Biology subjects

Jonikas, M. C.

Publications and source records attributed to Jonikas, M. C..

At least 19 recordsLinked to original sources

bioRxiv2026

SBE1 drives the circumferential growth of the starch sheath around the Chlamydomonas reinhardtii pyrenoid

Pyrenoids are CO2-fixing organelles responsible for approximately one-third of global CO2 fixation. The pyrenoids of many algae are surrounded by a starch sheath proposed to perform the critical function of slowing leakage of concentrated CO2 out of the pyrenoid. How the cell shapes starch granules into curved starch plates that encase the pyrenoid to enable efficient CO2 fixation is currently unknown. Here, we elucidate how starch transitions from granules into a fully formed pyrenoid starch sheath using confocal microscopy of the model green alga Chlamydomonas reinhardtii. We observe that after initiation, starch granules grow circumferentially along the surface of the pyrenoid matrix to encapsulate it. We show that the starch branching enzyme SBE1 localizes to the pyrenoid and is essential for this circumferential starch granule growth. Our data suggest that SBE1 promotes the circumferential growth of pyrenoid-associated starch granules by branching starch at the matrix-granule-stroma interface. Our findings advance the understanding of pyrenoid starch sheath assembly and, more broadly, starch-shaping mechanisms. Significance StatementThe shape of the starch granules that surround the pyrenoid is critical for efficient carbon fixation, but how these granules are shaped into curved plates is currently not understood. We find that starch granules spread along the pyrenoid surface rather than growing equally in all directions, and that this bias requires SBE1, a branching enzyme localized to the surface of the spherical pyrenoid matrix condensate. The findings support a model in which localizing a metabolic enzyme directs polymer growth to create curvature. This mechanism connects branching enzyme placement to starch granule geometry and contributes to the understanding of how cells convert local biosynthetic activity into large-scale organelle architecture.

molecular biology↗

bioRxiv2026

Rapid reconstitution and kinase-controlled regulation of algal pyrenoid condensates in engineered Nicotiana benthamiana

The pyrenoid is an organelle found in nearly all algae that concentrates CO2 around Rubisco to enhance photosynthetic carbon fixation. Engineering a functional pyrenoid into C plants is a promising route to improve plant photosynthetic performance and yields. However, progress has been limited by the lack of a plant platform for rapidly testing candidate components. Here, we engineered N. benthamiana to make its chloroplast Rubisco holoenzyme compatible with C. reinhardtii pyrenoid proteins. Disruption of native N. benthamiana RBCS genes and complementation with the C. reinhardtii RBCS2 generated a Rubisco-recombinant background where transient expression of the Rubisco linker protein EPYC1 was sufficient to drive the formation of Rubisco-EPYC1 condensates. Co-expression with the algal pyrenoid kinase KEY1 shifted the multi-condensate state toward a single condensate per chloroplast by altering EPYC1 phosphorylation in planta, recapitulating C. reinhardtii pyrenoid regulation. Together, these results establish engineered N. benthamiana as a tractable platform for rapidly characterizing candidate pyrenoid proteins and provide a step toward reconstructing pyrenoid architecture and regulation in plants.

plant biology↗

bioRxiv2026

A bifunctional coiled-coil protein generates the membrane-within-condensate architecture of the CO2-fixing pyrenoid

How membranes are integrated into biomolecular condensates is a fundamental question in cell biology. In the algal pyrenoid, an organelle responsible for one-third of global carbon fixation, CO2-delivering thylakoid membranes must penetrate a phase-separated condensate of the CO2-fixing enzyme Rubisco, but the mechanism governing membrane recruitment into the condensate remains unknown. Here, we demonstrate that the Chlamydomonas reinhardtii protein MITH1 acts as a molecular anchor that brings membrane into the pyrenoid condensate. MITH1 dimerizes into an extended coiled coil with an N-terminal amphipathic helix that binds thylakoid membrane. The coiled coil contains multiple novel binding sites for the Rubisco large subunit, allowing the condensate to wet onto the membrane. The coiled coil extends away from the membrane and promotes membrane organization within the condensate. These findings solve a longstanding mechanistic question in pyrenoid biogenesis and reveal general principles for how membranes are integrated into biological condensates.

