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.

We have identi¢ed a novel protein on the outer membrane of Arabidopsis thaliana mitochondria. This protein displays 67% sequence identity with the 64 kDa translocase of the outer envelope membrane of chloroplasts (Toc). A mitochondrial localisation for this protein was determined by (i) its presence in the proteome of highly puri¢ed Arabidopsis mitochondria, (ii) Western blot analysis with antibodies to Toc64 from pea that indicate its presence in Arabidopsis and pea mitochondria, (iii) green £uorescent protein fusion proteins that indicate an exclusive mitochondrial localisation for this protein, and (iv) expression pro¢les in various tissue types and during development that are more similar to translocase of the outer mitochondrial membrane components than to chloroplastic Toc components. Thus Arabidopsis mitochondria contain a protein with high sequence identity to a plastid protein import receptor.

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The insertion of the outer envelope protein Toc34 from chloroplasts was studied. Toc34 was chosen as a model protein because it contains one predicted transmembrane helix at the C-terminus and a large hydrophilic N-terminal located GTPase domain, which is exposed to the cytosol. Unlike proteins located in internal chloroplast compartments, Toc34 neither contains a cleavable presequence nor uses the general import pathway. The protein can insert into the outer envelope of chloroplasts but not into the outer membrane of mitochondria. Using protein-free liposomes we showed that Toc34 is able to insert directly into the lipid bilayer. This insertion is stimulated by GTP and the presence of nonbilayer lipids, but is independent of the presence or absence of charged lipids. The topology of the protein inserted into protein-free liposomes was not exclusively directed by the positive-inside rule but by the size of the hydrophilic domain.

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