A Substrate Preference for the Rough Endoplasmic Reticulum Resident Protein FKBP22 during Collagen Biosynthesis

The biosynthesis of collagens occurs in the rough endoplasmic reticulum and requires a large numbers of molecular chaperones, foldases, and post-translational modification enzymes. Collagens contain a large number of proline residues that are post-translationally modified to 3-hydroxyproline or 4-hydroxyproline, and the rate-limiting step in formation of the triple helix is the cis-trans isomerization of peptidyl-proline bonds. This step is catalyzed by peptidyl-prolyl cis-trans isomerases. There are seven peptidyl-prolyl cis-trans isomerases in the rER, and so far, two of these enzymes, cyclophilin B and FKBP65, have been shown to be involved in collagen biosynthesis. The absence of either cyclophilin B or FKBP65 leads to a recessive form of osteogenesis imperfecta. The absence of FKBP22 leads to a kyphoscoliotic type of Ehlers-Danlos syndrome (EDS), and this type of EDS is classified as EDS type VI, which can also be caused by a deficiency in lysyl-hydroxylase 1. However, the lack of FKBP22 shows a wider spectrum of clinical phenotypes than the absence of lysyl-hydroxylase 1 and additionally includes myopathy, hearing loss, and aortic rupture. Here we show that FKBP22 catalyzes the folding of type III collagen and interacts with type III collagen, type VI collagen, and type X collagen, but not with type I collagen, type II collagen, or type V collagen. These restrictive interactions might help explain the broader phenotype observed in patients that lack FKBP22.     

Developmental regulation and coordinate reexpression of FKBP65 with extracellular matrix proteins after lung injury suggest a specialized function for this endoplasmic reticulum immunophilin

AFKBP65 (65-kDa FK506-binding protein) is an endoplasmic reticulum (ER)-localized peptidyl-prolyl cis-trans isomerase predicted to play a role in the folding and trafficking of secretory proteins. In previous studies, we have shown that FKBP65 is developmentally regulated and associates with the extracellular matrix protein, tropoelastin, during its maturation and transport through the ER. In this study, we show that FKBP65 is expressed in the lung with the same developmental pattern as tropoelastin and other matrix proteins. To test the hypothesis that FKBP65 is upregulated at times when extracellular matrix proteins are being actively synthesized and assembled, adult mice were treated with bleomycin to cause reinitiation of matrix protein production during the ensuing development of pulmonary fibrosis. After bleomycin instillation, FKBP65 expression was reactivated in the lung with a pattern similar to that observed for tropoelastin and type I collagen. Using human lung fibroblast cultures, we showed that FKBP65 does not undergo the unfolded protein response, a response associated with an upregulation of resident ER proteins that occurs after increased ER stress. When fibroblasts were treated with transforming growth factor (TGF)-beta1, which is upregulated during the development of pulmonary fibrosis and known to induce matrix production, FKBP65 expression and synthesis was also increased. Similar to type I collagen and tropoelastin, this response was completely inhibited in a dose-dependent manner by GGTI-298, a geranylgeranyl transferase I inhibitor. Treatment of fibroblasts with an inhibitor of ribonucleic acid (RNA) polymerase II after TGF-beta1 treatment showed that the effect of TGF-beta1 was not because of increased stabilization of the FKBP65 messenger RNA. In summary, we have shown that FKBP65 is highly expressed in lung development, downregulated in the adult, and can be reactivated in a coordinated manner with extracellular matrix proteins after lung injury. The expression pattern of FKBP65, which is atypical for general ER foldases, suggests that FKBP65 has a distinct set of developmentally regulated protein ligands. The response to injury, which may be in part a direct response to TGF-beta1, assures the presence of FKBP65 in the ER of cells actively producing components of the extracellular matrix.     

Developmental regulation of FKBP65. An ER-localized extracellular matrix binding-protein

FKBP65 (65-kDa FK506-binding protein) is a member of the highly conserved family of intracellular receptors called immunophilins. All have the property of peptidyl-prolyl cis-trans isomerization, and most have been implicated in folding and trafficking events. In an earlier study, we identified that FKBP65 associates with the extracellular matrix protein tropoelastin during its transport through the cell. In the present study, we have carried out a detailed investigation of the subcellular localization of FKBP65 and its relationship to tropoelastin. Using subcellular fractionation, Triton X-114 phase separation, protease protection assays, and immunofluorescence microscopy (IF), we have identified that FKBP65 is contained within the lumen of the endoplasmic reticulum (ER). Subsequent IF studies colocalized FKBP65 with tropoelastin and showed that the two proteins dissociate before reaching the Golgi apparatus. Immunohistochemical localization of FKBP65 in developing lung showed strong staining of vascular and airway smooth muscle cells. Similar areas stained positive for the presence of elastic fibers in the extracellular matrix. The expression of FKBP65 was investigated during development as tropoelastin is not expressed in adult tissues. Tissue-specific expression of FKBP65 was observed in 12-d old mouse tissues; however, the pattern of expression of FKBP65 was not restricted to those tissues expressing tropoelastin. This suggests that additional ligands for FKBP65 likely exist within the ER. Remarkably, in the adult tissues examined, FKBP65 expression was absent or barely detectable. Taken together, these results support an ER-localized FKBP65-tropoelastin interaction that occurs specifically during growth and development of tissues.     