molecular biology↗

bioRxiv2026

SAGA1 and SAGA2 localize the starch sheath to the pyrenoid in Chlamydomonas reinhardtii

Most algae enhance their CO2 assimilation by concentrating CO2 within the pyrenoid, a biomolecular condensate that contains the CO2-fixing enzyme Rubisco. Many pyrenoids are surrounded by a starch sheath that is thought to slow the escape of CO2 from the pyrenoid, but how the starch sheath is localized to the pyrenoid remains poorly understood. Here, in the leading model alga Chlamydomonas reinhardtii, we find that the protein SAGA2 is necessary for early pyrenoid starch sheath biogenesis and works redundantly with its homolog, SAGA1, to localize the starch sheath to the pyrenoid. SAGA2 and SAGA1 were enriched in different regions of the pyrenoid-starch sheath interface, suggesting that they play complementary roles. Both saga2 and saga1 mutants showed defects in starch sheath coverage early during pyrenoid formation that were improved at a later timepoint. Strikingly, a saga1;saga2 double mutant did not have a starch sheath around the pyrenoid and showed decreased overall starch content. SAGA1 and SAGA2 starch-binding domains bound to starch, the starch mimic molecule {beta}-cyclodextrin, and the starch precursor molecule maltoheptaose, suggesting a role for SAGA1 and SAGA2 in starch granule initiation. We propose a model where SAGA1 and SAGA2 each locally prime starch sheath initiation in a distinct region of the pyrenoid surface by enriching starch precursor molecules around the pyrenoid. These findings advance the understanding of algal starch sheath biogenesis and provide insights into the associations between biomolecular condensates and other cellular structures. Significance StatementEukaryotic algae enhance their carbon assimilation using an organelle called the pyrenoid, where concentrated CO2 is supplied to the CO2-fixing enzyme Rubisco. In many algae, a starch sheath surrounding the pyrenoid is thought to enhance CO2 fixation, but how starch is localized to pyrenoids is unknown. Here, we show that two proteins, SAGA1 and SAGA2, each bind to starch precursor molecules and redundantly localize starch to the pyrenoid in the alga Chlamydomonas reinhardtii. Our results suggest that SAGA1 and SAGA2 promote starch sheath initiation at the pyrenoid, rather than merely tethering starch, as previously thought. This work advances the understanding of the proteins and molecular mechanisms involved in pyrenoid starch sheath biogenesis and lays the foundations for their further study.

molecular biology↗

bioRxiv2025

Kinase KEY1 controls pyrenoid condensate size throughout the cell cycle by disrupting phase separation interactions

Biomolecular condensates spatially organize cellular functions, but the regulation of their size, number, dissolution, and re-condensation is poorly understood. The pyrenoid, an algal biomolecular condensate that mediates one-third of global CO2 fixation, typically exists as one large condensate per chloroplast, but during cell division it transiently dissolves and reconfigures into multiple smaller condensates. Here, we identify a kinase, KEY1, in the model alga Chlamydomonas reinhardtii that regulates pyrenoid condensate size and number dynamics throughout the cell cycle and is necessary for normal pyrenoid function and growth. Unlike wild type, key1 mutant cells have multiple smaller condensates throughout the cell cycle that fail to dissolve during cell division. We show that KEY1 localizes to the condensates and promotes their dissolution by disrupting interactions between their core constituents, the CO2-fixing enzyme Rubisco and its linker protein EPYC1, through EPYC1 phosphorylation. We develop a biophysical model that recapitulates KEY1-mediated condensate size and number regulation and suggests a mechanism for controlling condensate position. These data provide a foundation for the mechanistic understanding of the regulation of size, number, position, and dissolution in pyrenoids and other biomolecular condensates.

cell biology↗

bioRxiv2024

The Chlamydomonas reinhardtii CLiP2 mutant collection expands genome coverage with high-confidence disrupting alleles

