Cloning and characterization of chloroplast and cytosolic forms of cyclophilin from Arabidopsis thaliana

Cyclophilin (CyP), a protein with peptidyl-prolyl cis-trans isomerase (rotamase) activity, is the specific cellular target of cyclosporin A. We have isolated cDNA clones of two genes (designated ROC1 and ROC4) encoding CyP homologs from Arabidopsis thaliana (L.). The protein products of these genes are distinct from a previously identified Arabidopsis CyP. ROC1 is expressed in all tested plant organs and encodes a protein which is highly similar to previously described cytosolic CyP isoforms of other plants. In contrast, ROC4 is expressed only in photosynthetic organs and encodes a protein which includes an amino-terminal extension with properties of known chloroplast transit peptides. In vitro import experiments using the putative precursor protein to ROC4 showed that the protein is imported into chloroplasts where it is processed to the predicted mature size. Rotamase assays and immunoblot analysis of subcellular fractions indicate the presence of a CyP isoform in the stroma of chloroplasts but not in the thylakoid membranes or thylakoid lumen. Together, these data show that ROC4 is a novel CyP isoform which is located in the stroma of chloroplasts. In vitro chloroplast import of precursors of other chloroplast proteins was unaffected by concentrations of cyclosporin A which completely inhibit rotamase activity of chloroplast stromal CyP. Thus, this activity is not essential for protein import into chloroplasts.