Abstract
Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer-dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO2-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO2-fixing modules to improve photosynthesis.
Highlights
Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria
To study the roles of Rubisco assembly factor 1 (Raf1) in cyanobacteria, a raf1 deletion Synechococcus elongatus PCC7942 (Syn7942) mutant (Δraf1) was generated (SI Appendix, Fig. S1A and Materials and Methods), and the genomic homogeneity and full deletion of raf1 were confirmed by PCR analysis (SI Appendix, Fig. S1B)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis using an anti-RbcL antibody revealed an ∼33% reduction in RbcL content in the soluble fraction of the total cell lysate in the Δraf1 mutant compared with WT (Fig. 1D)
Summary
Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. CcaA was proposed to localize in the inner shell surface and to dehydrate bicarbonate that diffuses through the shell to CO2 within the carboxysome lumen These naturally occurring architectural features of carboxysomes permit generation of elevated levels of CO2 around Rubisco, thereby enhancing carbon fixation [18]. Cyanobacteria are keystone organisms in global carbon fixation Their great carbon-assimilation capability arises from a specialized virus-like protein organelle, the carboxysome, which comprises hundreds of proteins that form a shell to encapsulate the CO2-fixing enzymes Rubisco and carbonic anhydrase. How do these proteins self-assemble to construct the defined architecture? Our results suggest a model of the Raf1-mediated biogenesis of carboxysomes and provide advanced knowledge of carboxysome assembly and function, informing synthetic engineering of functional CO2-fixing organelles for biotechnological applications
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
