Tuesday, April 9, 2019 - 4:00pm
Nanyang Technological University, Singapore
The distinct phase separations underlying diverse pyrenoids- the CO2-fixing membraneless organelles of green algae and diatoms
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 algae and carboxysomes in prokaryotes. Inside these compartments actively transported bicarbonate is converted into CO2 gas, which saturates the carboxylase with its substrate. The pyrenoid of the model green algae Chlamydomonas reinhardtii has recently been demonstrated to behave as a liquid non-membraneous compartment1. Using pure components we have shown that algal Rubisco and the disordered tandem repeat protein EPYC12 are necessary and sufficient to phase separate by complex coacervation3. The droplets exhibit mixing behavior similar to that reported in vivo. The phase-separated Rubisco was catalytically functional. Detailed characterization of the system revealed that plant Rubiscos do not phase separate with EPYC1 and permitted evaluation of the role of the EPYC1 tandem repeats3. Pyrenoids are ubiquitous and have evolved multiple times convergently. Diatoms are responsible for up to half of marine photosynthetic carbon fixation. Using chemical cross-linking and co-immunoprecipitation we have identified a largely disordered repeat protein (Pyrenoid factor 1-PF1) that localizes to the diatom pyrenoid in vivo. Full length PF1 and single repeats specifically bind diatom Rubisco. Unlike EPYC1, PF1 phase separates homotypically into condensates that recruit Rubisco. We have identified binding motifs on both PF1 and the small subunit of Rubisco. By systematically characterizing diverse pyrenoid-forming modules we aim to inform synthetic biology strategies towards enhancing the CO2-fixing efficiency of future food and energy crops. In addition the pyrenoid and the corresponding reconstituted droplets are an ideal system to study the effect of demixing on enzymatic activities, including the behavior of molecular chaperones (e.g. Rubisco activases) under crowded conditions.
- 1. Freeman Rosenzweig et al. Cell (2017), 171(1):148-162
- 2. MacKinder et al. PNAS (2016), 113(21):5958-63
- 3. Wunder et al. Nat. Commun.(2018), 9(1):5076