CPR4 (systematic name YCR069W; originally cloned as SCC3) is one of eight cyclophilins in Saccharomyces cerevisiae and the founder of the "cyclophilin C (CypC)"-type secretory-pathway subgroup, with CPR8 as its closest paralog. It is a peptidyl-prolyl cis-trans isomerase (PPIase/rotamase, EC 5.2.1.8) of the cyclophilin family, catalyzing cis-trans isomerization of peptidyl-prolyl bonds, a rate-limiting step in protein folding. CPR4 has an unusual domain architecture among cyclophilins: an N-terminal cleavable signal peptide, a central cyclophilin PPIase domain (with a predicted N-glycosylation site), and a single C-terminal transmembrane helix. This dual topology (signal peptide plus C-terminal membrane anchor) is shared with Drosophila NinaA, a membrane-anchored secretory-pathway cyclophilin required for rhodopsin folding and transport, and places CPR4 in the endoplasmic reticulum / endomembrane system; systematic and review-level localization data assign it to the vacuole, consistent with a protein that transits the secretory pathway. CPR4 is non-essential. Its catalytic residues are conserved, but the tryptophan equivalent to human cyclophilin A W121 that lines the cyclosporin A binding pocket is not conserved, predicting atypical cyclosporin A binding. No physiological in vivo substrate or specific biological role has been established.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003755 peptidyl-prolyl cis-trans isomerase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) assignment of cyclophilin PPIase activity, the defining molecular function of the family and of CPR4. Reason: CPR4 carries an intact cyclophilin-type PPIase domain (UniProt DOMAIN 55..225; Pfam PF00160; CDD cd00317) and the conserved cyclophilin catalytic/substrate-binding motifs are present and correctly positioned (inline sequence analysis, this review). PPIase is the well-supported core molecular function. This is the correct core MF term. Supporting Evidence: PMID:1803821 an open reading frame of 954 nucleotides with coding potential for a protein with high similarity to the ubiquitous cyclophilins which are both peptidyl-prolyl cis-trans isomerases and cyclosporin A-binding proteins |
| GO:0005783 endoplasmic reticulum | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic (IBA) assignment to the endoplasmic reticulum, consistent with CPR4's N-terminal signal peptide and entry into the secretory pathway. Reason: CPR4's signal peptide (UniProt SIGNAL 1..20) and C-terminal TM helix direct it into the ER / secretory pathway, so ER is a defensible localization. However, the more specific experimentally-supported steady-state localization for Cpr4 (and its paralog Cpr8) is the vacuole; the ER assignment reflects the transit compartment / family topology. Retained as a valid but non-core (localization, not molecular function) annotation; not contradicted. Supporting Evidence: PMID:15998457 Cpr4 and Cpr8 contain a single CLD domain plus a long amino-terminal signal peptide and are located in vacuoles |
| GO:0006457 protein folding | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) assignment to protein folding, the general biological process associated with cyclophilin PPIase activity. Reason: Protein folding is the family-level process for cyclophilins (PPIase accelerates the rate-limiting proline-isomerization step of folding). This general BP is appropriate for CPR4 on homology grounds. No CPR4-specific in-vivo substrate or more specific process is known (see knowledge_gaps), so the general term is retained without further specialization. Supporting Evidence: PMID:15998457 both de novo protein folding and the refolding processes following cellular membrane traffic necessitate isomerization to the cis form |
| GO:0003755 peptidyl-prolyl cis-trans isomerase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automated (InterPro/EC to GO) assignment of PPIase activity, redundant with the IBA and ISS PPIase annotations. Reason: InterPro IPR002130 (cyclophilin-type PPIase domain) and EC 5.2.1.8 map to GO:0003755; CPR4 carries this domain with intact catalytic residues. Consistent with the core molecular function; a redundant but correct electronic confirmation. Supporting Evidence: PMID:1803821 high similarity to the ubiquitous cyclophilins which are both peptidyl-prolyl cis-trans isomerases and cyclosporin A-binding proteins |
