Stages of biomolecular condensate formation in pro-β-carboxysome assembly

nature plants Article https://doi.org/10.1038/s41477-026-02227-6 Stages of biomolecular condensate formation in pro-β-carboxysome assembly Received: 29 June 2025 Accepted: 9 January 2026 Published online: 10 February 2026 Check for updates Kun Zang 1,7, Xiaoyu Hong1,7, Nghiem D. Nguyen2, Loraine M. Rourke3, Jiwon Lee4, Benedict M. Long 5,6, G. Dean Price3 & Manajit Hayer-Hartl 1 Cyanobacteria have evolved a CO2-concentrating mechanism (CCM) in the form of a microcompartment with a proteinaceous shell called carboxysome, harbouring the photosynthetic enzyme Rubisco and carbonic anhydrase (CA). β-Carboxysome assembly proceeds by an inside-out process, in which Rubisco, CA and the shell adaptor protein ApN (also known as CcmN) first form the pro-carboxysome biomolecular condensate mediated by the scaffolding protein CM (also known as CcmM). How ApN assembles into the pro-carboxysome as a prerequisite for shell formation has remained unclear. Here we show that ApN is recruited to the periphery of the pro-carboxysome as a hetero-complex of three ApN protomers and one CM protomer. The association of (ApN)3:CM at the rim of the pro-carboxysome ensures that shell formation and maturation of the carboxysome proceeds only after assembly of the two enzymes, Rubisco and CA, to form the pro-carboxysome core. These results provide mechanistic insight into a critical step of β-carboxysome assembly, informing efforts to introduce a cyanobacterial CCM into plants. Bacterial microcompartments (BMCs) are specialized proteinaceous organelles that house specific metabolic pathways1–3. A well-studied and highly biologically relevant example is the carboxysome of all cyanobacteria and many chemoautotrophic bacteria4–6. Carboxysomes enclose the key photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) together with carbonic anhydrase (CA), creating a high-CO2 microenvironment to enhance the efficiency of carbon fixation by Rubisco7–9. Understanding carboxysome biogenesis is key to both unravelling the principles of cellular compartmentalization6,10 and engineering carboxysomes into chloroplasts to enhance plant carbon fixation9,11–13. The Rubisco enzyme of α- and β-carboxysomes is a complex of ~550 kDa consisting of eight large (RbcL; ~55 kDa) and eight small (RbcS; ~13 kDa) subunits. However, the two carboxysome types differ in the sequence of their Rubisco RbcL subunits14: α-carboxysomes in proteobacteria and α-cyanobacteria contain form 1A, while β-carboxysomes found only in β-cyanobacteria contain prokaryotic form 1B Rubisco15. The prokaryotic form 1B Rubisco closely resembles the eukaryotic form of plants and green algae, making it a relevant model for chloroplast adaptation. The α- and β-carboxysomes also differ in their protein components and assembly mechanism6,16–18. The proteinaceous shell allows HCO3− entry but retards the escape of CO2 generated by CA inside the oxidizing carboxysome19–21 (Fig. 1a). This concentrates CO2 in the vicinity of Rubisco (so-called CO2-concentrating mechanism, CCM), competitively inhibiting Rubisco oxygenation, which leads to energetically costly photorespiration7,22,23. The cyanobacterial CCM also entails a mechanism to elevate HCO3− in the cytosol via active transporters in the cell membranes24,25. In addition, carboxysomal Rubisco enzymes have carboxylation properties, including high catalytic turnover rates, suited to improving carbon fixation in C3 crops Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany. 2Biochemical Science and Biochemistry Division, Research School of Biology, Australian National University, Acton, Australian Capital Territory, Australia. 