Molecular Physiology of the Chlamydomonas Pyrenoid

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.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2015

Author dc:creator

• Caspari, Oliver Dominik

Advisor dc:contributor.advisor

• Griffiths, Howard

• CO2-concentrating mechanism
• Rubisco
• Chlamydomonas reinhardti
• Pyrenoids
• Chloroplast
• Rubisco aggregation
• Photosynthesis

Licence

• https://www.repository.cam.ac.uk/bitstreams/91ec65af-b190-4afe-86c8-e8524b305077/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.100658

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/355811