Operation of the CO₂-concentrating mechanism (CCM) in the model green alga Chlamydomonas reinhardtii relies on confining Rubisco in a proteinaceous microcompartment, the chloroplastic pyrenoid. Despite a long history of research, this enigmatic structure lacks a full definition both in terms of physiological function as well as molecular composition.

The work presented here used a unique set of pyrenoid-less mutants (which differ from wild-type only with respect to the genes coding for the small subunit of Rubisco) to address two key questions: (i) the function of the pyrenoid in photosynthesis in enabling the CCM to supply Rubisco with inorganic carbon under CO₂ limiting conditions; (ii) the mechanism of Rubisco aggregation underlying pyrenoid formation.

Firstly, cells that are unable to aggregate Rubisco were found severely limited by access to CO₂, yet fully able to compensate any structural changes within the chloroplast. Secondly, in silico and in vitro analyses of Rubisco protein interactions established that a linking agent is required for Rubisco aggregation. A Rubisco interactome was characterised using native gel electrophoresis and co-IP assays followed by mass spectrometry. In a complementary approach, a forward genetic screen based on high-throughput immunofluorescence localization of Rubisco aimed to identify key pyrenoid assembly factors.

The present study combined and developed a wide range of molecular, physiological and computational techniques, to show that Rubisco aggregation forming the pyrenoid is achieved through a complex network of protein interactions in order to effectively supply CO₂ via the CCM.