Functions of FKBP12 and Mitochondrial Cyclophilin Active Site Residues In Vitro and In Vivo inSaccharomyces cerevisiae

Abstract

Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites 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 biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5-20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 "active-site" mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.

Rapamycin Blocks Sexual Development in Fission Yeast through Inhibition of the Cellular Function of an FKBP12 Homolog

“…The F99Y mutant supported the ryanodine channel function (67), and the F106Y mutant complemented the slow growth phenotype observed in ⌬fpr1 (68). It is also interesting to note that the HuFKBP12 F99Y mutant and the S. cerevisiae F106Y mutant show reduced PPIase activity in the in vitro peptide cleavage assay (67,68). More recently, however, it was demonstrated that the S. cerevisiae Phe-106 mutant retained PPIase activity in a different assay that uses ribonuclease T1 as a substrate (68).…”

“…Like the F100L mutation in Fkh1, neither the F99Y mutation nor the F106Y mutation affected the protein function in vivo. The F99Y mutant supported the ryanodine channel function (67), and the F106Y mutant complemented the slow growth phenotype observed in ⌬fpr1 (68). It is also interesting to note that the HuFKBP12 F99Y mutant and the S. cerevisiae F106Y mutant show reduced PPIase activity in the in vitro peptide cleavage assay (67,68).…”

“…More recently, however, it was demonstrated that the S. cerevisiae Phe-106 mutant retained PPIase activity in a different assay that uses ribonuclease T1 as a substrate (68). It has thus been suggested by Dolinski et al (68) 3 R. Weisman, S. Finkelstein, and M. Choder, unpublished data. ϩ , or fkh1 bearing the indicated mutations were streaked on plates containing rapamycin as indicated.…”

“…In particular, mutation at the Phe-100 amino acid residue did not impair the cellular activity of Fkh1 in sexual development, despite its being one of the most conserved amino acid residues in FKBP12 sequences (21). The effects of mutations at positions corresponding to Phe-100 in human FKBP12 (F99Y) and in S. cerevisiae FPR1 (F106Y) have been studied previously (67,68). Like the F100L mutation in Fkh1, neither the F99Y mutation nor the F106Y mutation affected the protein function in vivo.…”

“…It is also interesting to note that the HuFKBP12 F99Y mutant and the S. cerevisiae F106Y mutant show reduced PPIase activity in the in vitro peptide cleavage assay (67,68). More recently, however, it was demonstrated that the S. cerevisiae Phe-106 mutant retained PPIase activity in a different assay that uses ribonuclease T1 as a substrate (68). It has thus been suggested by Dolinski et al (68) 3 R. Weisman, S. Finkelstein, and M. Choder, unpublished data.…”