Wheat FKBP73 functions in vitro as a molecular chaperone independently of its peptidyl prolyl cis-trans isomerase activity

Peptidyl-prolyl cis-trans isomerases (PPIases) catalyse protein folding by accelerating the slow step of cis-trans isomerisation of peptidyl-prolyl bonds. Wheat (Triticum aestivum L.) FKBP73 (wFKBP73) is a peptidyl-prolyl cis-trans isomerase belonging to the FK506-binding protein (FKBP) family. It comprises three FKBP12-like domains, tetratricopeptide repeats and a calmodulin-binding domain (CaMbd). In vitro studies indicated that wFKBP73 possesses PPIase activity, binds calmodulin and forms a heterocomplex with mammalian p23 and wheat Hsp90 in wheat-germ lysate. To further study the role of wFKBP73 we have analysed its chaperone properties. Using the thermal unfolding and aggregation of citrate synthase (CS) as a model system, we have shown that the plant wFKBP73 exhibits chaperone activity, being able to suppress CS aggregation independently of its PPIase activity. The wFKBP73 interacts transiently with non-native CS and slows down its inactivation kinetics, whereas the mammalian homologue, hFKBP52 binds tightly to CS and does not affect its rate of inactivation. Hence, the first plant FKBP shown to function as a molecular chaperone has a mode of action different from that of the mammalian FKBP52.     

Hmo1p, a high mobility group 1/2 homolog, genetically and physically interacts with the yeast FKBP12 prolyl isomerase

The immunosuppressive drugs FK506 and rapamycin bind to the cellular protein FKBP12, and the resulting FKBP12-drug complexes inhibit signal transduction. FKBP12 is a ubiquitous, highly conserved, abundant enzyme that catalyzes a rate-limiting step in protein folding: peptidyl-prolyl cis-trans isomerization. However, FKBP12 is dispensible for viability in both yeast and mice, and therefore does not play an essential role in protein folding. The functions of FKBP12 may involve interactions with a number of partner proteins, and a few proteins that interact with FKBP12 in the absence of FK506 or rapamycin have been identified, including the ryanodine receptor, aspartokinase, and the type II TGF-beta receptor; however, none of these are conserved from yeast to humans. To identify other targets and functions of FKBP12, we have screened for mutations that are synthetically lethal with an FKBP12 mutation in yeast. We find that mutations in HMO1, which encodes a high mobility group 1/2 homolog, are synthetically lethal with mutations in the yeast FPR1 gene encoding FKBP12. Deltahmo1 and Deltafpr1 mutants share two phenotypes: an increased rate of plasmid loss and slow growth. In addition, Hmo1p and FKBP12 physically interact in FKBP12 affinity chromatography experiments, and two-hybrid experiments suggest that FKBP12 regulates Hmo1p-Hmo1p or Hmo1p-DNA interactions. Because HMG1/2 proteins are conserved from yeast to humans, our findings suggest that FKBP12-HMG1/2 interactions could represent the first conserved function of FKBP12 other than mediating FK506 and rapamycin actions.     

Neurospora crassa FKBP22 is a novel ER chaperone and functionally cooperates with BiP

FK506 binding proteins (FKBPs) belong to the family of peptidyl prolyl cis-trans isomerases (PPIases) catalyzing the cis/trans isomerisation of Xaa-Pro bonds in oligopeptides and proteins. FKBPs are involved in folding, assembly and trafficking of proteins. However, only limited knowledge is available about the roles of FKBPs in the endoplasmic reticulum (ER) and their interaction with other proteins. Here we show the ER located Neurospora crassa FKBP22 to be a dimeric protein with PPIase and a novel chaperone activity. While the homodimerization of FKBP22 is mediated by its carboxy-terminal domain, the amino-terminal domain is a functional FKBP domain. The chaperone activity is mediated by the FKBP domain but is exhibited only by the full-length protein. We further demonstrate a direct interaction between FKBP22 and BiP, the major Hsp70 chaperone in the ER. The binding to BiP is mediated by the FKBP domain of FKBP22. Interestingly BiP enhances the chaperone activity of FKBP22. Both proteins form a stable complex with an unfolded substrate protein and thereby prevent its aggregation. These results suggest that BiP and FKBP22 form a folding helper complex with a high chaperoning capacity in the ER of Neurospora crassa.     