Chlamydomonas reinhardtii (Chlamydomonas hereafter) is a powerful model organism for studies of photosynthesis, ciliary motility, and other cellular processes [1-4]. The CLiP library of mapped nuclear random insertion mutants [5,6] has accelerated progress for hundreds of laboratories in these fields by providing mutants in genes of interest. However, its value was limited by its modest coverage of the genome with high-confidence disruption alleles (46% of nuclear protein-coding genes with 1+ high-confidence allele in exons/introns; 12% of genes with 3+ alleles in exons/introns). Here we introduce the CLiP2 (Chlamydomonas Library Project 2) library, which greatly expands the number of available mapped high-confidence insertional mutants. The CLiP2 library includes 71,700 strains, covering 79% of nuclear protein-coding genes with 1+ high-confidence allele in exons/introns and 49% of genes with 3+ alleles in exons/introns. The mutants are available to the community via the Chlamydomonas Resource Center.

plant biology↗

bioRxiv2024

Proteomic analysis of the pyrenoid-traversing membranes of Chlamydomonas reinhardtii reveals novel components

O_LIPyrenoids are algal CO2-fixing organelles that mediate approximately one-third of global carbon fixation. Most pyrenoids are traversed by membranes that are thought to supply them with concentrated CO2. Despite the critical nature of these membranes for pyrenoid function, they are poorly understood, with few protein components known in any species. C_LIO_LIHere, we identify protein components of the pyrenoid-traversing membranes from the leading model alga, Chlamydomonas reinhardtii, by affinity purification and mass spectrometry of membrane fragments. Our proteome includes previously-known proteins as well as novel candidates. C_LIO_LIWe further characterize two of the novel pyrenoid-traversing membrane-resident proteins: Cre10.g452250, which we name Pyrenoid Membrane Enriched 1 (PME1), and Cre02.g143550, also known as Low-CO2-Induced 16 (LCI16). We confirm the pyrenoid-traversing membrane localization of LCI16 and observe that PME1 and LCI16 physically interact. We find that neither protein is required for normal membrane morphology or growth under CO2-limiting conditions, but that both mutants show a similar proteomic profile to those of established pyrenoid mutants. C_LIO_LITaken together, our study identifies the proteome of the pyrenoid-traversing membranes and initiates the characterization of a novel pyrenoid-traversing membrane complex, building toward a mechanistic understanding of the pyrenoid. C_LI

molecular biology↗

bioRxiv2024

Biogenesis, engineering and function of membranes in the CO2-fixing pyrenoid

Summary ParagraphApproximately one-third of global CO2 assimilation is performed by the pyrenoid1, a liquid-like organelle found in most algae and some plants2. Specialized membranes are hypothesized to drive CO2 assimilation in the pyrenoid by delivering concentrated CO23,4, but their biogenesis and function have not been experimentally characterized. Here, we show that homologous proteins SAGA1 and MITH1 mediate the biogenesis of the pyrenoid membrane tubules in the model alga Chlamydomonas reinhardtii and are sufficient to reconstitute pyrenoid-traversing membranes in a heterologous system, the plant Arabidopsis thaliana. SAGA1 localizes to the regions where thylakoid membranes transition into tubules and is necessary to initiate tubule formation. MITH1 localizes to the tubules and is necessary for their extension through the pyrenoid. Tubule-deficient mutants exhibit growth defects under CO2-limiting conditions, providing evidence for the function of membrane tubules in CO2 delivery to the pyrenoid. Furthermore, these mutants form multiple aberrant condensates of pyrenoid matrix, indicating that a normal tubule network promotes the coalescence of a single pyrenoid. The reconstitution of pyrenoid-traversing membranes in a plant represents a key milestone toward engineering a functional pyrenoid into crops for improving crop yields. More broadly, our study demonstrates the functional importance of pyrenoid membranes, identifies key biogenesis factors, and paves the way for the molecular characterization of pyrenoid membranes across the tree of life.

molecular biology↗

bioRxiv2023

Chloroplast Methyltransferase Homolog RMT2 is Involved in Photosystem I Biogenesis

