FUNCTIONS OF FKBP12 AND MITOCHONDRIAL CYCLOPHILIN ACTIVE-SITE RESIDUES IN-VITRO AND IN-VIVO IN SACCHAROMYCES-CEREVISIAE

Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans pept idyl-prolyl isomerization and bind to and mediate the effects of the i mmunosuppressants cyclosporin A and FK506. The normal cellular functio ns of these proteins, however, are unknown. We altered the active site s of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biolog ically active in vivo. In accord with previous reports, all of the mut ant enzymes had little or no detectable prolyl isomerase activity in t he standard peptide substrate-chymotrypsin coupled in vitro assay. How ever, in a variation of this assay in which the protease is omitted, t he mutant enzymes exhibited substantial levels of prolyl isomerase act ivity (5-20% of wild-type), revealing that these mutations confer sens itivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant e nzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelera ted by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 ''a ctive-site'' mutants previously identified are largely active but prot ease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R7 3A), and also the wild-type human FKBP12 enzyme, catalyze protein fold ing in vitro but lack biological activity in vivo in yeast. Our findin gs provide evidence that both prolyl isomerase activity and other stru ctural features are linked to FKBP and cyclophilin in vivo functions a nd suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.