Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum

Photosynthetic organisms have evolved a wide range of strategies to circumvent the limitations of Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). A carbon concentrating mechanism (CCM) is a strategy that collectively describes the organism’s method to increase substrate CO2 for Rubisco to...

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Main Author: Ang, Warren Shou Leong
Other Authors: Oliver Mueller-Cajar
Format: Thesis-Doctor of Philosophy
Language:English
Published: Nanyang Technological University 2023
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Online Access:https://hdl.handle.net/10356/164697
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spelling sg-ntu-dr.10356-1646972023-03-06T07:30:04Z Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum Ang, Warren Shou Leong Oliver Mueller-Cajar School of Biological Sciences cajar@ntu.edu.sg Science::Biological sciences Photosynthetic organisms have evolved a wide range of strategies to circumvent the limitations of Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). A carbon concentrating mechanism (CCM) is a strategy that collectively describes the organism’s method to increase substrate CO2 for Rubisco to achieve maximum functional capacity. Herein, we characterize part of the CCM of the diatom Phaeodactylum tricornutum through in vitro reconstitution of its pyrenoid. Studies prior to this project resulted in the discovery of an intrinsically disordered pyrenoid scaffold protein comprised of six tandem repeat sequences, Pyrenoid component 1 (PYCO1) which can form a dense phase to specifically recruit P. tricornutum Rubisco (PtRubisco). It was established that these interactions occur through “KWSPR/Q” motifs on PYCO1 with PtRubisco small subunits. In this study, more detailed characterization of these biomolecular condensates were carried out which elucidated several key findings. Rubisco was found to be enriched up to 200-fold in these condensates. Each PYCO1 molecule sequesters up to 3 units of Rubisco holoenzymes in fully-saturated condensates. The mobility of components in these condensates also change according to Rubisco occupancy. Cryo-EM structural analysis confirmed the interaction of the “KWSPR” motif with Rubisco. Additional motifs on the C-terminus of PYCO1 also interact with Rubisco through an alpha helical motif conserved within P. tricornutum. The data suggests that the “KWSPR” stickers must dimerize prior to binding the SSU of Rubisco. Specific aromatic residues on PYCO1 are required for both heterotypic and homotypic phase separation of the system. Additional pulldown experiments on pyrenoid components led to the identification of a novel protein with the accession number Phatr3_J47612. Phatr3_J47612 and its fragments partition into PYCO1 condensates and antagonize the recruitment of PtRubisco. The likely next steps in this project would be directed towards a systematic study of the interplay of several pyrenoidal proteins to develop our understanding of the diatom CCM. Doctor of Philosophy 2023-02-13T02:00:12Z 2023-02-13T02:00:12Z 2022 Thesis-Doctor of Philosophy Ang, W. S. L. (2022). Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/164697 https://hdl.handle.net/10356/164697 10.32657/10356/164697 en This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). application/pdf Nanyang Technological University
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
spellingShingle Science::Biological sciences
Ang, Warren Shou Leong
Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum
description Photosynthetic organisms have evolved a wide range of strategies to circumvent the limitations of Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). A carbon concentrating mechanism (CCM) is a strategy that collectively describes the organism’s method to increase substrate CO2 for Rubisco to achieve maximum functional capacity. Herein, we characterize part of the CCM of the diatom Phaeodactylum tricornutum through in vitro reconstitution of its pyrenoid. Studies prior to this project resulted in the discovery of an intrinsically disordered pyrenoid scaffold protein comprised of six tandem repeat sequences, Pyrenoid component 1 (PYCO1) which can form a dense phase to specifically recruit P. tricornutum Rubisco (PtRubisco). It was established that these interactions occur through “KWSPR/Q” motifs on PYCO1 with PtRubisco small subunits. In this study, more detailed characterization of these biomolecular condensates were carried out which elucidated several key findings. Rubisco was found to be enriched up to 200-fold in these condensates. Each PYCO1 molecule sequesters up to 3 units of Rubisco holoenzymes in fully-saturated condensates. The mobility of components in these condensates also change according to Rubisco occupancy. Cryo-EM structural analysis confirmed the interaction of the “KWSPR” motif with Rubisco. Additional motifs on the C-terminus of PYCO1 also interact with Rubisco through an alpha helical motif conserved within P. tricornutum. The data suggests that the “KWSPR” stickers must dimerize prior to binding the SSU of Rubisco. Specific aromatic residues on PYCO1 are required for both heterotypic and homotypic phase separation of the system. Additional pulldown experiments on pyrenoid components led to the identification of a novel protein with the accession number Phatr3_J47612. Phatr3_J47612 and its fragments partition into PYCO1 condensates and antagonize the recruitment of PtRubisco. The likely next steps in this project would be directed towards a systematic study of the interplay of several pyrenoidal proteins to develop our understanding of the diatom CCM.
author2 Oliver Mueller-Cajar
author_facet Oliver Mueller-Cajar
Ang, Warren Shou Leong
format Thesis-Doctor of Philosophy
author Ang, Warren Shou Leong
author_sort Ang, Warren Shou Leong
title Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum
title_short Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum
title_full Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum
title_fullStr Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum
title_full_unstemmed Characterization of a rubisco condensate derived from the red type pyrenoid of Phaeodactylum tricornutum
title_sort characterization of a rubisco condensate derived from the red type pyrenoid of phaeodactylum tricornutum
publisher Nanyang Technological University
publishDate 2023
url https://hdl.handle.net/10356/164697
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