2.1 Work performed. In carrying out this project, (i) I have successfully generated transgenic lines of the model plant Arabidopsis thaliana (in total, three lines) with an HA-tag inserted at multiple positions of the TOC channel component, Toc75, by agrobacterium-mediated transformation. Such HA-tags facilitate the purification of TOC complexes. (ii) As noted earlier, the TOC complex is fragile, and thus, it is important to design a purification protocol to protect the fragile nature of the protein complex. We have developed a methodology to rapidly purify the complex under mild solubilization conditions from chloroplast envelope membranes. This methodology includes a short solubilization time and a mild detergent. (iii) To reveal the biogenesis and assembly of the TOC complex, it is necessary to understand a specific stage where this process operates at its maximum. We have found that early plant greening is the suitable stage for this investigation. (iv) To detect the newly synthesized components in the assembly of TOC, I have labelled young plant seedlings with 35S radio-isotopes. Then, pull-down experiments were conducted to isolate and study radio-labelled (newly formed) TOC complexes from young plant seedlings. (v) To reveal the structure of TOC complex, I have successfully purified the intact and pure TOC complex, and this complex is under investigation by cryo-electron microscope.
2.2 Results achieved so far: This project provided novel insights and mechanistic details concerning the biogenesis and assembly of the TOC complex. We revealed that the central core component of the TOC complex is the master player in the biogenesis of the TOC complex, controlling the assembly and stability of all the peripheral receptor proteins; it forms a photosynthetic-type TOC configuration (TOC-P) in green chloroplasts, and a non-photosynthetic-type TOC configuration (TOC-NP) that is abundant in non-green plastids. Functionally, the preference for different substrates for protein import is the key difference between TOC-P and TOC-NP. We proposed the step-wise assembly of the TOC complex in chloroplasts, in which the central core component is the initial component in the assembly of the TOC complex, followed by its stabilization with a small peripheral subunit. In the final step, a large peripheral protein is then integrated to form a fully mature TOC complex. Our structural investigation of the plant TOC complex by cryo-electron microscopy is extended beyond the timeline of this fellowship; however, I have developed an advanced pipeline.
2.3 Exploitation and dissemination. The results of this project were thoroughly discussed within the group and departmental seminars organized monthly or annually at the Department of Biology, Oxford. Prof Paul Jarvis (PI) and I have presented the results of this project at national (Plastid Preview – 2023, Oxford) and international (EMBO meeting on Protein translocation – 2022, Spain) conferences. A further major route of dissemination is via publication; I have already published a review article in J. Cell Sci. (2023), and I am preparing a manuscript to submit to Nature Commun. or PNAS. I will update my discoveries on the lab’s webpages and/or social media, which are well viewed; and I will communicate the work to the general public by interacting with the mainstream media when opportunities arise (e.g. via the University’s News and Information Office). All research publications arising from the project will be deposited in the Oxford Research Archive, which is freely accessible. With regard to exploitation, I will take care to protect (e.g. by patent applications) new intellectual property coming from the project using procedures already established in the host laboratory. Because of the vital role that chloroplasts (and by extension chloroplast protein import) play in plant growth, productivity, and stress tolerance, the new information my work generates may enable novel crop improvement strategies.
