PT  - JOURNAL ARTICLE
AU  - Lauren Ann Metskas
AU  - Davi Ortega
AU  - Luke M. Oltrogge
AU  - Cecilia Blikstad
AU  - Tom Laughlin
AU  - David F. Savage
AU  - Grant J. Jensen
TI  - Rubisco forms a lattice inside alpha-carboxysomes
AID  - 10.1101/2022.01.24.477598
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.01.24.477598
4099  - http://biorxiv.org/content/early/2022/01/25/2022.01.24.477598.short
4100  - http://biorxiv.org/content/early/2022/01/25/2022.01.24.477598.full
AB  - Bacteria employ microcompartments to sequester enzymatic processes, either for purposes of protecting cellular contents from reactive intermediates or as a way of increasing reaction efficiency. In these structures, a cargo of enzymes and accessory proteins is encased within a semi-permeable protein shell that permits passage of substrates and products but restricts movement of intermediates. In addition to their importance as a component of many bacterial species’ metabolisms, microcompartments have recently become a target of protein engineering. The shells can be reassembled from purified proteins, and the full operons can be functionally expressed outside their native prokaryotes and can remain functional following purification. Despite the importance of microcompartments in prokaryotic biology and bioengineering, structural heterogeneity has prevented a complete understanding of their architecture, ultrastructure, and spatial organization. Here, we employ cryo electron tomography to image α-carboxysomes, a pseudo-icosahedral microcompartment responsible for carbon fixation. We have solved a high-resolution subtomogram average of the Rubisco cargo in situ, and determined a novel arrangement of the enzyme. We find that the H. neapolitanus Rubisco polymerizes in vivo, mediated by the small Rubisco subunit. These fibrils can further pack to form a lattice with six-fold pseudo-symmetry. This arrangement preserves freedom of motion and accessibility around the Rubisco active site and the binding sites for two other carboxysome proteins, CsoSCA (a carbonic anhydrase) and the disordered CsoS2, even at Rubisco concentrations exceeding 800 μM. This characterization of Rubisco cargo inside the α-carboxysome provides new insight into the balance between order and disorder in microcompartment organization.Competing Interest StatementThe authors have declared no competing interest.