Towards a functional characterization of a 23 kDa glycine-rich protein as integral component of the chloroplast inner envelope membrane translocase (TIC)

• Applicants: Dr. Birgit Agne; Professor Dr. Sacha Baginsky
• Subject Area: Plant Biochemistry and Biophysics ; Plant Cell and Developmental Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 570179326

We have provided evidence that Grp23 is a component of the 1-MDa TIC complex that is conserved from algae to plants. Despite its conservation and importance for chloroplast biogenesis, the molecular function of the 1-MDa TIC complex subunits -including Grp23- are largely unknown. Since grp23-1 lines do not fully lack Grp23, a functional characterization is potentially hampered by residual functional protein. We therefore generated amiRNAi lines to deplete Grp23 expression. We propose characterizing these lines in detail since they provide a unique tool to assess details of TIC functioning including moonlighting functions, TIC/TOC complex assembly, stability and alternative import pathways that may operate independently of the 1-MDa TIC complex. We will assess TOC/TIC complex composition in Grp23-silencing plants compared to non-silenced control plants by BN-PAGE analysis combined with quantitative protein complex (complexome) profiling by mass spectrometry. Furthermore, we will assess protein import competence of plastids lacking Grp23 and determine the quantitative proteome of silenced and non-silenced lines to assess the effect of Grp23-depletion on protein accumulation in plastids and in the cytosol. Therefore, the latter will be done with full cell extracts. To obtain information about redundancy in the import system, we will express the amiGrp23 construct in the tic20-IV and the tic40 single mutants or cross stable silencing lines into the single mutant background and perform phenotypic and molecular characterizations of the resulting plant lines. This approach has the potential to reveal functional overlap between the different TIC components.

• DFG Programme: Research Grants