A phase-separated CO2-fixing pyrenoid proteome determined by TurboID in Chlamydomonas reinhardtii

Chun Sing Lau; Adam Dowle; Gavin H Thomas; Philipp Girr; Luke C M Mackinder

doi:10.1093/plcell/koad131

journal article Open Access May 17, 2023

A phase-separated CO2-fixing pyrenoid proteome determined by TurboID in Chlamydomonas reinhardtii

Chun Sing Lau

Adam Dowle Gavin H Thomas

Philipp Girr

Luke C M Mackinder

The Plant Cell Vol. 35 No. 9 pp. 3260-3279 · Oxford University Press (OUP)

View at Publisher Save 10.1093/plcell/koad131

Abstract

Abstract
Phase separation underpins many biologically important cellular events such as RNA metabolism, signaling, and CO2 fixation. However, determining the composition of a phase-separated organelle is often challenging due to its sensitivity to environmental conditions, which limits the application of traditional proteomic techniques like organellar purification or affinity purification mass spectrometry to understand their composition. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial phase-separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, we developed a TurboID-based proximity labeling technique in which proximal proteins in Chlamydomonas chloroplasts are labeled by biotin radicals generated from the TurboID-tagged protein. By fusing 2 core pyrenoid components with the TurboID tag, we generated a high-confidence pyrenoid proxiome that contains most known pyrenoid proteins, in addition to new pyrenoid candidates. Fluorescence protein tagging of 7 previously uncharacterized TurboID-identified proteins showed that 6 localized to a range of subpyrenoid regions. The resulting proxiome also suggests new secondary functions for the pyrenoid in RNA-associated processes and redox-sensitive iron–sulfur cluster metabolism. This developed pipeline can be used to investigate a broad range of biological processes in Chlamydomonas, especially at a temporally resolved suborganellar resolution.

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Details

Published: May 17, 2023
Vol/Issue: 35(9)
Pages: 3260-3279
License: View

Authors

C

Chun Sing Lau

Centre for Novel Agricultural Products, Department of Biology, University of York , York YO10 5DD , UK

A

Adam Dowle

Department of Biology, University of York , York YO10 5DD , UK

G

Gavin H Thomas

Department of Biology, University of York , York YO10 5DD , UK

P

Philipp Girr

Centre for Novel Agricultural Products, Department of Biology, University of York , York YO10 5DD , UK

L

Luke C M Mackinder

Centre for Novel Agricultural Products, Department of Biology, University of York , York YO10 5DD , UK

Cite This Article

Chun Sing Lau, Adam Dowle, Gavin H Thomas, et al. (2023). A phase-separated CO2-fixing pyrenoid proteome determined by TurboID in Chlamydomonas reinhardtii. The Plant Cell, 35(9), 3260-3279. https://doi.org/10.1093/plcell/koad131

A phase-separated CO2-fixing pyrenoid proteome determined by TurboID in Chlamydomonas reinhardtii

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