Oxygen (O2), a dominant element in the atmosphere and essential for most life on Earth, is produced by the photosynthetic oxidation of water. However, metabolic activity can cause accumulation of reactive O2 species (ROS) and severe cell damage. To identify and characterize mechanisms enabling cells to cope with ROS, we performed a high-throughput O2 sensitivity screen on a genome-wide insertional mutant library of the unicellular alga Chlamydomonas reinhardtii. This screen led to identification of a gene encoding a protein designated Rubisco methyltransferase 2 (RMT2). Although homologous to methyltransferases, RMT2 has not been experimentally demonstrated to have methyltransferase activity. Furthermore, the rmt2 mutant was not compromised for Rubisco (first enzyme of Calvin-Benson Cycle) levels but did exhibit a marked decrease in accumulation/activity of photosystem I (PSI), which causes light sensitivity, with much less of an impact on other photosynthetic complexes. This mutant also shows increased accumulation of Ycf3 and Ycf4, proteins critical for PSI assembly. Rescue of the mutant phenotype with a wild-type (WT) copy of RMT2 fused to the mNeonGreen fluorophore indicates that the protein localizes to the chloroplast and appears to be enriched in/around the pyrenoid, an intrachloroplast compartment present in many algae that is packed with Rubisco and potentially hypoxic. These results indicate that RMT2 serves an important role in PSI biogenesis which, although still speculative, may be enriched around or within the pyrenoid. Significance StatementA high-throughput genetic screen was used to identify O2 sensitive mutants of Chlamydomonas reinhardtii (Chlamydomonas throughout) that experience elevated oxidative stress in the light relative to WT cells. Identification of genes altered in these mutants offers opportunities to discover activities that a) protect photosynthetic cells from oxidative damage, b) participate in rapid assembly of photosynthetic complexes, which would limit accessibility of intermediates to O2, and/or c) facilitate repair of damaged cellular complexes. A mutant from this screen disrupted for RMT2, originally described as encoding a Rubisco methyltransferase, was defective for PSI biogenesis. Additionally, RMT2 appears to be enriched in/around the pyrenoid, a chloroplast localized compartment harboring much of the Chlamydomonas Rubisco, raising the possibility that this compartment plays a role in PSI biogenesis.

plant biology↗

bioRxiv2023

The chloroplastic phosphate transporter CrPHT4-7 supports phosphate homeostasis and photosynthesis in Chlamydomonas

In eukaryotic cells, phosphorus is assimilated and utilized primarily as phosphate (Pi). Pi homeostasis is mediated by transporters that have not yet been adequately characterized in green algae. This study reports on CrPHT4-7 from Chlamydomonas reinhardtii, a member of the PHT4 transporter family, which exhibits remarkable similarity to AtPHT4;4 from Arabidopsis thaliana, a chloroplastic ascorbate transporter. Using fluorescent protein tagging we show that CrPHT4-7 resides in the chloroplast envelope membrane. Crpht4-7 mutants, generated by the CRISPR/Cas12a-mediated single-strand templated repair, show retarded growth especially in high light, enhanced sensitivity to phosphorus limitation, reduced ATP level, strong ascorbate accumulation and diminished non-photochemical quenching in high light. Conversely, CrPHT4-7 overexpressing lines exhibit enhanced biomass accumulation under high light conditions in comparison with the wild-type strain. Expressing CrPHT4-7 in a yeast strain lacking Pi transporters substantially recovered its slow growth phenotype demonstrating that it transports Pi. Even though CrPHT4-7 shows a high degree of similarity to AtPHT4;4, it does not display any significant ascorbate transport activity in yeast or intact algal cells. Thus, the results demonstrate that CrPHT4-7 functions as a chloroplastic Pi transporter essential for maintaining Pi homeostasis and photosynthesis in Chlamydomonas reinhardtii. One-sentence summaryWe demonstrate that the CrPHT4-7 transporter of Chlamydomonas reinhardtii is located in the chloroplast envelope membrane and contributes to maintaining phosphate homeostasis and photosynthesis.

