Oral Presentation 49th Lorne Conference on Protein Structure and Function 2024

Understanding functional phase separations by reconstituting pyrenoidal and carboxysomal Rubisco condensates (#50)

Oliver Mueller-Cajar 1 , Tobias Wunder 1 , Zhen Guo Oh 1 , Warren Shou Leong Ang 1 , Cheng Wei Poh 1 , Jian Ann How 1 , Jian Boon How 1 , Alexander Schober 1 , Yisiang Ng 1 , Shashi Bhushan 1
  1. School of Biological Sciences, Nanyang Technological University, Singapore

Almost all photosynthetic aquatic organisms utilize the same strategy to overcome the limitations of their CO2-fixing enzyme Rubisco. They sequester the enzyme in a minimal volume. Equilibration of an elevated bicarbonate pool at the same site by carbonic anhydrase permits the CO2 concentration to be increased near the Rubisco active sites. High substrate concentrations permit carboxylation velocity to be maximized and the suppresses the wasteful oxygenase reaction. The Rubisco condensates (Rubiscondensates), pyrenoids in eukaryotic microalgae and carboxysomes in prokaryotes, form via liquid-liquid phase separation (1). This involves intrinsically disordered linker proteins that multivalently bind the enzyme to form a system-spanning network.

Using bottom-up biochemical reconstitution, we have studied the assembly of multiple Rubiscondensates (2,3). The dense phase formed by the green algal EPYC1 linker protein and Rubisco is highly dynamic and its protein concentration exceeds 100 mg/ml. In contrast the diatom (red lineage phytoplankton) PYCO1-Rubisco condensate presents as much more viscous, but relatively dilute material. These material properties are encoded by the sequence of the linkers.

In both systems additional condensate components are recruited via multivalent interactions with Rubisco. The well-folded enzyme is proving to be an excellent structural template, permitting  different bound sticker motifs to be resolved by cryo-electron microscopy. For example our structure of diatom Rubisco bound to PYCO1 revealed that repeating PYCO1 stickers oligomerized to bind the Rubisco small subunits. (3)

In our efforts to reconstitute a carboxysomal Rubiscondensate we have found that shell proteins can dissolve the micron-sized Rubisco-linker droplets. This behaviour supports a possible role as a proteinaceous surfactant enabling the formation of Rubiscondensate emulsions. 

Rubiscondensates are emerging as outstanding models to study functionally well-defined biological phase separation. In addition their characterization will guide their the introduction of biophysical CCMs into higher plants to overcome the energetic penalty of photorespiration.

  1. Ang WSL, How JA, How JB, Mueller-Cajar O. The stickers and spacers of Rubiscondensation: assembling the centrepiece of biophysical CO2-concentrating mechanisms. J Exp Bot. 2023 Jan 11;74(2):612-626
  2. Wunder T, Cheng SLH, Lai SK, Li HY, Mueller-Cajar O. The phase separation underlying the pyrenoid-based microalgal Rubisco supercharger. Nat Commun. 2018 Nov 29;9(1):5076.
  3. Oh ZG, Ang WSL, Poh CW, Lai SK, Sze SK, Li HY, Bhushan S, Wunder T, Mueller-Cajar O. A linker protein from a red-type pyrenoid phase separates with Rubisco via oligomerizing sticker motifs. Proc Natl Acad Sci U S A. 2023 Jun 20;120(25):e2304833120