Proximity-based proteomics reveals the thylakoid lumen proteome in the cyanobacterium Synechococcus sp. PCC 7002

Abstract

Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined different features of cyanobacteria to learn about the intracellular structures and their respective functions. While these studies have made great progress in understanding cyanobacterial physiology, the conventional fractionation methods used to purify cellular structures have limitations; specifically, certain regions of cells cannot be purified with existing fractionation methods. Proximity-based proteomics techniques were developed to overcome the limitations of biochemical fractionation for proteomics. Proximity-based proteomics relies on spatiotemporal protein labeling followed by mass spectrometry of the labeled proteins to determine the proteome of the region of interest. We performed proximity-based proteomics in the cyanobacterium Synechococcus sp. PCC 7002 with the APEX2 enzyme, an engineered ascorbate peroxidase. We determined the proteome of the thylakoid lumen, a region of the cell that has remained challenging to study with existing methods, using a translational fusion between APEX2 and PsbU, a lumenal subunit of photosystem II. Our results demonstrate the power of APEX2 as a tool to study the cell biology of intracellular features and processes, including photosystem II assembly in cyanobacteria, with enhanced spatiotemporal resolution.

Advances in the Understanding of the Lifecycle of Photosystem II

“…The membrane fraction which contains PratA was proposed to be the site of the ‘thylakoid convergence zones’ (also called variously biogenesis centers, thylakoid centers, or thylapses [ 142 ]), as it also contained other PSII and chlorophyll biogenesis proteins. These studies were performed by separating membrane fractions by density, but an alternative approach was recently taken by Dahlgren et al [ 145 ], which is a promising avenue for this type of investigation. The authors of that study utilized a proximity-based proteomics approach to identify components of different intracellular compartments.…”

The phase separated CO2-fixing pyrenoid proteome determined by TurboID

“…We set out to establish proximity labeling in the LLPS pyrenoid within the chloroplast of Chlamydomonas (Figure 1A). TurboID has been established in Arabidopsis (Mair and Bergmann, 2022; Zhang et al, 2019) and APEX2 in cyanobacteria (Dahlgren et al, 2021) and diatoms (Turnšek et al, 2021). To determine which approach is best suited for Chlamydomonas , we designed constructs to test both APEX2 and TurboID (Supplemental Data Set 1).…”

“…We have established TurboID-based proximity labeling in the chloroplast of the model green alga Chlamydomonas reinhardtii . Proximity labeling has proven powerful in unraveling a broad range of cellular processes and suborganelle composition in a diverse range of organisms including plants (Mair and Bergmann, 2022; Zhang et al, 2019), diatoms (Turnšek et al, 2021) and cyanobacteria (Dahlgren et al, 2021). However, until now it has not been established in plastids or Chlamydomonas .…”

Algae in Recreational Waters: An Overview within a One Health Perspective

“…Ultrasonic cell lysis consists of varying sonic pressure causing cavitation, which is able to break down both cell walls and membranes [219,220]. Physical methodologies to lyse algal and cyanobacterial cells consist of mechanical (e.g., bead beating, commonly using ceramic glass beads) and cryogenic strategies (mainly lyophilization and freeze-thawing) [218,221]. The advantage of using physical methods or ultrasonication is to avoid chemical contamination issues.…”