Partitioning of Rubisco activase into the pyrenoidal Rubisco condensate is mediated by a functional protein-protein...

Most unicellular microalgae achieve robust photosynthetic CO₂-fixation by sequestering their carboxylase in chloroplastic Rubisco condensates (Rubiscondensates) termed pyrenoids. Only a fraction of the stromal proteome partitions to the Rubiscondensate. One critical pyrenoid component is Rubisco activase (Rca). Here we show that Chlamydomonas reinhardtii Rca partitions into the minimal two component EPYC1-Rubisco condensate. We identify the N-terminal domain (NTD) of Rca as containing the responsible sticker motifs. Mutagenesis reveals that single amino acid substitutions previously shown to abolish Rca function also eliminate partitioning. Blue fluorescent protein is excluded from EPYC1-Rubisco condensates, but partitions when fused to the Rca NTD. Expression of an NTD-GFP fusion in the Chlamydomonas chloroplast is consistent with the in vitro finding. We hypothesize that partitioning of proteins to biomolecular condensates will frequently utilize pre-existing functionally relevant protein-protein interactions. Identification of the sticker motif provides additional avenues to localize synthetic or phylogenetically remote pyrenoid components to the compartment.