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Molecular Physiology of the Chlamydomonas Pyrenoid https://doi.org/10.17863/CAM.100658 Caspari, Oliver Dominik Griffiths, Howard 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. University of Cambridge 2015-09 Thesis Doctoral Doctor of Philosophy (PhD) eng BBSRC Studienstiftung https://www.repository.cam.ac.uk/handle/1810/355811 https://www.repository.cam.ac.uk/bitstreams/91ec65af-b190-4afe-86c8-e8524b305077/download eb9d2d75cbfff5683a827dd2229be5c0 https://www.repository.cam.ac.uk/bitstreams/234b7470-5874-40e8-a786-2f047513744e/download 567311c7fd0ce320cb74044040d1372b https://www.rioxx.net/licenses/all-rights-reserved/ CO2-concentrating mechanism Rubisco Chlamydomonas reinhardti Pyrenoids Chloroplast Rubisco aggregation Photosynthesis