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

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.