Abstract
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.
Highlights
Pletely inhibit rotamase activity of chloroplast stromal Studies in transgenic flies show that the Drosophila NinaA
cyclosporin A (CsA) slows an early step in transferrin folding in human hepatoma cells, and high concentrations (10 p ~ b)lock exit of Cyclophilin (CyP)’ isan abundant, highly conserved protein this protein from the rough endoplasmic reticulum, suggesting present in virtually all organisms (Koletsky et al, 1986)
For CsAinhibition experi- resulted in the isolation of three independent clones encoding ments, CsA was added prior t o reaction initiation
Summary
Plant Material-A. thaliana (L.) Heynh. ecotypes Landsberg erectu and Columbiawere grown at 20-25 “C under continuous fluorescent and incandescent light as described by Kranz and Kirchheim (1987). Ecotypes Landsberg erectu and Columbiawere grown at 20-25 “C under continuous fluorescent and incandescent light as described by Kranz and Kirchheim (1987). Progress #91 weregrown as described by Ettinger and Theg (1991).
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