plant biology↗

bioRxiv2023

Effects of linker length on phase separation: lessons from the Rubisco-EPYC1 system of the algal pyrenoid

Biomolecular condensates are membraneless organelles formed via phase separation of macromolecules, typically consisting of bond-forming "stickers" connected by flexible "linkers". Linkers have diverse roles, such as occupying space and facilitating interactions. To understand how linker length relative to other lengths affects condensation, we focus on the pyrenoid, which enhances photosynthesis in green algae. Specifically, we apply coarse-grained simulations and analytical theory to the pyrenoid proteins of Chlamydomonas reinhardtii: the rigid holoenzyme Rubisco and its flexible partner EPYC1. Remarkably, halving EPYC1 linker lengths decreases critical concentrations by ten-fold. We attribute this difference to the molecular "fit" between EPYC1 and Rubisco. Varying Rubisco sticker locations reveals that the native sites yield the poorest fit, thus optimizing phase separation. Surprisingly, shorter linkers mediate a transition to a gas of rods as Rubisco stickers approach the poles. These findings illustrate how intrinsically disordered proteins affect phase separation through the interplay of molecular length scales.

biophysics↗

bioRxiv2022

Systematic identification and characterization of novel genes in the regulation and biogenesis of photosynthetic machinery

Photosynthesis is central to food production and the Earths biogeochemistry, yet the molecular basis for its regulation remains poorly understood. Here, using high-throughput genetics in the model eukaryotic alga Chlamydomonas reinhardtii, we identify with high confidence (FDR<0.11) 70 previously-uncharacterized genes required for photosynthesis. We then provide a resource of mutant proteomes that enables functional characterization of these novel genes by revealing their relationship to known genes. The data allow assignment of 34 novel genes to the biogenesis or regulation of one or more specific photosynthetic complexes. Additional analysis uncovers at least seven novel critical regulatory proteins, including five Photosystem I mRNA maturation factors and two master regulators: MTF1, which impacts chloroplast gene expression directly; and PMR1, which impacts expression via nuclear-expressed factors. Our work provides a rich resource identifying novel regulatory and functional genes and placing them into pathways, thereby opening the door to a system-level understanding of photosynthesis. HighlightsO_LIHigh-confidence identification of 70 previously-uncharacterized genes required for photosynthesis C_LIO_LIProteomic analysis of mutants allows assignment of function to novel genes C_LIO_LICharacterization of 5 novel Photosystem I mRNA maturation factors validates this resource C_LIO_LIMTF1 and PMR1 identified as master regulators of photosynthesis C_LI

cell biology↗

bioRxiv2022

Phase-separating pyrenoid proteins form complexes in the dilute phase

While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play a significant role in the stability, dynamics, and regulation of condensates.

biophysics↗

bioRxiv2021

Identification of green lineage osmotic stress pathways

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the green alga Chlamydomonas reinhardtii to establish a foundational understanding of evolutionarily conserved osmotic-stress signaling pathways in the green lineage through transcriptomics, phosphoproteomics, and functional genomics approaches. Five genes acting across diverse cellular pathways were found to be essential for osmotic-stress tolerance in Chlamydomonas including cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs of these genes in the multicellular land plant Arabidopsis thaliana have conserved functional roles in stress tolerance and reveal a novel PROFILIN-dependent actin remodeling stage of acclimation that ensures cell survival and tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.

plant biology↗

bioRxiv2021

Diffusion barriers and adaptive carbon uptake strategies enhance the modeled performance of the algal CO2-concentrating mechanism

