All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae

Abstract

The cyclophilins and FK506 binding proteins (FKBPs) bind to cyclosporin A, FK506, and rapamycin and mediate their immunosuppressive and toxic effects, but the physiological functions of these proteins are largely unknown. Cyclophilins and FKBPs are ubiquitous and highly conserved enzymes that catalyze peptidyl-prolyl isomerization, a rate-limiting step during in vitro protein folding. We have addressed their functions by a genetic approach in the yeast Saccharomyces cerevisiae. Five cyclophilins and three FKBPs previously were identified in yeast. We identified four additional enzymes: Cpr6 and Cpr7, which are homologs of mammalian cyclophilin 40 that have also recently been independently isolated by others, Cpr8, a homolog of the secretory pathway cyclophilin Cpr4, and Fpr4, a homolog of the nucleolar FKBP, Fpr3. None of the eight cyclophilins or four FKBPs were essential. Surprisingly, yeast mutants lacking all 12 immunophilins were viable, and the phenotype of the dodecuplet mutant resulted from simple addition of the subtle phenotypes of each individual mutation. We conclude that cyclophilins and FKBPs do not play an essential general role in protein folding and find little evidence of functional overlap between the different enzymes. We propose that each cyclophilin and FKBP instead regulates a restricted number of unique partner proteins that remain to be identified.

Figure 3

Disruption of all 12 immunophilin genes in the dodecuplet mutant yeast strain. Genomic DNA was isolated from the isogenic wild type (JK93da) and the dodecuplet mutant strain (SMY136–1) and analyzed by PCR with primers flanking the immunophilin gene indicated above each lane. (A) PCR analysis of the cyclophilins. (B) PCR analysis of the FKBPs.

Figure 4

Transcription of several immunophilins is induced by heat shock and unfolded proteins. Wild type (WT) and mutant (Δ, which is Δcpr1 through Δcpr6, and Δfpr1 through Δfpr3) immunophilin strains were analyzed by Northern blot with probes specific to the gene indicated on the left. Fold induction was determined by normalizing the amounts of immunophilin RNA to actin RNA by PhosphorImaging. The same membrane was used for all blots except for lanes 1–4 of the FPR4 blot. Lanes 1 and 3, untreated (−). Lanes 2 and 4, treated with 10 μg/ml tunicamycin (+) for 3 hr. Lanes 5–9, a wild-type strain (JK93da) was grown at 24°C, heat-shocked at 37°C, and RNA samples were analyzed after 0, 2, 5, 10, and 30 min growth at 37°C.

Figure 1

Conservation among novel cyclophilins and FKBPs. (A) Alignment of Cpr8, Cpr4, and Cpr1. (B) Alignment of Fpr3, Fpr4, and Fpr1. Identical residues, bold.

Figure 2

The immunophilins are not required for viability. A series of yeast strains isogenic with the wild-type strain JK93d but lacking one or more of the 12 immunophilin genes were grown on yeast extract/peptone/dextrose medium for 48 hr at 30°C. (A) Strains lacking individual cyclophilins (Δcpr1 through Δcpr8) or FKBPs (Δfpr1 through Δfpr4). (B) Strains lacking all of the cyclophilins (ΔCYPs: Δcpr1 Δcpr2 Δcpr3 Δcpr4 Δcpr5 Δcpr6 Δcpr7 Δcpr8); all the FKBPs (ΔFKBPs: Δfpr1 Δfpr2 Δfpr3 Δfpr4); all cytoplasmic immunophilins (Δcytoplasmics: Δcpr1 Δcpr6 Δcpr7 Δfpr1); all of the immunophilins localized to the secretory pathway (ΔERs: Δcpr2 Δcpr4 Δcpr5 Δcpr8 Δfpr2); the two nucleolar FKBPs (Δnucleolars: Δfpr3 Δfpr4); or all 12 cyclophilins and FKBPs (Δ12: Δcpr1 Δcpr2 Δcpr3 Δcpr4 Δcpr5 Δcpr6 Δcpr7 Δcpr8 Δfpr1 Δfpr2 Δfpr3 Δfpr4); WT, isogenic wild-type strain JK93da.