{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/355811"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/355811","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Molecular Physiology of the Chlamydomonas Pyrenoid","abstract":"Operation of the CO<sub>2</sub>-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 CO<sub>2</sub> 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 CO<sub>2</sub>, 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 CO<sub>2</sub> via the CCM.","abstract_html":"Operation of the CO&lt;sub&gt;2&lt;/sub&gt;-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 CO&lt;sub&gt;2&lt;/sub&gt; 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 CO&lt;sub&gt;2&lt;/sub&gt;, 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 CO&lt;sub&gt;2&lt;/sub&gt; via the CCM.","abstract_has_math":false,"creators":["Caspari, Oliver Dominik"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Griffiths, Howard"],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09","date_published":"2015-09","updated_at":"2026-07-22T22:23:57Z","subjects":["CO2-concentrating mechanism","Rubisco","Chlamydomonas reinhardti","Pyrenoids","Chloroplast","Rubisco aggregation","Photosynthesis"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/91ec65af-b190-4afe-86c8-e8524b305077/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.100658","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Griffiths, Howard"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["BBSRC Studienstiftung"]},{"key":"dc:creator","label":"Author","values":["Caspari, Oliver Dominik"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2015-09"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/355811"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["CO2-concentrating mechanism","Rubisco","Chlamydomonas reinhardti","Pyrenoids","Chloroplast","Rubisco aggregation","Photosynthesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/91ec65af-b190-4afe-86c8-e8524b305077/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.100658"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/234b7470-5874-40e8-a786-2f047513744e/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Operation of the CO<sub>2</sub>-concentrating mechanism (CCM) in the model green alga *Chlamydomonas reinhardtii* relies on confining Rubisco in a proteinaceous microcompartment, the chloroplastic pyrenoid. 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