Abstract - The slow kinetics and poor substrate specificity of the key photosynthetic CO₂-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco containing compartments known as pyrenoids in diverse algal lineages and carboxysomes in prokaryotes. Their formation has recently been linked to the condensation of Rubisco with intrinsically disordered linker proteins. Inside these Rubisco condensates actively transported bicarbonate is converted into CO₂ gas, which saturates the carboxylase with its substrate.

Using co-immunoprecipitation experiments in the marine diatom Phaeodactylum tricornutum we have identified the Rubisco linker protein PYCO1. Fluorescent PYCO1 fusion proteins localize as a rod shaped structure in the diatom chloroplast, consistent with the shape of the pyrenoid defined by transmission electron microscopy. Recombinant PYCO1 protein undergoes homotypic liquid liquid phase separation in a salt dependent manner. Diatom Rubisco specifically partitions into PYCO1 condensates. PYCO1 is highly mobile in homotypic condensates. In contrast PYCO1 condensates saturated with diatom Rubisco have greatly reduced dynamics, with both PYCO1 and Rubisco becoming immobile. Consistently, FRAP experiments indicate that PYCO1 is not mobile in vivo. A combination of Cryo-electron microscopy and site-directed mutagenesis data revealed the “sticker” motifs required for phase separation and Rubisco recruitment on both PYCO1 and Rubisco.

We speculate that the unusual material properties of the PYCO1-Rubisco condensate are necessary to support the unusual non-spherical shape of the Phaeodactylum pyrenoid. Careful characterization of multiple diverse Rubisco condensates will strengthen translational approaches aiming to introduce pyrenoids and other metabolic condensates into new host organisms.

Biography - Oliver completed his PhD here at the ANU under the supervision of Spencer Whitney, where he investigated the artificial evolution of the photosynthetic CO2-fixing enzyme Rubisco. Following his PhD Oliver joined the laboratory of Dr. Manajit Hayer-Hartl at the Max-Planck Institute of Biochemistry near Munich, Germany, where he discovered a novel molecular motor that activates Rubisco. And in 2012 Oliver started his group at NTU (Nanyang technological university) in Singapore, where he continues to investigate Rubisco and its molecular chaperones.