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Stengel, Anna (2009): Characterisation of components and mechanisms involved in redox-regulation of protein import into chloroplasts. Dissertation, LMU München: Faculty of Biology
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Abstract

The vast majority of chloroplast proteins is encoded in the nucleus and thus has to be posttranslationally imported into the organelle, a process that is facilitated by two multimeric protein machineries, the Toc and Tic complexes (translocon at the outer/inner envelope of chloroplasts). Regulation of protein import, e.g. by redox signals, is a crucial step to adapt the protein content to the biochemical requirements of the organelle. In particular, one subunit of the Tic complex, Tic62, has been proposed as a redox sensor, whose possible function is to regulate protein import by sensing and reacting to the redox state of the organelle. To elucidate a potential redox regulation of protein import, structural features, redox-dependent properties and the evolutional origin of Tic62 were investigated. The results show that Tic62 consists of two very different modules: the N-terminal part was found to be mainly -helical and possesses dehydrogenase activity in vitro. It is furthermore an evolutionary ancient domain, as it is highly conserved in all photosynthetic organisms from flowering plants to cyanobacteria and even green sulfur bacteria. In contrast to this, the C-terminus is largely disordered and interacts specifically with ferredoxin-NADP+ oxidoreductase (FNR), a key enzyme in photosynthetic electron transfer reactions. Moreover, this domain was found to exist only in flowering plants, and thus the full-length Tic62 protein seems to be one of the evolutionary youngest Tic components. The results of this study make also clear that Tic62 is a target of redox regulation itself, as its localization and interaction properties depend on the metabolic redox state: oxidized conditions lead to fast membrane binding and interaction with the Tic complex, whereas reduced conditions cause solubilization of Tic62 into the stroma and increased interaction with FNR. This novel shuttling behaviour indicates a dynamic composition of the Tic complex. The NADP+/NADPH ratio was furthermore found to be able to influence the import efficiency of many precursor proteins. Interestingly, the import of not all preproteins depends on the stromal redox state. Hence it was proposed that not a single stable Tic translocon exists, but several Tic subcomplexes with different subunit compositions, which might mediate the import of different precursor groups in a redox-dependent or -independent fashion. Another redox signal that was analyzed in regard to an impact on protein import is the reversible reduction of disulfide bridges, which was found to affect the channel and receptor proteins of the Toc complex. The import of all proteins that use the Toc translocon for entering the chloroplast was shown to be influenced by disulfide bridge formation. Thus it can be concluded that a variety of redox signals, acting both on the Toc and Tic complexes, are able to influence chloroplast protein import.