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. 2018 Nov 2;17(11):3628-3643.
doi: 10.1021/acs.jproteome.8b00170. Epub 2018 Oct 3.

Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142

Free PMC article

Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142

Uma K Aryal et al. J Proteome Res. .
Free PMC article

Abstract

The unicellular cyanobacterium Cyanothece ATCC 51142 is capable of oxygenic photosynthesis and biological N2 fixation (BNF), a process highly sensitive to oxygen. Previous work has focused on determining protein expression levels under different growth conditions. A major gap of our knowledge is an understanding on how these expressed proteins are assembled into complexes and organized into metabolic pathways, an area that has not been thoroughly investigated. Here, we combined size-exclusion chromatography (SEC) with label-free quantitative mass spectrometry (MS) and bioinformatics to characterize many protein complexes from Cyanothece 51142 cells grown under a 12 h light-dark cycle. We identified 1386 proteins in duplicate biological replicates, and 64% of those proteins were identified as putative complexes. Pairwise computational prediction of protein-protein interaction (PPI) identified 74 822 putative interactions, of which 2337 interactions were highly correlated with published protein coexpressions. Many sequential glycolytic and TCA cycle enzymes were identified as putative complexes. We also identified many membrane complexes that contain cytoplasmic domains. Subunits of NDH-1 complex eluted in a fraction with an approximate mass of ∼669 kDa, and subunits composition revealed coexistence of distinct forms of NDH-1 complex subunits responsible for respiration, electron flow, and CO2 uptake. The complex form of the phycocyanin beta subunit was nonphosphorylated, and the monomer form was phosphorylated at Ser20, suggesting phosphorylation-dependent deoligomerization of the phycocyanin beta subunit. This study provides an analytical platform for future studies to reveal how these complexes assemble and disassemble as a function of diurnal and circadian rhythms.

Keywords: Cyanothece 51142; mass spectrometry; protein complexes; protein−protein interaction prediction; proteomics; size exclusion chromatography.

Conflict of interest statement

Conflict of Interest:

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Experimental workflow. (A) Proteins extracted under native condition were fractionated by SEC, and analyzed by Q Exactive Orbitrap HF mass spectrometer. Data were analyzed using MaxQuant (–35) for protein identification and label free MS1 quantitation. Peak elution fraction of each identified protein, Mapp, and Rapp were determined as described previously (19). Mapp, apparent molecular mass; Mmono, predicted molecular mass of monomer. Rapp, the ratio of the Mapp to the Mmono (Mapp / Mmono). Proteins with an Rapp ≥ 2 in both the replicate were considered to be in a complex.
Figure 2.
Figure 2.
LC-MS reproducibility. (A) Venn diagram showing the overlap of proteins identified between two biological replicates. (B) Heat map showing the Pearson’s correlation coefficients (PCC) of protein abundances (MS1 intensity) across SEC fractions. The correlations coefficients were calculated using Data Analysis and Extension Tool (DAnTE) (36). (C) Box plot showing the median distribution of protein intensities. (D) Shift in peak elution fraction of proteins in two SEC separations. ~90% proteins were identified within 0–2 fractions shift indicating good SEC reproducibility.
Figure 3.
Figure 3.
Determination of protein oligomerization states. (A) Hierarchical clustering of protein elution profiles. Proteins were clustered using Euclidean distance and average linkage hierarchical clustering method. In this plot, each row represents a protein and each column represents the index of protein elution fraction. Numbers on the top show molecular masses of protein standards, and the peak elution fraction for each of the standard was used to determine the Mapp of proteins. (B), Histogram showing the distribution of the monomeric (blue) and experimentally determined apparent masses (green and red) of proteins that were identified in both the biological replicates. (C), Scatter plots showing Rapp distribution of proteins between the two biological replicates. Each circle represents Rapp values for Bio1 and Bio 2. Circles along the black solid line represent proteins without any fraction shift in elution peak (same Rapp values) in both the replicates. Circles along the black dotted lines represent proteins with 1 fraction shift and circles along the blue dotted lines represent proteins with 2 fraction shifts between the replicates. Bio1; biological replicate 1, Bio2; biological replicate 2.
Figure 4.
Figure 4.
Elution profiles of phycobilisomes (PBS) and other complexes. (A, B), Elution profiles of phycocyanin (Cpc) and allophycocyanin (Apc) subunits. Elution profiles varied among the individual polypeptide. (C), Elution profiles of Rubisco large (RbcL) and small (RbcS) subunits. Both RbcL and RbcS peaked at fraction 12 with calculated Mapp of 105 kDa. (D), Elution profiles of CO2 concentrating mechanism (Ccm) proteins. CcmM showed major elution peak as a complex while others showed major peaks as monomers.
Figure 5.
Figure 5.
NDH-1 complex. (A-D), Elution profiles and structure of multiple forms of NDH-1 complex subunits. All the subunits eluted in high molecular weight (669 kDa) fraction. The existence of NDH-1L (respiratory), and NDH-1MS and NDH-1MS’ (CO2 uptake) forms of NDH-1 complexes were determined by comparing SEC co-elution profiles and known functional and structural multiplicity in the literature (64, 65). Hydrophilic domain subunits showed higher abundance than the membrane domain subunits. We identified both hydrophilic (I, J, K, H) and hydrophobic domain subunits (A, B, C, D1, F1, D3, F3, D4) as well as Oxygenic-Photosynthesis-Specific (OPS)-domain subunits (O, M, N). Results show the existence of functional multiplicity of NDH-1 complexes in Cyanothece 51142 cells that are responsible for a variety of functions including respiration, cyclic electron flow and CO2 uptake.
Figure 6.
Figure 6.
The plot of Euclidean distance of protein elution profiles vs. Pearson’s correlation coefficient of the mRNA-level co-expression information (14). The dots are colored based on the number of common GO terms. Grey indicates no common GO terms. Blue to black color indicates the number of common GO terms is from 1 to 10.
Figure 7.
Figure 7.
(A). Elution profiles of phycocyanin α and β subunits as a complex (blue) and as a monomer (orange). Proteins eluting as a complex were non-phosphorylated & proteins eluting as a monomer were phosphorylated. (B). MS/MS spectra showing the phosphorylated peptide mapped to β subunit. (C), Structure of α and β subunits showing the phosphorylated S20 site. Results indicate phosphorylation dependent de-oligomerization of phycocyanin.
Figure 8.
Figure 8.
(A) Profiles of glycolytic enzymes organized to reflect their order in the pathway. (B) Biochemical pathways and enzymes involved in carbon metabolism. Pathways were generated by mapping proteins onto known pathways. Each arrow indicates the direction of the reaction. Symbols in red indicate proteins identified as putative complexes (Rapp ≥ 2) and in blue as monomers (Rapp ≤ 1). The numbers in parenthesis correspond to the Rapp value. GlgP1; glycogen phosphorylase (cce_1269), Zwf; glucose 6-phosphate dehydrogenase (cce_2536); Pgi; glucose-6-phosphate isomerase (Pgi1; cce_0666, Pgi2; cce_5178), PfkA1; 6-phosphofructokinase (cce_0669), Fda; fructose-bisphosphate aldolase class I (cce_4254), Gap; glyceraldehyde-3-phosphate dehydrogenase (cce_3612), Pgk; phosphoglycerate kinase (cce_4219), Gpm; phosphoglycerate mutase (GpmA; cce_1542, GpmB; cce_2454), Eno; enolase (Eno1; cce_2156, Eno2; cce_5179), Ppc; phosphoenolpyruvate carboxylase (cce_3822), Gnd; 6-phosphogluconate dehydrogenase (cce_3746), RpiA; ribose 5-phosphate isomerase (cce_0103), Rpe; ribulose-phosphate 3-epimerase (cce_0798), TalA; transaldolase (cce_4687), TktA; transketolase (cce_4627), Pkt; phosphoketolase (cce_2225), GltA; citrate synthase (cce_1900), AcnB; aconitate hydratase 2 (cce_3280), Icd; isocitrate dehydrogenase (cce_3202), GabD; succinate-semialdehyde dehydrogenase (cce_4228), SucC; succi nyl-CoA synthetase (cce_2357), SdhB; succinate dehydrogenase iron-sulfur protein subunit (cce_3244), FumC; fumarate hydratase (cce_0396), Mdh; malate dehydrogenase (cce_1850). G6P; glucose-6-phosphate; F6P; fructose-6-phosphate, F1,6P; fructose 1,6-bisphosphate, Gap; glyceraldehyde-3-phosphate, 3PGA; 3-phosphoglycerate’ 2PGA; 2-phosphoglycerate, PEP; phosphoenolpyruvate.

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