THE YEAST CYCLOPHILIN MULTIGENE FAMILY - PURIFICATION, CLONING AND CHARACTERIZATION OF A NEW ISOFORM

Cyclophilins (Cyps) constitute a highly conserved family of proteins present in a wide variety of organisms. Historically, Cyps were first identified by their ability to bind the immunosuppressive agent cyclosporin A (CsA) with high affinity; they later were found to have peptidyl-prolyl cis-trans isomerase (PPIase) activity, which catalyzes the folding of oligopeptides at proline-peptide bonds in vitro and may be important for protein folding in vivo. Cells of Saccharomyces cerevisiae contain at least two distinct Cyp-related PPIases encoded by the genes CYP1 and CYP2. A yeast strain (GL81) containing genomic disruptions of three known yeast PPIase-encoding genes [CYP1, CYP2 and RBP1 (for rapamycin-binding protein); Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] was previously constructed and found to be viable. Soluble fractions of these cells possess residual CsA-sensitive PPIase activity (2-5% of that present in wild-type cells as assayed in vitro). We have purified an approx. 18-kDa protein exhibiting PPIase activity from a soluble fraction of GL81 cells and determined that its N-terminal amino acid (aa) sequence exhibits significant homology (but nonidentity) to the Cyp1 and Cyp2 proteins. We designate the gene for this new protein, CYP3. Using a degenerate oligodeoxyribonucleotide (oligo) based on the N-terminal aa sequence, plus an internal oligo homologous to a conserved region within the portion of CYP1 and CYP2 that had been deleted in the genome, a CYP3-specific DNA fragment was generated by the polymerase chain reaction (PCR) using GL81 genomic DNA as a substrate. This PCR fragment was used as a probe to isolate CYP3 genomic and cDNA clones. Chromosome blots indicate that CYP3 maps to chromosome VIII, and therefore is unlinked to either CYP1 or CYP2. The aa sequence of the deduced protein (Cyp3) exhibits 70% and 48% identity with Cyp1 and Cyp2, respectively. Like Cyp2, Cyp3 contains an N-terminal extension which may play a role in subcellular localization. Haploid cells carrying a genomic disruption of CYP3 are viable, indicating that it is a nonessential gene. Furthermore, haploid cells carrying a genomic disruption of CYP1, CYP2, CYP3 and RBP1 are also viable, suggesting either that these enzymes do not carry out an essential function, or that additional PPIases exist which can compensate for their specific absence.