the mitochondrial proteome of M. truncatula (Jemalong A17) was published recently. In the frame of this study, mitochondrial protein complexes were characterized using Two-dimensional (2D) Blue native (BN)/SDS-PAGE. From 139 detected spots, the "first hit" (=most abundant) proteins of 59 spots were identified by mass spectrometry. Here, we present a comprehensive analysis of the mitochondrial "complexome" (the "protein complex proteome") of M. truncatula via 2D BN/SDS-PAGE in combination with highly sensitive MS protein identification. In total, 1,485 proteins were identified within 158 gel spots, representing 467 unique proteins. Data evaluation by the novel GelMap annotation tool allowed recognition of protein complexes of low abundance. Overall, at least 36 mitochondrial protein complexes were found. To our knowledge several of these complexes were described for the first time in Medicago. The data set is accessible under http://www.gelmap.de/medicago/. The mitochondrial protein complex proteomes of Arabidopsis available at http://www.gelmap.de/arabidopsis/ and Medicago are compared.

Arabidopsis peroxisome proteomics.

The analytical depth of investigation of the peroxisomal proteome of the model plant Arabidopsis thaliana has not yet reached that of other major cellular organelles such as chloroplasts or mitochondria. This is primarily due to the difficulties associated with isolating and obtaining purified samples of peroxisomes from Arabidopsis. So far only a handful of research groups have been successful in obtaining such fractions. To make things worse, enriched peroxisome fractions frequently suffer from significant organellar contamination, lowering confidence in localization assignment of the identified proteins. As with other cellular compartments, identification of peroxisomal proteins forms the basis for investigations of the dynamics of the peroxisomal proteome. It is therefore not surprising that, in terms of functional analyses by proteomic means, peroxisomes are lagging considerably behind chloroplasts or mitochondria. Alternative strategies are needed to overcome the obstacle of hard-to-obtain organellar fractions. This will help to close the knowledge gap between peroxisomes and other organelles and provide a full picture of the physiological pathways shared between organelles. In this review, we briefly summarize the status quo and discuss some of the methodological alternatives to classic organelle proteomic approaches.

The 'protein complex proteome' of chloroplasts in Arabidopsis thaliana.

Here, a first GelMap of the chloroplast "protein complex proteome" of Arabidopsis thaliana is presented. The GelMap software tool allows assigning multiple proteins to gel spots, thereby taking advantage of the high sensitivity of state-of-the-art mass spectrometry systems. Furthermore, the software allows functional annotation of all identified proteins. If applied to a 2D blue native (BN)/SDS gel, GelMap can selectively display protein complexes of low abundance. For the chloroplast GelMap, highly purified organelles were separated by 2D BN/SDS PAGE and spots were automatically detected using Delta 2D software. Within 287 spots, a total of 1841 proteins were identified (on average 6.4 proteins per spot), representing a set of 436 non redundant proteins. Most of these proteins form part of protein complexes. The quality of the map is reflected by its inclusion of a more or less complete set of protein complexes described for chloroplasts in the literature. The GelMap is publically available at www.gelmap.de/arabidopsis-chloro and may be used as a resource for identifying novel protein complexes within any of its functional categories. BIOLOGICAL SIGNIFICANCE: The chloroplast GelMap represents a data resource for the definition of protein complexes in the model plant A. thaliana. It should be useful for in depth understanding of chloroplast biochemistry, as illustrated by the discovery of so far unknown protein complexes. The GelMap is publically available at www.gelmap.de/arabidopsis-chloro.

A possible role for the chloroplast pyruvate dehydrogenase complex in plant glycolate and glyoxylate metabolism.

Glyoxylate is a peroxisomal intermediate of photorespiration, the recycling pathway for 2-phosphoglycolate (2-PG) produced by the oxygenase activity of Rubisco. Under hot and dry growth conditions, photorespiratory intermediates can accumulate and must be detoxified by alternative pathways, including plastidal reactions. Moreover, there is evidence that chloroplasts are capable of actively producing glyoxylate from glycolate. Further metabolic steps are unknown, but probably include a CO2 release step. Here, we report that CO2 production from glycolate and glyoxylate in isolated tobacco chloroplasts can be inhibited by pyruvate, but not related compounds. We isolated a protein fraction that was enriched for the chloroplast pyruvate dehydrogenase complex (PDC). The fraction contained a protein complex of several MDa in size that included all predicted subunits of the chloroplast PDC and a so far unidentified HSP93-V/ClpC1 heat shock protein. Glyoxylate competitively inhibited NADH formation from pyruvate in this fraction. The Km for pyruvate and the Ki for glyoxylate were 330 and 270 µM, respectively. Glyoxylate decarboxylation was also enriched in this fraction and could be in turn inhibited by pyruvate. Based on these data, we suggest that the chloroplast PDC might be part of a pathway for glycolate and/or glyoxylate oxidation in chloroplasts.