Monitoring light/dark association dynamics of multi-protein complexes in cyanobacteria using size exclusion chromatography-based proteomics

Guerreiro, Ana C. L.; Penning, Renske; Raaijmakers, Linsey M.; Axman, Ilka M.; Heck, Albert J. R.; Altelaar, Maarten

doi:10.1016/j.jprot.2016.04.030

Cited by 13 publications

(14 citation statements)

References 54 publications

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“…In S. elongatus, diurnal fluctuations appear to be less pronounced at the protein level than at the transcript levels (Guerreiro et al, 2014). However, important differences between light and dark conditions affect mega Dalton protein assemblies (Guerreiro et al, 2016), supporting the importance of protein complexes and protein interaction networks in cyclic regulation of cellular processes.…”

Section: Introductionmentioning

confidence: 96%

Energy drives the dynamic localization of cyanobacterial nitrogen regulators during diurnal cycles

Espinosa

Labella

Cantos

et al. 2018

Environmental Microbiology

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Cyanobacteria, phototrophic organisms performing oxygenic photosynthesis, must adapt their metabolic processes to the challenges imposed by the succession of days and nights. Two conserved cyanobacterial proteins, PII and PipX, function as hubs of the nitrogen interaction network, forming complexes with a variety of diverse targets. While PII proteins are found in all three domains of life as integrators of signals of the nitrogen and carbon balance, PipX proteins are unique to cyanobacteria, where they provide a mechanistic link between PII signalling and the control of gene expression by the global nitrogen regulator NtcA. Here we demonstrate that PII and PipX display distinct localization patterns during diurnal cycles, co-localizing into the same foci at the periphery and poles of the cells during dark periods, a circadian-independent process requiring a low ATP/ADP ratio. Genetic, cellular biology and biochemical approaches used here provide new insights into the nitrogen regulatory network, calling attention to the roles of PII as energy sensors and its interactions with PipX in the context of essential signalling pathways. This study expands the contribution of the nitrogen regulators PII and PipX to integrate and transduce key environmental signals that allow cyanobacteria to thrive in our planet.

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Section: Introductionmentioning

confidence: 96%

Energy drives the dynamic localization of cyanobacterial nitrogen regulators during diurnal cycles

Espinosa

Labella

Cantos

et al. 2018

Environmental Microbiology

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“…An alternative size-based fractionation of native proteins via an SEC column combined with the high-resolution LC-MS/MS has been recently introduced (27). SEC combined with LC-MS was applied to a non-N 2 -fixing cyanobacterium Synechococcus elongates PCC 7942 (28), Arabidopsis cytosol (19, 29) and chloroplast (30) as well as human cell lysates (31, 32). In this study, we combined size fractionation of native proteins using Superdex 6 column with label-free LC-MS profiling and bioinformatic analysis to identify subunits of protein complexes in Cyanothece 51142.…”

Section: Introductionmentioning

confidence: 99%

Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142

et al. 2018

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The unicellular cyanobacterium Cyanothece ATCC 51142 is capable of oxygenic photosynthesis and biological N 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 CO 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.

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“…The stability of known complexes involving PipX and the relative abundance of the proteins implicated (Llacer et al ., ; Forcada‐Nadal et al ., ; Guerreiro et al ., ; Labella et al ., ) provide support for the idea that once PII–PipX and NtcA–PipX complexes are formed, PipX would be trapped. Fluctuation of C/N and energy signals would drive partner swapping, changing the relative proportion of PII–PipX and NtcA–PipX (Espinosa et al ., , ), but still preventing PipX from making interactions with additional partners.…”

Section: Resultsmentioning

confidence: 99%

“…Fluctuation of C/N and energy signals would drive partner swapping, changing the relative proportion of PII–PipX and NtcA–PipX (Espinosa et al ., , ), but still preventing PipX from making interactions with additional partners. Additional support for this idea comes from the finding that PII, which appears to be 14 times more abundant than PipX (Guerreiro et al ., ; Labella et al ., ) prevents PipX toxicity in S. elongatus (Espinosa et al ., , ; Chang et al ., ), a phenomenon further suggesting that PII binding is counteracting the interaction of PipX with yet unidentified low affinity partners.…”

Section: Resultsmentioning

confidence: 99%

“…PII-PipX complexes interact with the transcriptional regulator PlmA, presumably to decrease transcriptional activity (Labella et al, 2016). When considering the affinities between PipX and partners NtcA (Kd of 85 nM) (Forcada-Nadal et al, 2014) and PII (Kd of 7 μM in the absence of 2-OG and nucleotides) (Llacer et al, 2010) and the relative abundances of the individual proteins in S. elongatus (in molar terms of subunits PII is 14 times more abundant than PipX which is four times more abundant than NtcA) (Guerreiro et al, 2016;Labella et al, 2016), it appears that, under a wide range of conditions, most of the PipX protein would be forming part of proteins complexes with PII and/or NtcA.…”

Section: Introductionmentioning

confidence: 99%

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The nitrogen regulator PipX acts in cis to prevent operon polarity

Cantos

Labella

Espinosa

et al. 2018

Environ Microbiol Rep

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Cyanobacteria, phototrophic organisms performing oxygenic photosynthesis, must adapt their metabolic processes to important environmental challenges, like those imposed by the succession of days and nights. Not surprisingly, certain regulatory proteins are found exclusively in this phylum. One of these unique factors, PipX, provides a mechanistic link between signals of carbon/nitrogen and of energy, transduced by the signalling protein PII, and the control of gene expression by the global nitrogen regulator NtcA. Here we report a new regulatory function of PipX: enhancement in cis of pipY expression, a gene encoding a universally conserved protein involved in amino/keto acid and Pyridoxal phosphate homeostasis. In Synechococcus elongatus and many other cyanobacteria these genes are expressed as a bicistronic pipXY operon. Despite being cis-acting, polarity suppression by PipX is nevertheless reminiscent of the function of NusG paralogues typified by RfaH, which are non-essential operon-specific bacterial factors acting in trans to upregulate horizontally-acquired genes. Furthermore, PipX and members of the NusG superfamily share a TLD/KOW structural domain, suggesting regulatory interactions of PipX with the translation machinery. Our results also suggest that the cis-acting function of PipX is a sophisticated regulatory strategy for maintaining appropriate PipX-PipY stoichiometry.

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Monitoring light/dark association dynamics of multi-protein complexes in cyanobacteria using size exclusion chromatography-based proteomics

Cited by 13 publications

References 54 publications

Energy drives the dynamic localization of cyanobacterial nitrogen regulators during diurnal cycles

Energy drives the dynamic localization of cyanobacterial nitrogen regulators during diurnal cycles

Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142

The nitrogen regulator PipX acts in cis to prevent operon polarity

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