TPRc1 :A novel chaperone recepetor at the chloroplast outer membrane.

The correct targeting of proteins to their organelles is crucial for the organisation and viability of a cell. Although mitochondria and chloroplasts have their own genome, over 90% of their protein content is encoded by nuclear DNA and needs to be imported from the cytosol after translation. In this case a cytosolic preprotein-complex is recognized by receptors, which are localized at the outer membrane of the organelle. Cytosolic chaperones like the heat shock protein 70 (Hsc70) and 90 (Hsp90) have been found to be part of the preprotein-complex to keep the proteins in an unfolded, targeting compatible state. The general import receptors, Tom70 at the outer membrane of mitochondria, and Toc64 at the chloroplast envelope, have been shown to bind these chaperones via a tetratricopeptide repeat (TPR) domain i.e. are chaperone receptors. A search for membrane proteins containing a TPR domain resulted in an uncharacterised protein from Arabidopsis thaliana named TPRc1. Its sequence includes an N-terminal TPR domain and a C-terminal membrane anchor suggesting that TPRc1 is a chaperone receptor. In this work, phylogenetic comparison of the TPR domain from TPRc1 showed that TPRc1 is most closely related to uncharacterised plant proteins. Comparison of the TPR domain from TPRc1 to Arabidopsis proteins resulted in a close similarity between the TPR domain of TPRc1 and the Hsp90 binding TPR domains of PPIases. A comparison with TPR domains from other chaperone receptors showed that the TPR domain of TPRc1 is most closely related to the TPR domain of Toc64. According to quantitative real time RNA analysis and western blotting TPRc1 is expressed in all tissues, but highest protein levels can be detected in buds, flowers, siliques and roots. Evidence from confocal microscopy and targeting assays supports localisation to the chloroplast envelope, with the N-terminus including the TPR domain, facing the cytosol. Pulldown assays suggest that the TPR domain of TPRc1 is able to pull down Hsc70 via interaction with the C-terminal end of Hsc70, and that TPRc1 is able to interact specifically with chloroplast precursor complexes. Crosslinking and immunoprecipitation of radiolabeled, into chloroplasts imported TPRc1 resulted in no adducts, suggesting that TPRc1 is isolated in the membrane. Phenotyping of knockout mutants has not been possible so far, since a T-DNA insertion line with an insertion inside an exon of the TPRc1 gene is not available. Taken together, we propose TPRc1 to be a novel Hsc70 binding plant chaperone receptor, which is involved into preprotein targeting to chloroplast similar to Toc64.