"Strikingly, a fourth protein, previously identified as a low-CO 2 –induced nuclear-encoded protein (LCI5; Cre10.g436550) ( 24 ), was found in the low-CO 2 pyrenoid fraction with comparable abundance to Rubisco"

"Venus-tagged EPYC1 showed clear colocalization with mCherry-tagged RBCS in the pyrenoid"

"the epyc1 mutant showed defective photoautotrophic growth in low CO 2 , which was rescued by high CO 2 and by reintroducing the EPYC1 gene"

"We conclude that EPYC1 is required for Rubisco localization to the pyrenoid not only at low CO 2 , but also at high CO 2 ."

"We conclude that EPYC1 and Rubisco are part of the same supramolecular complex in the pyrenoid."

"Here we find that Rubisco accumulation in the pyrenoid of the model alga Chlamydomonas reinhardtii is mediated by a disordered repeat protein, which we term Essential Pyrenoid Component 1 (EPYC1)."

"a protein called Essential Pyrenoid Component 1 (EPYC1; also known as LCI5) links Rubisco holoenzymes together to form the pyrenoid matrix"

"A portion of the RbcS1-Venus and EPYC1-Venus signals rapidly dispersed from the pyrenoid matrix into the stroma for ~20 minutes near the end of chloroplast and pyrenoid division"

"Here we use biochemical reconstitution to demonstrate that Rubisco and EPYC1 are the two components necessary and sufficient to bring about a liquid-liquid phase separation (LLPS) that recapitulates the liquid-like behavior reported for the microalgal pyrenoid"

"Rubisco enzyme activity (indicated as 3PG production) is unaffected by droplet formation."

"In contrast to the scaffold hypothesis we find EPYC1 is relatively soluble and only homotypically phase separates at concentrations of 100 µM or more in the presence of a crowding agent."

"Together, our data demonstrate that EPYC1’s Rubisco-binding regions bind to the Rubisco small subunit α-helices via salt-bridge interactions and a hydrophobic interface, enabling the condensation of Rubisco into the phase separated matrix."

"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."

"Expression of EPYC1 in the Arabidopsis line S2Cr results in condensate formation."

"Addition of the first EPYC1 repeat region was sufficient to detect interaction, and each subsequent repeat region correlated with growth at increased concentrations of 3-AT, indicating that EPYC1 is a modular protein and each repeat has an additive effect on SSU interaction."

"EPYC1 is a ~35-kDa intrinsically disordered protein that phase-separates with Rubisco to form the pyrenoid matrix (13, 15, 16)."

"First, in a mutant lacking EPYC1, a pyrenoid-like structure still assembles around the tubules, containing some Rubisco enclosed by a starch sheath, although the canonical matrix is absent (13)."

"essential pyrenoid component 1 (EPYC1), a Rubisco linker protein"

"EPYC1 interacts with two 14-3-3 proteins FTT1 and FTT2."

"these results establish that KEY1 is the primary kinase of EPYC1 and suggest that KEY1 preferentially phosphorylates the Rubisco-binding regions of EPYC1"

"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."

"The majority of these complexes contain exactly one Rubisco molecule."

"In the following, we tested all extant and ancestral sequences with respect to carboxylation rate enhancement and maximum amount of 3-PG produced using the radioisotope assay described above."

"higher plant-like helices knock out the pyrenoid, whereas native algal helices establish a pyrenoid"

"Pyrenoids are formed by liquid-liquid phase separation (LLPS) of Rubisco, the primary CO2 fixing enzyme, with an intrinsically disordered multivalent Rubisco-binding protein."

"EPYC1 appears to interact exclusively with the small subunit of Rubisco (SSU) via Rubisco binding motifs (RBMs) that are common to several other pyrenoid components in Chlamydomonas"

"The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein."

"Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light."

"Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes."

"Although the function of LCI5 is unknown at present, posttranslational modification of proteins during the acclimation process to LC conditions, which cannot be identified by a DNA array analysis, could be associated with the CCM induction."