Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.
A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.
McNew
J A
JA
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Sykes
K
K
Goodman
J M
JM
D000591:Amino Acid Isomerases / Q000378:metabolism*; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002451:Cell Compartmentation; D003432:Cross-Linking Reagents; D016572:Cyclosporine / Q000494:pharmacology; D005800:Genes, Fungal; D008830:Microbodies / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000737:chemistry; D019696:Peptidylprolyl Isomerase; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D010455:Peptides; D016572:Cyclosporine; D011392:Proline; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase