Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

Liberton, Michelle; Saha, Rajib; Jacobs, Jon; Nguyen, Amelia Y.; Gritsenko, Marina; Smith, Richard; Koppenaal, David W.; Pakrasi, Himadri B.

doi:10.1074/mcp.m115.057240

Cited by 32 publications

(46 citation statements)

References 38 publications

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“…8A). Slr1796 has been detected as a component of the thylakoid membrane in proteomics studies of isolated membrane fractions (29,30), and our result is consistent with these studies. Considering the presence of a transmembrane fragment in Slr1796, as predicted by TMHMM, it is likely an integral thylakoid protein.…”

Section: Resultssupporting

confidence: 82%

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A Novel Redoxin in the Thylakoid Membrane Regulates the Titer of Photosystem I

Zhu

Liberton

Pakrasi

2016

Journal of Biological Chemistry

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In photosynthetic organisms like cyanobacteria and plants, the main engines of oxygenic photosynthesis are the pigmentprotein complexes photosystem I (PSI) and photosystem II (PSII) located in the thylakoid membrane. In the cyanobacterium Synechocystis sp. PCC 6803, the slr1796 gene encodes a single cysteine thioredoxin-like protein, orthologs of which are found in multiple cyanobacterial strains as well as chloroplasts of higher plants. Targeted inactivation of slr1796 in Synechocystis 6803 resulted in compromised photoautotrophic growth. The mutant displayed decreased chlorophyll a content. These changes correlated with a decrease in the PSI titer of the mutant cells, whereas the PSII content was unaffected. In the mutant, the transcript levels of genes for PSI structural and accessory proteins remained unaffected, whereas the levels of PSI structural proteins were severely diminished, indicating that Slr1796 acts at a posttranscriptional level. Biochemical analysis indicated that Slr1796 is an integral thylakoid membrane protein.We conclude that Slr1796 is a novel regulatory factor that modulates PSI titer.Oxygenic photosynthesis is performed by pigment-protein complexes located in the thylakoid membranes of cyanobacteria and chloroplasts. This process generates atmospheric oxygen and chemical energy in the form of carbohydrates and is thus the ultimate source of food, feed, and fuel on the planet. Photosystem II (PSII) 2 performs light-driven oxidation of water (generating a strong oxidant, P680 ϩ ) and initiates the electron flow to photosystem I (PSI) via the cytochrome b 6 f complex during linear electron flow, whereas PSI catalyzes the last step, the oxidation of plastocyanin in the thylakoid lumen and reduction of ferredoxin to generate ATP and NADPH (a strong reductant) for CO 2 fixation. Thus, oxygenic photosynthesis operates in both highly oxidizing and reducing redox environments, conditions that inevitably lead to the production of reactive oxygen species.In photosynthetic organisms, a number of proteins are present that are involved in redox activity (1, 2). These include thioredoxins (Trxs), small proteins that can regulate enzyme activity by acting as reversible redox switches. Trxs belong to the thioredoxin superfamily of proteins, which contain a common thioredoxin fold with a characteristic two-cysteine CXXC active site motif (where X is any amino acid). The CXXC motifs have different variants, including Trx folds with only a single cysteine, which are comparatively less well studied (3). Redox regulation by Trx fold proteins via their oxidation, reduction, or disulfide exchange activities, depending on their redox environments, is an extensive system in almost all life forms, including oxygenic photosynthetic organisms.PSI is among the largest and most complex multisubunit pigment-protein structures in nature (4). Most cyanobacterial PSI functions as a trimer with each monomer containing 12 subunits (PsaA-F, PsaI-M, and PsaX) and 127 cofactors, including 96 chlorophylls, 22 ␤-carotene pigmen...

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Section: Resultssupporting

confidence: 82%

“…Disruption of slr1796 Leads to Higher PSII/PSI Ratio-The protein Slr1796 was identified as a thylakoid membrane component by proteomics studies (29,30), but the role of the protein remained unknown. The Slr1796 protein has three cysteine residues (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Novel Redoxin in the Thylakoid Membrane Regulates the Titer of Photosystem I

Zhu

Liberton

Pakrasi

2016

Journal of Biological Chemistry

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“…NdbC was clearly located only in the PM, while no signal was detected from the thylakoid membrane fraction or from the soluble proteins (Fig. 5C), in line with results obtained by proteomic studies (Pisareva et al, 2011;Liberton et al, 2016).…”

Section: Western-blot Analysis Of Selected Proteins Provided Further supporting

confidence: 77%

“…Thus, NdbC is involved in the accumulation of NDH-1 4 , but not the other way around. Most probably, NdbC affects NDH-1 4 indirectly, since they are located in different membrane compartments (Liberton et al, 2016).…”

