[P008] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P04-552] Linking structure to function: a pyrenoid-associated protein controls CO₂ concentration in a marine diatom

○Masakazu Toyoshima¹, Ginga Shimakawa², Yusuke Matsuda¹ (1. Kwansei Gakuin University (Japan), 2. Osaka University (Japan))

Keywords:

diatom,CO2-concentrating mechanism,pyrenoid,photosynthesis,PyShell

The marine diatom Phaeodactylum tricornutum thrives in environments where CO₂ is often limited. To overcome this constraint, it employs a CO₂-concentrating mechanism (CCM) that increases the local CO₂ concentration around the key photosynthetic enzyme Rubisco. This process takes place in a specialized compartment within the chloroplast called the pyrenoid. Recent studies suggest that the pyrenoid behaves like a liquid-like assembly, enabling dynamic organization of proteins to support efficient carbon fixation.Surrounding the pyrenoid, additional protein layers are thought to regulate its structure and function. In 2024, our laboratory identified a novel structure termed the Pyrenoid Shell (PyShell), which forms a distinct boundary around the pyrenoid. We hypothesized that this structure plays an important role in delivering CO₂ to the pyrenoid, but the underlying molecular connections remained unclear. In this study, we aimed to identify proteins that link the outer PyShell structure to the inner enzyme-rich core. To do so, we used a proximity labeling approach (TurboID), which allows detection of proteins located near PyShell in living cells. This analysis identified several candidate proteins, among which we focused on two previously uncharacterized factors, Pt46709 and Pt46710. Fluorescence imaging of GFP-tagged proteins revealed that both candidates localize to the pyrenoid, suggesting that they are part of the pyrenoid-associated protein network. To assess their function, we generated gene knockout strains using CRISPR/Cas9 nickase. Under ambient CO₂ conditions, disruption of Pt46710 led to a clear growth defect, whereas loss of Pt46709 had only a minor effect.To further evaluate photosynthetic performance, we measured the response to dissolved inorganic carbon (DIC). The Pt46710 mutant showed a marked decrease in apparent affinity for inorganic carbon, indicating impaired CCM function. These results suggest that Pt46710 is required for efficient delivery or retention of CO₂ within the pyrenoid. Overall, our findings identify Pt46710 as a key factor linking pyrenoid structure to function in a marine diatom. This work provides new insight into how microscopic cellular structures enhance photosynthetic efficiency and may inform future efforts to improve carbon fixation in other organisms.