A Proteomic Strategy for Global Analysis of Plant Protein Complexes

Abstract

Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.

Cytonuclear interactions remain stable during allopolyploid evolution despite repeated whole‐genome duplications in Brassica

“…In particular, genes exhibiting the retention of three and two copies were more frequent, whereas genes with a single copy were less abundant in our data set, compared with genome‐wide retention rates (Chalhoub et al ., ). In order to determine whether the conservation of genes in duplicate or triplicate was a general trend for genes involved in protein complexes, we determined the number of copies retained for genes involved in cytosolic complexes (Aryal et al ., ). We were able to assign retention rates for 86 and 84 genes implicated in cytosolic complexes for B. rapa and B. oleracea , respectively (Table S3).…”

“…In Brassica , we tested whether this retention of redundant genes may be linked to their involvement in protein complexes and compared our data set including nuclear genes involved in plastid protein complexes with genes encoding cytosolic protein complexes (Aryal et al ., ). Surprisingly, we found that genes implicated in these latter complexes have a strong tendency towards a return to single copy status, and that the proportions of copy retention were identical to those found genome wide (Wang et al ., ).…”

“…For this purpose we retrieved 270 Arabidopsis genes implicated in cytosolic protein complexes from Aryal et al . (). Using identifiers, we recovered the amino‐acid sequences from TAIR.…”

Spatial Organization of Metabolic Enzyme Complexes in Cells

“…37–40 In isolated mitochondria from Arabidopsis , all 10 glycolytic enzymes were detected by in vitro enzymatic assays, thus indicating that glycolytic enzymes are concurrent with mitochondria. 39,41 The association of glycolytic enzymes to Arabidopsis mitochondria suggests the possibility of substrate channeling of metabolic intermediates between cytoplasmic glycolysis and mitochondria.…”

Global Landscape of Native Protein Complexes in Synechocystis sp. PCC 6803

“…After fractionation, samples were analyzed using LC/MS/MS. The value of Rapp, the ratio of Mapp (proteins apparent molecular mass, Figure S2) to Mmono (the predicted monomeric mass), was evaluated to estimate whether a protein involved a stable complex on SEC column [26], [27]. Rapp>=2 is used to classify proteins to be not oligomeric but within a complex, while Rapp≤0.5 suggests protein degradation during the protein extraction process (Figure 1B).…”