Many photosynthetic organisms enhance the performance of their CO2-fixing enzyme Rubisco by operating a CO2-concentrating mechanism (CCM). Most CCMs in eukaryotic algae supply concentrated CO2 to Rubisco in an organelle called the pyrenoid. Ongoing efforts seek to engineer an algal CCM into crops that lack a CCM to increase yields. To advance our basic understanding of the algal CCM, we develop a chloroplast-scale reaction-diffusion model to analyze the efficacy and the energy efficiency of the CCM in the green alga Chlamydomonas reinhardtii. We show that achieving an effective and energetically efficient CCM requires a physical barrier such as thylakoid stacks or a starch sheath to reduce CO2 leakage out of the pyrenoid matrix. Our model provides insights into the relative performance of two distinct inorganic carbon uptake strategies: at air-level CO2, a CCM can operate effectively by taking up passively diffusing external CO2 and catalyzing its conversion to HCO3-, which is then trapped in the chloroplast; however, at lower external CO2 levels, effective CO2 concentration requires active import of HCO3-. We also find that proper localization of carbonic anhydrases can reduce futile carbon cycling between CO2 and HCO3-, thus enhancing CCM performance. We propose a four-step engineering path that increases predicted CO2 saturation of Rubisco up to seven-fold at a theoretical cost of only 1.5 ATP per CO2 fixed. Our system-level analysis establishes biophysical principles underlying the CCM that are broadly applicable to other algae and provides a framework to guide efforts to engineer an algal CCM into land plants. Significance StatementEukaryotic algae mediate approximately one-third of CO2 fixation in the global carbon cycle. Many algae enhance their CO2-fixing ability by operating a CO2-concentrating mechanism (CCM). Our model of the algal CCM lays a solid biophysical groundwork for understanding its operation. The models consistency with experimental observations supports existing hypotheses about the operating principles of the algal CCM and the functions of its component proteins. We provide a quantitative estimate of the CCMs energy efficiency and compare the performance of two distinct CO2 assimilation strategies under varied conditions. The model offers a quantitative framework to guide the engineering of an algal CCM into land plants and supports the feasibility of this endeavor.

biophysics↗

bioRxiv2020

Systematic characterization of gene function in a photosynthetic organism

Photosynthetic organisms are essential for human life, yet most of their genes remain functionally uncharacterized. Single-celled photosynthetic model systems have the potential to accelerate our ability to connect genes to functions. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. 78% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles allow us to place known and previously uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism, and ciliogenesis. We illustrate the value of this resource by validating novel phenotypes and gene functions, including the discovery of three novel components of a defense pathway that counteracts actin cytoskeleton inhibitors released by other organisms. The data also inform phenotype discovery in land plants: mutants in Arabidopsis thaliana genes exhibit similar phenotypes to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

systems biology↗

bioRxiv2020

Arabidopsis bZIP11 is a susceptibility factor during Pseudomonas syringae infection

AO_SCPLOWBSTRACTC_SCPLOWThe induction of plant nutrient secretion systems is critical for successful pathogen infection. Some bacterial pathogens, e.g. Xanthomonas species, use TAL (transcription activator-like) effectors to induce transcription of SWEET sucrose efflux transporters. Pseudomonas syringae pathovar (pv.) tomato strain DC3000 lacks TAL effectors, yet is able to induce multiple SWEETs in Arabidopsis thaliana by unknown mechanisms. Since bacteria require other nutrients besides sugars for efficient reproduction, we hypothesized that Pseudomonas may depend on host transcription factors involved in secretory programs to increase access to essential nutrients. Bioinformatic analyses identified the Arabidopsis basic-leucine zipper transcription factor bZIP11 as a potential regulator of nutrient transporters, including SWEETs and UmamiT amino acid transporters. Inducible downregulation of bZIP11 expression in Arabidopsis resulted in reduced growth of P. syringae pv. tomato strain DC3000, whereas inducible overexpression of bZIP11 resulted in increased bacterial growth, supporting the hypothesis that bZIP11 regulated transcription programs are essential for maximal pathogen titer in leaves. Our data are consistent with a model in which a pathogen alters host transcription factor expression upstream of secretory transcription networks to promote nutrient efflux from host cells.

plant biology↗

bioRxiv2020

The Structural Basis of Rubisco Phase Separation in the Pyrenoid

Approximately one-third of global CO2 fixation occurs in a phase separated algal organelle called the pyrenoid. Existing data suggest that the pyrenoid forms by the phase-separation of the CO2-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 EPYC1 (Essential Pyrenoid Component 1) 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 one 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 where EPYC1 and Rubisco form a co-dependent 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.

molecular biology↗