Abstract
The slow and promiscuous properties of the CO2-fixing enzyme Rubisco constrain photosynthetic efficiency and have prompted the evolution of powerful CO2 concentrating mechanisms (CCMs). In eukaryotic microalgae a key strategy involves sequestration of the enzyme in the pyrenoid, a liquid non-membranous compartment of the chloroplast stroma. Here we show using pure components that two proteins, Rubisco and the linker protein Essential Pyrenoid Component 1 (EPYC1), are both necessary and sufficient to phase separate and form liquid droplets. The phase-separated Rubisco is functional. Droplet composition is dynamic and components rapidly exchange with the bulk solution. Heterologous and chimeric Rubiscos exhibit variability in their tendency to demix with EPYC1. The ability to dissect aspects of pyrenoid biochemistry in vitro will permit us to inform and guide synthetic biology ambitions aiming to engineer microalgal CCMs into crop plants.
Highlights
The slow and promiscuous properties of the CO2-fixing enzyme Rubisco constrain photosynthetic efficiency and have prompted the evolution of powerful CO2 concentrating mechanisms (CCMs)
We use biochemical reconstitution to demonstrate that Rubisco and Essential Pyrenoid Component 1 (EPYC1) are the two components necessary and sufficient to bring about a liquid-liquid phase separation (LLPS) that recapitulates the liquid-like behavior reported for the microalgal pyrenoid[9]
The EPYC1 protein consists of four quasi-identical 60 amino-acid tandem repeats, which are predicted to bind multivalently to the surface of Rubisco holoenzymes and drive a phase separation analogous to that described for other systems[19,20]
Summary
EPYC1 and Rubisco are necessary and sufficient for LLPS. The behavior of pyrenoid localized fluorescent fusion proteins in the C. reinhardtii chloroplast in conjunction with Rubisco distributions determined by in situ cryo-electron tomography[9,14,18] have culminated in a current LLPS model of pyrenoid formation. The turbidity was caused by the formation of spherical droplets from the bulk solution that could be labeled by including a fluorescent EPYC1-GFP fusion protein in the reaction (Fig. 1b) and occurred within seconds (Supplementary Fig. 2a). In contrast to other phase-separating scaffold proteins that contain unstructured regions[26,27,28,29], demixing of purified EPYC1 or Rubisco alone did not occur even at low temperatures and high concentrations (Supplementary Fig. 2h, i). To confirm that Rubisco structure and function was not negatively affected by concentrating the protein in the demixed droplets we assayed the ECM holoenzyme prior to, and after phase separation. Soluble and phase-separated Rubisco produced 3PG at the same rate, showing that the demixed enzyme is catalytically active (Fig. 2b). Microscopy and the droplet sedimentation assays confirmed that the Rubisco was droplet-bound during this experiment (Supplementary Fig. 3a, b)
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