MOLECULAR ANALYSIS OF CYCLOPHILIN FUNCTION IN THE YEAST SACCHAROMYCES CEREVISIAE

Abstract

The cyclophilins are a family of proteins first identified as receptors for cyclosporin A (CsA), a cyclic peptide of fungal origin. CsA inhibits T-lymphocyte activation, and is thus a potent immunosuppressant. Although cyclophilins are ubiquitous, and highly conserved, among eukaryotes, their normal physiological functions are unknown. As the receptors for CsA, cyclophilins might be involved in regulating signal transduction pathways. Cyclophilns also have peptidyl-prolyl, cis-trans isomerase (PPIase) activity in vitro, suggesting a role in protein folding in vivo. While CsA inhibits cyclophilin's PPIase activity, this inhibition is insufficient to account for the pharmacological activity of CsA. Therefore, previous results cannot be readily synthesized into a model for cyclophilin function. The goal of this project was to define and characterize physiological roles of cyclophilins using the yeast S. cerevisiae. Three S. cerevisiae cyclophilin genes were cloned and inactivated by insertional mutagenesis. I demonstrated that one, CPR3, is necessary for the efficient metabolism of non-fermentable carbon sources. The CPR] gene product, Cpr3, is localized to the mitochondrial matrix, and a truncated version of Cpr3 expressed in bacteria binds CsA. CPR3 inactivation does not significantly compromise the induction of transcription of two nuclear cytochrome genes. Thus, Cpr3 is not necessary for the signal transduction pathway governing cytochrome gene expression. To identify biochemical targets of Cpr3, I demonstrated that inactivation of a mitochondrial lactate dehydrogenase is insufficient to account for the growth defect of cpr3 mutants. An exhaustive search for high-copy suppressors of the growth defect of cpr3 mutants led to the identification of a novel gene, JEN1, that suppresses the growth defect at elevated temperature. JEN1 encodes a protein that is probably a lactate transporter, and thus not a direct biochemical target of Cpr3. A dominant mutation in a nuclear gene, JEN2, suppresses the growth defect of cpr3 mutants on lactate at 30°C and 37°C. JEN2 might encode a direct biochemical target of Cpr3. In summary, the cyclophilin, Cpr3, plays a general role in the efficient function of yeast mitochondria, and presents an excellent model system for studying cyclophilin function.