Identification of all FK506-binding proteins from Neurospora crassa

Immunophilins are intracellular receptors of immunosuppressive drugs, carrying peptidyl-prolyl cis-trans isomerase activity, with a general role in protein folding but also involved in specific regulatory mechanisms. Four immunophilins of the FKBP-type (FK506-binding proteins) were identified in the genome of Neurospora crassa. Previously, FKBP22 has been located in the endoplasmic reticulum as part of chaperone/folding complexes and FKBP13 has been found to have a dual location in the cytoplasm and mitochondria. FKBP11 is apparently located exclusively in the cytoplasm. It is not expressed during vegetative development of the fungus although its expression can be induced with calcium and during sexual development. Overexpression of the respective gene appears to confer a growth advantage to the fungus in media containing some divalent ions. FKBP50 is a nuclear protein and its genetic inactivation leads to a temperature-sensitive phenotype. None of these proteins is, alone or in combination, essential for N. crassa, as demonstrated by the isolation of a mutant strain lacking all four FKBPs.     

The cytosolic-binding protein for the immunosuppressant FK-506 is both a ubiquitous and highly conserved peptidyl-prolyl cis-trans isomerase.

We have recently isolated an abundant cytosolic protein from human T-cells which specifically binds the immunosuppressive agent, FK-506. The FK-506-binding protein (FKBP) is a member of a novel class of proteins possessing peptidyl-prolyl cis-trans isomerase activity. These proteins are believed to play an important role in accelerating the rate at which proteins fold into their native conformations. In the present study, we demonstrate that FKBP is not a lymphoid-specific protein, but is widely distributed and phylogenically conserved. FKBP, purified from three sources (a human T-lymphocyte cell line JURKAT, bovine calf thymus, and Saccharomyces cerevisiae) exhibit identical molecular weights, immunological cross-reactivities, and a high degree of NH2-terminal amino acid sequence homology. In addition, FKBP from all sources possesses peptidyl-prolyl cis-trans isomerase activity which can be specifically inhibited by FK-506. We conclude that FKBP may serve an important biological function in all eukaryotic cells.     

Cloning, mapping and mutation analysis of human geneGJB5 encoding gap junction protein β-5

By homologous EST searching and nested PCR a new human geneGJB5 encoding gap junction protein β-5 was identified.GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing ofGJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.     

Cyclosporin A slows collagen triple-helix formation in vivo: indirect evidence for a physiologic role of peptidyl-prolyl cis-trans-isomerase

Peptidyl-prolyl cis-trans-isomerase accelerates otherwise slow, rate-limiting isomerization steps during folding of proteins in vitro, but is not yet securely identified with any specific physiologic role. Peptidyl-prolyl cis-trans-isomerase and the cyclosporin A (CsA)-binding protein cyclophilin are identical, and peptidyl-prolyl cis-trans-isomerase activity is inhibited by the immunosuppressive drug CsA in vitro. To establish a possible physiologic role of peptidyl-prolyl cis-trans-isomerase, we have studied the folding of procollagen I in suspended chick embryo tendon fibroblasts. Folding of procollagen I is slowed by CsA: the time needed for 50% of the molecules to reach a completely helical confirmation is 8.5 min in the absence and 13.5 min in the presence of 5 microM CsA; and the calculated products, k x K, of the rate constant (k) and the equilibrium constant (K) of peptidyl-prolyl cis-trans isomerization are 2.10 and 1.30 s-1, respectively. In contrast, folding of purified collagen III in vitro is unaffected by CsA. In cultured human fibroblasts, CsA caused posttranslational overmodification (hydroxylation of lysine 32.1 versus 22.1%) and increased intracellular degradation (18.7 versus 12.5%), and hence decreased production (10.2 versus 13.2% of total protein synthesis) of collagens I and III, indicating that procollagen folding is slowed by CsA also in human fibroblasts. We conclude that peptidyl-prolyl cis-transisomerase (and hence cyclophilin) accelerates protein folding in living cells. Furthermore, the CsA-induced changes in collagen metabolism are reminiscent of those observed in several variants of osteogenesis imperfecta caused by structural abnormalities in the pro-collagen chains which impair helix formation.     

