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Vojta, Aleksandar (2006): Protein Translocon at the Outer Envelope of Chloroplasts. Dissertation, LMU München: Fakultät für Biologie



The first step of preprotein translocation across the membranes of chloroplasts is facilitated by the Toc translocon. Aim of this work was to elucidate the dynamics and the mechanism of action of this molecular machine. The central, stably associated part of the Toc translocon, the Toc core complex, consists of the pore forming Toc75 and two receptors with GTPase activity, Toc34 and Toc159. The question of Toc159 localization was addressed since controversal results on this topic were reported. In this study, membrane localization of Toc159 was confirmed, which has further implications on the mode of its action. To understand the necessity of multiple isoforms of Toc components as found in Arabidopsis thaliana, expression analysis and tissue-specific localization were conducted. Gathered data suggested the existence of several types of the complex, assembled from different types of subunits. These complexes have different preprotein specificities. Expression analysis provided further arguments for dynamic association of the intermembrane space complex with the Toc core complex. Comparison of gene expression and protein presence of translocon subunits contradicts the function of Tic20 as a general pore for stromal targeted proteins, but not as a protein conducting channel per se. For further analysis of the Toc translocon structure and function, its purification and reconstitution into proteoliposomes was reinvestigated. To this end, a technique for liposome size determination in a single spectrophotometric measurement was developed.