The goal of this project is to determine the function of the cyclophilins, a family of highly conserved, ubiquitously expressed eukaryotic proteins. Cyclophilins were first identified by their high affinity binding to the immunosuppressant drug, cyclosporin A. Elucidating the function of cyclophilins will provide information about a highly conserved aspect of cell physiology and may aid in the design of new immunosuppressive drugs. To this end we undertook to clone the cyclophilin genes from the yeast Saccharomyces cerevisiae and to determine the consequences of mutations in these genes. We cloned and sequenced two cyclophilin-encoding genes (Cpr1 and Cpr3): the predicted amino acid sequences both of which are highly homologous with known mammalian cyclophilins. The predicted Cpr3 protein has a signal sequence resembling known mitochondrial targeting sequences. Both genes were disrupted by the insertion of selectable markers in their coding sequences. Surprisingly, inactivation of neither gene affected viability under standard growth conditions. However, mutants carrying the disrupted allele of the Cpr3 gene were unable to grow on lactates the sole carbon source at 35 degrees Celsius. Growth of yeast on lactate requires the transcriptional induction of the gene encoding ferrocytochrome b2. Both mRNA and activity of this enzyme are unaffected in the mutant cells. Our working hypothesis is that the Cpr3 gene product is necessary for transport of lactate into the mitochondria. We have isolated revertants of the Cpr3 mutant which are capable of growth on lactate at 37 degrees Celsius. Cloning and analysis of this second site suppressors will provide further information on the function of cyclophilins.
