Analysis of pyrenoid proteome in the diatom Phaeodactylum tricornutum

Diatoms are a major primary producer accounting for 20% of the global primary production. Their massive carbon fixation is driven by the CO2-concentrating mechanisms (CCM), in which CO2 is actively supplied to the Rubisco localized in the pyrenoid, a membrane-less organelle of the plastid. To promote CO2 fixation, CO2 concentration in the pyrenoid matrix is assumed to be elevated while leakage of CO2 is minimized. However, due to the lack of information on the structural basis of diatom pyrenoid, the mechanism allowing efficient CO2 supply in conjunction with pyrenoid structure remains to be elucidated. While isolation of diatom pyrenoid is technically difficult due to structural instability, here we applied in vivo crosslinking using photo amino acids (pAA) which enabled us to obtain Rubisco enriched fractions. Based on LC-MS/MS analysis of the RbcL-enriched fraction, we identified a novel protein that surrounds the pyrenoid like a shell and hence named a Pyshell (Pyrenoid shell). Using genome-edited mutants, Pyshell was demonstrated to be a critical component required for growth under CO2-limiting conditions. The comprehensive analysis and identification of the pyrenoid component gave an important insight into the molecular basis driving the oceanic primary production.