A linker protein from a red-type pyrenoid phase separates with Rubisco via oligomerizing sticker motifs.

The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as pyrenoids in the majority of eukaryotic microalgae. Diatoms dominate marine photosynthesis, but the interactions underlying their pyrenoids are unknown. Here, we identify and characterize the Rubisco linker protein PYCO1 from Phaeodactylum tricornutum. PYCO1 is a tandem repeat protein containing prion-like domains that localizes to the pyrenoid. It undergoes homotypic liquid-liquid phase separation (LLPS) to form condensates that specifically partition diatom Rubisco. Saturation of PYCO1 condensates with Rubisco greatly reduces the mobility of droplet components. Cryo-electron microscopy and mutagenesis data revealed the sticker motifs required for homotypic and heterotypic phase separation. Our data indicate that the PYCO1-Rubisco network is cross-linked by PYCO1 stickers that oligomerize to bind to the small subunits lining the central solvent channel of the Rubisco holoenzyme. A second sticker motif binds to the large subunit. Pyrenoidal Rubisco condensates are highly diverse and tractable models of functional LLPS.

π-π Interactions Drive the Homotypic Phase Separation of the Prion-like Diatom Pyrenoid Scaffold PYCO1.

CO2 fixation in most unicellular algae relies on the pyrenoid, a biomolecular condensate, which sequesters the cell's carboxylase Rubisco. In the marine diatom Phaeodactylum tricornutum, the pyrenoid tandem repeat protein Pyrenoid Component 1 (PYCO1) multivalently binds Rubisco to form a heterotypic Rubisco condensate. PYCO1 contains prion-like domains and can phase-separate homotypically in a salt-dependent manner. Here we dissect PYCO1 homotypic liquid-liquid phase separation (LLPS) by evaluating protein fragments and the effect of site-directed mutagenesis. Two of PYCO1's six repeats are required for homotypic LLPS. Mutagenesis of a minimal phase-separating fragment reveals tremendous sensitivity to the substitution of aromatic residues. Removing positively charged lysines and arginines instead enhances the propensity of the fragment to condense. We conclude that PYCO1 homotypic LLPS is mostly driven by π-π interactions mediated by tyrosine and tryptophan stickers. In contrast π-cation interactions involving arginine or lysine are not significant drivers of LLPS in this system.