Function and Topology of Toc64,a subunit of the protein translocation machinery of the chloroplast outer envelope

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

The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64

Precursor protein targeting toward organellar surfaces is assisted by different cytosolic chaperones. We demonstrate that the chloroplast protein translocon subunit Toc64 is the docking site for Hsp90 affiliated preproteins. Thereby, Hsp90 is recognised by the clamp type TPR domain of Toc64. The subsequent transfer of the preprotein from Toc64 to the major receptor of the Toc complex, namely Toc34, is affinity driven and nucleotide dependent. We propose that Toc64 acts as an initial docking site for Hsp90 associated precursor proteins. We outline a mechanism in which chaperones are recruited for a specific targeting event by a membrane-inserted receptor