Section: The Deletion Of Ndbc Alters the Expression Of Multiple Transmentioning

confidence: 99%

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis

et al. 2017

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H dehydrogenases comprise type 1 (NDH-1) and type 2 (NDH-2s) enzymes. Even though the NDH-1 complex is a wellcharacterized protein complex in the thylakoid membrane of Synechocystis sp. PCC 6803 (hereafter Synechocystis), the exact roles of different NDH-2s remain poorly understood. To elucidate this question, we studied the function of NdbC, one of the three NDH-2s in Synechocystis, by constructing a deletion mutant (DndbC) for a corresponding protein and submitting the mutant to physiological and biochemical characterization as well as to comprehensive proteomics analysis. We demonstrate that the deletion of NdbC, localized to the plasma membrane, affects several metabolic pathways in Synechocystis in autotrophic growth conditions without prominent effects on photosynthesis. Foremost, the deletion of NdbC leads, directly or indirectly, to compromised sugar catabolism, to glycogen accumulation, and to distorted cell division. Deficiencies in several sugar catabolic routes were supported by severe retardation of growth of the DndbC mutant under light-activated heterotrophic growth conditions but not under mixotrophy. Thus, NdbC has a significant function in regulating carbon allocation between storage and the biosynthesis pathways. In addition, the deletion of NdbC increases the amount of cyclic electron transfer, possibly via the NDH-1 2 complex, and decreases the expression of several transporters in ambient CO 2 growth conditions.

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“…We used recent proteomics (34) and fluorescence microscopy data (35) to reconcile membrane localization of electron transport complexes that previous cyanobacterial models had included inaccurately in the cytoplasmic membrane. Additionally, the ferredoxin:plastoquinone oxidoreductase complex gene associations were updated to include additional subunits and the use of ferredoxin as the electron donor (36,37).…”

Section: Resultsmentioning

confidence: 99%

Unique attributes of cyanobacterial metabolism revealed by improved genome-scale metabolic modeling and essential gene analysis

Broddrick

Rubin

Welkie

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

117

118

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The model cyanobacterium, Synechococcus elongatus PCC 7942, is a genetically tractable obligate phototroph that is being developed for the bioproduction of high-value chemicals. Genome-scale models (GEMs) have been successfully used to assess and engineer cellular metabolism; however, GEMs of phototrophic metabolism have been limited by the lack of experimental datasets for model validation and the challenges of incorporating photon uptake. Here, we develop a GEM of metabolism in S. elongatus using random barcode transposon site sequencing (RB-TnSeq) essential gene and physiological data specific to photoautotrophic metabolism. The model explicitly describes photon absorption and accounts for shading, resulting in the characteristic linear growth curve of photoautotrophs. GEM predictions of gene essentiality were compared with data obtained from recent dense-transposon mutagenesis experiments. This dataset allowed major improvements to the accuracy of the model. Furthermore, discrepancies between GEM predictions and the in vivo dataset revealed biological characteristics, such as the importance of a truncated, linear TCA pathway, low flux toward amino acid synthesis from photorespiration, and knowledge gaps within nucleotide metabolism. Coupling of strong experimental support and photoautotrophic modeling methods thus resulted in a highly accurate model of S. elongatus metabolism that highlights previously unknown areas of S. elongatus biology.cyanobacteria | constraint-based modeling | TCA cycle | photosynthesis | Synechococcus elongatus T he unicellular cyanobacterium Synechococcus elongatus PCC 7942 is being developed as a photosynthetic bioproduction platform for an array of industrial products (1-3). This model strain is attractive for this purpose because of its genetic tractability (4) and its reliance on mainly CO 2 , H 2 O, and light for metabolism, reducing the environmental and economic costs of cultivation. For low-cost, high-volume products, such as biofuels, however, one of the biggest challenges is attaining profitable product yields (5, 6). Genome-scale models (GEMs) of metabolism provide a valuable tool for increasing product titers by optimizing yield in silico and then, reproducing the changes in vivo (7). For instance, GEMs were used to select the optimal synthetic pathway for 3-hydroxypropanoate biosynthesis in Saccharomyces cerevisiae (8). In Escherichia coli, GEM optimization was used to realize heterologous production of 1,4-butanediol synthesis and increase titers three orders of magnitude (9). Although there have been numerous modeling efforts in Synechocystis sp. PCC 6803 (here in referred to as PCC 6803), this organism is highly divergent from S. elongatus, where limited modeling has been done (10).This deficit can partially be explained by the lack of in vivo validation datasets, such as 13 C metabolic flux analysis (MFA), for obligate phototrophs (11). Development of metabolic models of S. elongatus with strong experimental support is necessary to exploit the organism as a bioproduc...

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Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

Cited by 32 publications

References 38 publications

A Novel Redoxin in the Thylakoid Membrane Regulates the Titer of Photosystem I

A Novel Redoxin in the Thylakoid Membrane Regulates the Titer of Photosystem I

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis

Unique attributes of cyanobacterial metabolism revealed by improved genome-scale metabolic modeling and essential gene analysis

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