Dataset Information

A Spatial Interactome Reveals the Protein Organization of the Algal CO2 Concentrating Mechanism

ABSTRACT: One-third of global CO2 fixation is mediated by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2 concentrating mechanism (CCM), built around a mysterious organelle called the pyrenoid. Here, we leveraged new tools to determine the localizations of 146 candidate CCM proteins in the model alga Chlamydomonas reinhardtii. Twelve of the tagged proteins localized to the pyrenoid in six distinct sub-pyrenoid localizations: matrix, membrane tubules, plates, a mesh layer, and two punctate layers. Many stromal proteins can access the pyrenoid matrix. The proteins with access to the matrix tend to be small, suggesting that the pyrenoid may show protein size selectivity. Contrary to current models, the carbonic anhydrase CAH6 is in the flagella. Affinity purification mass spectrometry of 38 CCM-related proteins reveals dozens of additional components, including novel Rubisco interacting proteins, photosystem I assembly factor candidates and inorganic carbon flux components. Together, our spatial interactome reveals new protein components, sub-compartments and biophysical characteristics that open doors to a detailed molecular characterization of the algal CCM.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Chlamydomonas Reinhardtii

SUBMITTER: Ryan Leib

LAB HEAD: Martin C Jonikas

PROVIDER: PXD007664 | Pride | 2018-01-24

REPOSITORIES: Pride

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			Action	DRS
	150831_LMackinder_Jonikas_LM_3873_T1E11.raw	Raw
	150831_LMackinder_Jonikas_LM_3873_T1E11.raw_20150902_Byonic.mgf	Mgf
	150831_LMackinder_Jonikas_LM_3873_T1E11.raw_20150902_Byonic.mzid	Mzid
	150831_LMackinder_Jonikas_LM_3873_T1E11.raw_20150902_Byonic.xlsx	Xlsx
	150831_LMackinder_Jonikas_LM_3873_T1E11.raw_20150902_Byonic_Light.mgf	Mgf

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Publications

A Spatial Interactome Reveals the Protein Organization of the Algal CO2-Concentrating Mechanism.

Mackinder Luke C M LCM Chen Chris C Leib Ryan D RD Patena Weronika W Blum Sean R SR Rodman Matthew M Ramundo Silvia S Adams Christopher M CM Jonikas Martin C MC

Cell 20170901 1

Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins and physical interactors of 38 of these proteins. Our data reveal the iden ...[more]

PMID: 28938113

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Project description:Diatoms are responsible for ~40% of marine primary productivity1, fueling the oceanic carbon cycle and contributing to natural carbon sequestration in the deep ocean2. Diatoms rely on energetically expensive carbon concentrating mechanisms (CCMs) to fix carbon efficiently at modern levels of CO23–5. How diatoms may respond over the short and long-term to rising atmospheric CO2 remains an open question. Here we use nitrate-limited chemostats to show that the model diatom Thalassiosira pseudonana rapidly responds to increasing CO2 by differentially expressing gene clusters that regulate transcription and chromosome folding and subsequently reduces transcription of photosynthesis and respiration gene clusters under steady-state elevated CO2. These results suggest that exposure to elevated CO2 first causes a shift in regulation and then a metabolic rearrangement. Genes in one CO2-responsive cluster included CCM and photorespiration genes that share a putative cyclic-AMP responsive cis-regulatory sequence, implying these genes are co-regulated in response to CO2 with cAMP as an intermediate messenger. We verified cAMP-induced down-regulation of CCM gene ?-CA3 in nutrient-replete diatom cultures by inhibiting the hydrolysis of cAMP. These results indicate an important role for cAMP in down-regulating CCM and photorespiration genes under elevated CO2 and provide insights into mechanisms of diatom acclimation in response to climate change. In steady-state experiments: axenic T. pseudonana cells in four biological replicates (duplicate chemostats x 2 experimental runs) were acclimated to nitrate-limitation at 70% (1.5 day-1) of max growth rate for >10 days (>15 generations) under continuous light of 80 µmol photons·m?2·s?1. Cell biomass was maintained at ~2 x 105 cells·mL?1 by 10 ?M nitrate and carbonate chemistry stabilized to 300, 475 or 800 ?atm CO2, verified by pH and DIC measurements. Transition samples and carbonate chemistry were collected daily from chemostat cultures as CO2 levels were increased from ~300-800 ?atm at a rate ? 0.2 ?atm·min?1 over four consecutive days (6 generations) after pre-acclimation to 300 ?atm CO2 and nitrate-limitation.

Dataset Information

A Spatial Interactome Reveals the Protein Organization of the Algal CO2 Concentrating Mechanism

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Publications

A Spatial Interactome Reveals the Protein Organization of the Algal CO<sub>2</sub>-Concentrating Mechanism.

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