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Becker, Thomas (2005): Preprotein recognition and translocation by the Toc complex. Dissertation, LMU München: Faculty of Biology
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Abstract

Preproptein recognition and translocation across the outer envelope membrane of plastids is catalysed by a proteinaceous machinery, called Toc translocon. The Toc core complex is composed of the pore forming Toc75 and the two GTP-regulated receptors Toc159 and Toc34. A main issue of this work is the nature of preprotein recognition and transfer by this Toc apparatus. It is still under debate, whether Toc34 or Toc159 is the initial receptor. Here, using proteoliposomes with reconstituted either Toc core complex or Toc159 and Toc75 and several in vitro binding analysis Toc34 was shown to act upstream of Toc159. Moreover, a certain set of preproteins engages Toc64 before passing the Toc core complex. The receptor function and the dynamic association with the Toc core complex of this protein are established. Toc64 is the central component of the intermembrane space translocon. This complex migrates at approximately 700 kDa in a BN-PAGE and contains Toc64, Tic22, Toc12 and isHsp70. Toc12 is a novel identified Toc component, which exposes a J-domain toward the intermembrane space. This domain recruits isHsp70 to the intermembrane space translocon in an ATP and preprotein dependent manner. Finally, the work addresses the molecular identity of this isHsp70. Therefore, isHsp70 was purified by chromatographic approaches and analysed by mass spectroscopy. Several peptide masses were obtained, which reveal high similarity of P. sativum isHsp70 to S. oleracea Com70.