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Kovács-Bogdán, Erika (2011): Characterization of protein import channel-forming proteins in chloroplasts. Dissertation, LMU München: Faculty of Biology
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Abstract

Most chloroplast proteins are encoded for in the nucleus and have to be transported into the organelle after translation in the cytoplasm. The TOC and TIC machineries (Translocon at the Outer/Inner envelope membrane of Chloroplasts) mediate the import of these proteins across the chloroplast membranes. The major aim of this work was to characterize two TIC translocon components: Tic110, the main protein translocation channel in the inner envelope, and Tic20, which was proposed to also form a protein import channel. After a detailed study of Tic110, a topological model could be established, demonstrating that the protein is inserted into the membrane with two hydrophobic and four amphipathic helices, placing residues both to the intermembrane space and to the stromal side. The presence of highly conserved cysteine residues and experiments demonstrating that Tic110 possess a redox active disulfide bridge, which could be reduced by stromal thioredoxins in vitro, indicated that Tic110 might be a possible target for thioredoxin regulation. To explore which cysteines are involved in disulfide bridge formation, mutations were generated for conserved cysteines in Tic110. As a result, Cys492 and Cys890 were identified as possible candidates. To define the functional role of disulfide bridge(s), components of the TIC motor complex were overexpressed and purified and their interaction was analysed with Tic110 via different approaches. The second part of this work focuses on the channel activity of Tic20. Although both Tic110 and Tic20 are clearly important for plant viability and preprotein translocation, there were neither electrophysiological nor biochemical data supporting that Tic20 can form a channel. After inserting the heterologously overexpressed and purified protein into liposomes, swelling assays and electrophysiological measurements provided the first experimental evidence for the channel activity of Tic20, being a cation selective channel with a pore size of about 8-14 Å. Therefore, it was concluded that the TIC translocon consists of at least two distinct translocation channels: Firstly, Tic110 forms the main translocation pore and therefore facilitates import of most of the chloroplast-targeted preproteins. Secondly, Tic20 might be specifically required for the translocation of some possibly essential proteins. To gain further insight into the structure and function of both proteins, preliminary tests were performed for crystallization in lipidic phases. The large sample of grown crystals observed under different conditions will presumably enable to crystallize these proteins and resolve their crystal structures in the future.