Dataset Information

Characterization of the pyrenoid proteome in the green microalga Chlamydomonas reinhardtii

ABSTRACT: The pyrenoid, a single microcompartment of the chloroplasts of most algae and hornworts, is the major site of CO2 fixation. To compensate the limited availability of CO2 in aquatic environments due to slower CO2 diffusion in water than air, the RubisCO-containing pyrenoid is involved in a carbon concentrating mechanism (CCM) and promotes the carboxylase activity rather than the oxygenase activity of RubisCO, the Calvin-Benson cycle enzyme catalyzing the first step of CO2 fixation. Data in the literature suggest that pyrenoid can have other functions in addition to CO2 fixation. To decipher its functions, the characterization of the pyrenoid proteome in the microalga Chlamydomonas reinhardtii was undertaken. Pyrenoid-enriched fractions from two independent biological cultures (sample1 and sample2) were obtained from cells (WT-cell) or isolated chloroplasts (WT-cp) either from the wild-type (WT) strain or from pyrenoid-deficient Chlamydomonas strains (MX-CW15 and SS-AT) and analyzed by nLC-MS/MS. Substractive proteomic analysis allowed the identification of contaminant proteins and the characterization of the pyrenoid proteome.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Chlamydomonas Reinhardtii

TISSUE(S): Photosynthetic Cell

SUBMITTER: Christophe H. MARCHAND

LAB HEAD: Stéphane D. Lemaire

PROVIDER: PXD004509 | Pride | 2018-02-28

REPOSITORIES: Pride

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Publications

Pyrenoid functions revealed by proteomics in Chlamydomonas reinhardtii.

Zhan Yu Y Marchand Christophe H CH Maes Alexandre A Mauries Adeline A Sun Yi Y Dhaliwal James S JS Uniacke James J Arragain Simon S Jiang Heng H Gold Nicholas D ND Martin Vincent J J VJJ Lemaire Stéphane D SD Zerges William W

PloS one 20180226 2

Organelles are intracellular compartments which are themselves compartmentalized. Biogenic and metabolic processes are localized to specialized domains or microcompartments to enhance their efficiency and suppress deleterious side reactions. An example of intra-organellar compartmentalization is the pyrenoid in the chloroplasts of algae and hornworts. This microcompartment enhances the photosynthetic CO2-fixing activity of the Calvin-Benson cycle enzyme Rubisco, suppresses an energetically waste ...[more]

PMID: 29481573

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Project description:The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco containing compartments known as pyrenoids in diverse algal lineages and carboxysomes in prokaryotes. Inside these compartments actively transported bicarbonate is converted into CO2 gas, which saturates the carboxylase with its substrate. Using co-immunoprecipitation experiments in Phaeodactylum tricornutum we have identified the Rubisco linker protein PYCO1. Similar to the green algal Rubisco linker protein EPYC1, PYCO1 is intrinsically disordered, possesses repeats and is positively charged at physiological pH. However, it possesses no sequence similarity to EPYC1, as expected for convergent evolution of a red Rubisco containing pyrenoid. Fluorescent PYCO1 fusion proteins localize as a rod shaped structure in the diatom chloroplast, consistent with the shape of the pyrenoid defined by transmission electron microscopy. To test the hypothesis that PYCO1 is the diatom pyrenoid scaffold we produced pure protein in Escherichia coli. Recombinant PYCO1 protein undergoes homotypic liquid liquid phase separation in a salt dependent manner. Diatom Rubisco specifically partitions into PYCO1 condensates. Heterotypic PYCO1-Rubisco condensates can bind up to three Rubisco hexadecamers per PYCO1 protein. Rubisco carboxylase function is unaffected in the condensates. PYCO1 is highly mobile in homotypic condensates. In contrast PYCO1 condensates saturated with diatom Rubisco have greatly reduced dynamics, with both PYCO1 and Rubisco becoming immobile. Consistently, FRAP experiments indicate that PYCO1 is not mobile in vivo. A combination of Cryo-electron microscopy and site-directed mutagenesis data show that the KWSP motif found in PYCO1 repeats binds to small subunits at the entrance of the Rubisco hexadecamer’s solvent channel. Analysis of mutant PYCO1 proteins show that both the “KWSP” tryptophan and another repeating tyrosine are essential for homotypic phase separation. We speculate that the unusual material properties of the PYCO1-Rubisco condensate are necessary to support the unusual non-spherical shape of the Phaeodactylum pyrenoid. Careful characterization of multiple diverse Rubisco condensates will strengthen translational approaches aiming to introduce pyrenoids and other metabolic condensates into new host organisms.

Dataset Information

Characterization of the pyrenoid proteome in the green microalga Chlamydomonas reinhardtii

Publications

Pyrenoid functions revealed by proteomics in Chlamydomonas reinhardtii.

Similar Datasets

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