3Plant Science Division, Research School of Biology, Australian National University, Acton, Australian Capital Territory, Australia. 4Centre for Advanced Microscopy, The Australian National University, Acton, Australian Capital Territory, Australia. 5ARC Centre of Excellence in Synthetic Biology, Sydney, New South Wales, Australia. 6Discipline of Biological Sciences, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, New South Wales, Australia. 7These authors contributed equally: Kun Zang, Xiaoyu Hong. e-mail: 1 Nature Plants | Volume 12 | February 2026 | 447–464 447 Article https://doi.org/10.1038/s41477-026-02227-6 RuBP c CM ApN SSUL 2 341 423 455 mK 2 mL cc Rubisco ccmO 0.6 0.4 0.2 CMCt MW ~58 kDa 197 225 SSUL1 307 rbcL rbcS 341 SSUL2 423 455 f Absorbance (280 nm) 0.8 ApN ~63.5 kDa SIIApN ~69.3 kDa 537 e 1.0 5 539 SSUL3 1 × 10 Molar mass (Da) d ccmN Carbonic anhydrase (CA) ccaA Shell proteins HCO3– 196 γCAL domain (7-turn β-helix) SSUL1 225 307 161 1 5-turn β-helix EP MW ~16 kDa 114 143 1 Scaffolding Adaptor protein protein (CM) (ApN) ccmM ccmM – HCO3 cc cc mP mK 3 cc CO2 ccm operon mK 4 Selectively permeable proteinaceous shell Reducing agents Rubisco CA b cc 3PGA a B A MW ~37 kDa A N11 C114 537 B C114 C 539 SSUL3 N11 C A C114 N11 C N11 A B C 0 4 1 × 10 34 36 38 40 42 50 Å 44 B C114 Time (min) g Wall A A114 C B Wall C A114 R 85 C B Rubisco AF647 D39 E 19 AF488 (CA)4 + (CM)3 0.25 µM AF488 R 59 R 85 E 37 E 43 D12 R 13 + (ApN)4 0.5 µM 10 min I64 G60 G61 R 41 D39 E 19 D25 S 11 10 min S 113 L111 I82 A38 + Hydrophobicity scale Rubisco (CA)4 CMCt + + 0.5 µM 0.125 µM 2 µM AF568 G60 Interacting surfaces Wall A E 101 R 41 90° – Wall C E 101 I82 S 11 i Wall A A A h Interface Interface Wall C E 101 G109 V96 T 94 S 92 V78 L76 R 59 G74 V55 H57 E 37 G53 I35 L33 L52 G31 F 15 R 13 A24 R 13 Positively charged P 108 A91 A73 L52 P 30 D12 V105 L89 C88 L71 L70 Q86 I50 C49 V27 D25 R 13 Negatively charged FM AF405 CMCt & (CM)3 Fig. 1 | Analysis of the shell adaptor protein, ApN. a, CO2-concentrating mechanism (CCM) of β-cyanobacteria. 3PGA, 3-phosphoglycerate; RuBP, ribulose-1,5-bisphosphate. b, Cartoon representation of the key carboxysome genes of β-cyanobacterium Se7942. The genes for pro-β-carboxysome assembly: ccmM, scaffolding protein CM; ccmN, shell adaptor protein ApN; rbcL/rbcS, Rubisco; ccaA, carbonic anhydrase CA. c, Domain structures of the scaffolding protein (CM), its truncated form (CMCt) and the shell adaptor protein (ApN), with approximate molecular weights indicated. d, SEC–MALS analysis of ApN and SIIApN. The theoretical molar masses of tetrameric ApN and SIIApN are 65.31 and 70.85 kDa, respectively. The measured molar masses are indicated. Representative data of 2 independent experiments are shown (n = 2). e, CryoEM single-particle analysis of ApN tetramers. Shown are representative 2D class averages from Extended Data Fig. 1b. Scale bar, 50 Å. f, AlphaFold 3 (AF3) Nature Plants | Volume 12 | February 2026 | 447–464 (ApN)4 DIC structural model in ribbon representation of (ApN)4 complex (residues 11–114) shown in end-on view with (...truncated)