| GO:0016020 membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Automated (UniProt SubCell) assignment to membrane, based on the predicted C-terminal transmembrane helix. Reason: CPR4 has a single predicted C-terminal transmembrane helix (UniProt TRANSMEM 286..303), so membrane localization is supported. This is a general CC term; retained as a valid but non-core localization annotation. The specific membrane (ER/vacuolar endomembrane) is better captured by the ER and vacuole terms. Supporting Evidence: PMID:1803821 the other one at the carboxyl end with a structure similar to a transmembrane helix |
| GO:0000324 fungal-type vacuole | HDA PMID:26928762 One library to make them all: streamlining the creation of y... | KEEP AS NON CORE | Summary: High-throughput microscopy (HDA) assignment of CPR4 to the fungal-type vacuole. Reason: Experimental (HDA) localization from a systematic screen; concordant with the review-level statement that Cpr4 and its paralog Cpr8 are vacuolar. This is the most specific experimentally-supported steady-state localization for CPR4. Retained as a valid but non-core (localization) annotation. Note the source paper (PMID:26928762) is a genome-wide library/methods paper and does not discuss CPR4 individually; the vacuole is nonetheless supported independently by Wang & Heitman 2005. Supporting Evidence: PMID:15998457 Cpr4 and Cpr8 contain a single CLD domain plus a long amino-terminal signal peptide and are located in vacuoles |
| GO:0003755 peptidyl-prolyl cis-trans isomerase activity | ISS PMID:9371805 All cyclophilins and FK506 binding proteins are, individuall... | ACCEPT | Summary: Sequence-similarity (ISS) assignment of PPIase activity to CPR4, based on cyclophilin homology. Reason: ISS PPIase from SGD, consistent with the family assignment and CPR4's intact cyclophilin catalytic domain. Reinforces the core molecular function; correct. Supporting Evidence: PMID:9371805 Cpr8, a homolog of the secretory pathway cyclophilin Cpr4 |
| GO:0008150 biological_process | ND GO_REF:0000015 | ACCEPT | Summary: Root biological_process annotation with ND (no biological data), recording that no specific biological process has been experimentally determined for CPR4. Reason: This ND root annotation is an honest and correct statement of the current state of knowledge: despite the general protein-folding process inferred from homology, no CPR4-specific in-vivo biological role has been experimentally established (see knowledge_gaps). Root ND annotations are conventionally retained. Supporting Evidence: PMID:9371805 the physiological functions of these proteins are largely unknown |
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Download this section (compressed HTML)Q: Which secretory, membrane, or vacuolar proteins are the physiological substrates/clients of CPR4, and does their folding or transport depend on CPR4 (analogous to NinaA and rhodopsin)?
Q: Does CPR4 have measurable PPIase activity in vitro, and is that catalytic activity (versus a catalysis-independent chaperone role) required for any in vivo function?
Q: Do CPR4 and CPR8 share clients or buffer each other, revealing a conditional phenotype in the cpr4 cpr8 double mutant that is absent from either single mutant?
Experiment: Identify CPR4-proximal proteins and candidate substrates by proximity labeling (BioID/TurboID or APEX2) on tagged CPR4 in the ER/vacuolar membrane, and by co-immunoprecipitation, then test whether candidate clients misfold, mislocalize, or are destabilized in a cpr4 deletion (optionally cpr4 cpr8 double deletion) under standard and ER-stress conditions.
Hypothesis: CPR4 is a membrane-anchored, substrate-specific foldase of the secretory pathway (NinaA analog) that assists folding/transport of a restricted set of secretory or vacuolar proteins.
Experiment: Purify recombinant CPR4 (catalytic domain) and measure PPIase activity with a standard protease-coupled peptidyl-prolyl isomerization assay; determine cyclosporin A inhibition and compare to CPR1 (CypA-type). Test a catalytic-dead point mutant for loss of any cpr4-dependent phenotype to separate catalytic from chaperone roles.
