The structural basis of Rubisco phase separation in the pyrenoid

Approximately one-third of global CO 2 fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO 2 -fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this pha...

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Published inNature plants Vol. 6; no. 12; pp. 1480 - 1490
Main Authors He, Shan, Chou, Hui-Ting, Matthies, Doreen, Wunder, Tobias, Meyer, Moritz T., Atkinson, Nicky, Martinez-Sanchez, Antonio, Jeffrey, Philip D., Port, Sarah A., Patena, Weronika, He, Guanhua, Chen, Vivian K., Hughson, Frederick M., McCormick, Alistair J., Mueller-Cajar, Oliver, Engel, Benjamin D., Yu, Zhiheng, Jonikas, Martin C.
Format Journal Article
LanguageEnglish
Published London Nature Publishing Group UK 01.12.2020
Nature Publishing Group
Subjects
631/1647/2204
631/1647/2258/1258/1259
631/1647/334/2244
631/1647/767
631/449/1734/1790
Algae
Binding sites
Biomedical and Life Sciences
Carbon cycle
Carbon dioxide
Carbon dioxide fixation
Chlamydomonas reinhardtii - chemistry
Chlamydomonas reinhardtii - metabolism
Electron microscopy
Fixation
Life Sciences
Molecular interactions
Molecular Structure
Mutation
Phase separation
Photosynthesis - physiology
Plant Sciences
Proteins
Ribulose-bisphosphate carboxylase
Ribulose-Bisphosphate Carboxylase - chemistry
Ribulose-Bisphosphate Carboxylase - metabolism
Structure-function relationships
United States > US
Germany
Singapore
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Summary:Approximately one-third of global CO 2 fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO 2 -fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii . We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle. The structural basis of the interactions between Rubisco and its intrinsically disordered linker protein provides insight into phase separation within the algal pyrenoid, an organelle responsible for around a third of global CO 2 fixation.
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S.H., P.D.J., V.C., F.M.H., T.W., O.M.-C., B.D.E., and M.C.J. designed experiments. S.H. identified EPYC1’s Rubisco-binding regions on EPYC1 by peptide tiling array and SPR. S.H. and S.A.P. prepared the Rubisco and EPYC1 peptide sample for single-particle cryo-EM; S.H., S.A.P. and G.H. prepared the Rubisco samples for peptide tiling array and surface plasmon resonance. H.-T.C., D.M. and Z.Y. performed Cryo-EM grid preparation, sample screening, data acquisition, image processing, reconstruction and map generation. D.M. and P.D.J. carried out single-particle model building and fitting and refinement. S.H., H.-T.C., D.M., P.D.J., F.M.H. and M.C.J. analyzed the structures. S.H. and W.P. analyzed EPYC1 binding to Rubisco by peptide substitution array and SPR. T.W. performed in vitro reconstitution phase separation experiments. N.A. and A.J.M. performed yeast two-hybrid experiments. S.H. and M.T.M. made Rubisco small subunit point mutants. S.H. performed spot test experiments. M.T.M. performed TEM. A.M.-S. performed the cryo-ET data analysis and modeling. S.H. and M.C.J. wrote the manuscript. All authors read and commented on the manuscript.
Author contributions
ISSN:2055-0278
2055-0278
DOI:10.1038/s41477-020-00811-y