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Qbadou, Soumya (2006): Function and Topology of Toc64,a subunit of the protein translocation machinery of the chloroplast outer envelope. Dissertation, LMU München: Fakultät für Biologie



Precursor protein targeting toward surfaces of organelles is assisted by different cytosolic chaperones. The Toc translocon recognizes precursor proteins and facilitates their translocation across the outer envelope of chloroplasts. Toc64 is a subunit of the chloroplast protein import machinery. This work focuses on topological and functional properties of Toc64. The topological prediction of the protein by different programs revealed that Toc64 contains three transmembrane domains, which has been confirmed by the obtained biochemical an experimental results. It was demonstrated that the TPR domain of Toc64 is cytosolic exposed, whereas a second domain of about 30 kDa is exposed to the intermembrane space and protected by the chloroplast outer envelope, which is a part of the amidase and charged regions. Functional analysis demonstrated that Toc64 is a bi-functional preprotein receptor. First, the cytosolic exposed TPR is the docking site for Hsp90 bound precursor proteins. The Hsp90 is recognised by the clamp type TPR of Toc64. Hence, a novel mechanism in which chaperones are recruited for a specific targeting event by a membrane-inserted receptor is outlined. Second, the intermembrane space exposed domain allows the association of Toc64 with the Toc complex and is involved in precursor protein recognition and translocation across the intermembrane space. This domain also participates in the formation of the intermembrane space complex, which involves Toc12, isHsp70 and Tic22.