Hypothesis: CPR4 has cyclophilin PPIase catalytic activity but atypical (reduced) cyclosporin A binding owing to the non-conserved W121-equivalent residue.
Experiment: Compare growth, secretory-pathway integrity, and unfolded-protein-response activation across wild-type, cpr4, cpr8, and cpr4 cpr8 strains under tunicamycin, heat, and other proteostatic stresses; quantify CPR4 expression/induction under these conditions.
Hypothesis: CPR4 function is buffered by CPR8 and/or induced under ER/folding stress.
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: No physiological in vivo substrate or client protein of CPR4 has been identified. It is unknown which secretory/membrane/vacuolar proteins (if any) require CPR4-catalyzed peptidyl-prolyl isomerization for their folding, assembly, or transport.
OPEN BIOLOGY BP_DARK
What is known: Firmly established: CPR4 is a cyclophilin-family PPIase (EC 5.2.1.8) with an intact catalytic domain, a signal peptide, and a C-terminal transmembrane anchor, localizing to the ER/secretory pathway and vacuole. What is NOT established is any specific client protein or the reaction it performs on that client in vivo.
Significance: Cyclophilins act on restricted, unique partner sets rather than as general foldases; identifying CPR4's client(s) would convert a homology-level "protein folding" annotation into a specific, mechanistically meaningful biological role and test the NinaA-analogous foldase hypothesis in yeast.
Provenance (the field's own admissions):
Gap: It is unknown whether CPR4 has measurable PPIase activity and whether that catalytic activity is required in vivo for any process. No direct biochemical measurement of CPR4 PPIase kinetics, substrate specificity, or catalysis-dependent phenotype has been reported.
OPEN BIOLOGY MF_DARK
What is known: Firmly established: the cyclophilin catalytic residues are conserved in the CPR4 sequence, so catalytic competence is predicted. What is NOT established is any experimental enzyme measurement for the CPR4 protein or a catalysis-requiring phenotype.
Significance: Cyclophilins can act as chaperones independently of catalysis; distinguishing a catalytic from a catalysis-independent role for CPR4 is prerequisite to a mechanistic annotation.
Provenance (the field's own admissions):
Gap: The extent of functional redundancy between CPR4 and its closest paralog CPR8 (and other ER/secretory folding factors) is unknown. Viability data show non-essentiality but do not test substrate-level or conditional redundancy.
OPEN BIOLOGY BP_DARK
What is known: Firmly established: CPR4 and CPR8 are paralogous vacuolar/secretory cyclophilins (same PANTHER subfamily SF568, CypC-type), and none of the eight yeast cyclophilins — nor an octuple mutant — is essential, with little evidence of functional redundancy at the level of viability. What is NOT established is whether CPR4 and CPR8 (or other folding factors) share clients or buffer one another under specific stresses.
Significance: Redundancy would explain the lack of a deletion phenotype and would guide combinatorial-mutant experiments needed to reveal the pathway CPR4 serves.
Provenance (the field's own admissions):
Gap: Whether CPR4 binds cyclosporin A is untested and predicted to be atypical: the tryptophan equivalent to human cyclophilin A W121, which lines the cyclosporin A binding pocket, is not conserved in CPR4.
OPEN BIOLOGY MF_DARK
What is known: Firmly established (this review): the entire CPR4 cyclophilin domain contains no tryptophan, and the position homologous to human CypA W121 (in the motif TAKTE-W-LDGK) is not a Trp in CPR4; the deep-research synthesis independently reports a W121-to-glutamate substitution. What is NOT established is the actual cyclosporin A affinity/sensitivity of the CPR4 protein.
Significance: Cyclosporin A binding and PPIase catalysis are structurally separable in cyclophilins; a non-canonical CsA pocket would distinguish CPR4 pharmacologically from CypA-type cyclophilins and refine the family-propagated "cyclosporin A binding" inference for this protein.
Provenance (the field's own admissions):
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