Exploring the organelle assembly mechanism of the Chlamydomonas reinhardtii pyrenoid through Proximity Labelling

A significant fraction of global carbon fixation occurs in a phase-separated organelle called the pyrenoid. To achieve this, the pyrenoid participates in a process called the CO2 concentrating mechanism (CCM) that raises the cell’s internal inorganic carbon concentration by up to a 100-fold above the external concentration, thereby suppressing photorespiration and increasing the photosynthetic efficiency. The CCM of Chlamydomonas reinhardtii represents one of the best characterised pyrenoid-based CCM to date, however the assembly mechanism of the pyrenoid structural components: the starch sheath and thylakoid tubules is relatively unclear. To accelerate this process and identify proteins which might contribute to their biogenesis, I developed a TurboID-based proximity labelling pipeline which allows efficient pulldown of proteins based on their proximity to the tagged Protein-of-interest. By targeting Rubisco, and other pyrenoid proteins thought to be critical for the assembly of the starch sheath and thylakoid tubules, I obtained a dataset containing previously identified pyrenoid proteins and a list of high-confidence novel pyrenoid proteins. Within this list, PINS2 was taken forward for further characterisation due to its mesh-like localisation which parallels another starch-related protein LCI9. This homology and proximity in their chromosomal position led to the identification of LCI9/PINS1, PINS2, and PINS3 as a three-gene family sharing a similar domain arrangement. Via TEM imaging, I observed that the pyrenoid starch sheath in the pins2 mutant exhibits aberrant overlap that are uncommon in wildtype strains. Further, the pins2 mutant displays a high-CO2 requiring phenotype suggesting a defect in the CCM. The carbonic anhydrase LCIB alters its localisation from a diffuse stromal pattern to the pyrenoid periphery in sub-air level CO2. This re-localisation is proposed to be critical for maintaining CCM efficiency by scavenging the CO2 leaked from the pyrenoid. I observed that the LCIB re-localisation towards the pyrenoid is perturbed in the pins2 mutant. This highlights the importance of a correctly formed starch sheath to the LCIB-mediated CO2-scavenging system. Together, our results indicate that the PINS proteins collectively alter the pyrenoid starch sheath through their different localisation and interaction with starch synthesis enzymes on the starch sheath gap, this in turn builds a canonical starch sheath necessary for LCIB recruitment.