The cyclophilin homolog ninaA is required in the secretory pathway

In Drosophila, the major rhodopsin Rh1 is synthesized in endoplasmic reticulum (ER)-bound ribosomes of the R1-R6 photoreceptor cells and is then transported to the rhabdomeres where it functions in phototransduction. Mutations in the cyclophilin homolog ninaA lead to a 90% reduction in Rh1 opsin. Cyclophilins have been shown to be peptidyl-prolyl cis-trans isomerases and have been implicated in catalyzing protein folding. We now show that mutations in the ninaA gene severely inhibit opsin transport from the ER, leading to dramatic accumulations of ER cisternae in the photoreceptor cells. These results demonstrate that ninaA functions in the ER. Interestingly, ninaA and Rh1 also colocalize to secretory vesicles, suggesting that Rh1 may require ninaA as it travels through the distal compartments of the secretory pathway. These results are discussed in relation to the possible role of cyclophilins in protein folding and intracellular protein trafficking.     

A novel type of FKBP in the secretory pathway of Neurospora crassa

FKBPs define a subfamily of peptidyl-prolyl cis/trans isomerases (PPIases). PPIases are known to play roles in cellular protein folding, protein interactions and signal transduction. Here we describe NcFKBP22 from Neurospora crassa, a novel type of FKBP. NcFKBP22 is synthesized as a precursor protein with a cleavable signal sequence. In addition to a typical FKBP domain in the amino-terminal part mature NcFKBP22 contains a novel second domain which is unique amongst all known FKBPs. The amino acid composition of this carboxy-terminal domain is highly biased. Secondary structure predictions suggest that this domain may form an amphipathic -helix. The carboxy-terminus of NcFKBP22 is –HNEL, a potential endoplasmic reticulum (ER) retention signal, suggesting that NcFKBP22 is a resident protein of the ER.     

FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli

The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.     

Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein b-5

By homologous EST searching and nested PCR a new human gene GJB5encoding gap junction protein b-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.     

Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein β-5

By homologous EST searching and nested PCR a new human gene GJB5 encoding gap junction protein β-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.     

Extremely low penetrance of deafness associated with the mitochondrial 12S rRNA mutation in 16 Chinese families: implication for early detection and prevention of deafness

Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. We report here the clinical, genetic, and molecular characterization of 16 Chinese pedigrees (a total of 246 matrilineal relatives) with aminoglycoside-induced impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects, although these subjects share some common features: being bilateral and sensorineural hearing impairment. Strikingly, these Chinese pedigrees exhibited extremely low penetrance of hearing loss, ranging from 4% to 18%, with an average of 8%. In particular, nineteen of 246 matrilineal relatives in these pedigrees had aminoglycoside-induced hearing loss. Mutational analysis of the mtDNA in these pedigrees showed the presence of homoplasmic 12S rRNA A1555G mutation, which has been associated with hearing impairment in many families worldwide. The extremely low penetrance of hearing loss in these Chinese families carrying the A1555G mutation strongly supports the notion that the A1555G mutation itself is not sufficient to produce the clinical phenotype. Children carrying the A1555G mutation are susceptible to the exposure of aminoglycosides, thereby inducing or worsening hearing impairment, as in the case of these Chinese families. Using those genetic and molecular approaches, we are able to diagnose whether children carry the ototoxic mtDNA mutation. Therefore, these data have been providing valuable information and technology to predict which individuals are at risk for ototoxicity, to improve the safety of aminoglycoside therapy, and eventually to decrease the incidence of deafness.     

A novel type of FKBP in the secretory pathway of Neurospora crassa

FKBPs define a subfamily of peptidyl-prolyl cis/trans isomerases (PPIases). PPIases are known to play roles in cellular protein folding, protein interactions and signal transduction. Here we describe NcFKBP22 from Neurospora crassa, a novel type of FKBP. NcFKBP22 is synthesized as a precursor protein with a cleavable signal sequence. In addition to a typical FKBP domain in the amino-terminal part mature NcFKBP22 contains a novel second domain which is unique amongst all known FKBPs. The amino acid composition of this carboxy-terminal domain is highly biased. Secondary structure predictions suggest that this domain may form an amphipathic α-helix. The carboxy-terminus of NcFKBP22 is –HNEL, a potential endoplasmic reticulum (ER) retention signal, suggesting that NcFKBP22 is a resident protein of the ER.     

Ultrastructural and functional abnormalities of mitochondria in cultivated fibroblasts from α -mannosidosis patients

α-Mannosidosis is a lysosomal storage disorder caused by α-mannosidase deficiency. Clinical course of the disease ranges from severe infantile to milder juvenile type and includes mental retardation, skeletal deformities, coarse facies, hepatomegaly and hearing loss. The aim of the study was to analyse mitochondrial ultrastructure and function in cultivated fibroblasts from three patients with α-mannosidosis. All patients were homozygous for the c.2248C>T mutation in the MAN2B1 gene encoding lysosomal α-mannosidase. The mutation results in incorrect protein folding and severe decrease of α-mannosidase activity. The misfolded protein is retained by the control system of endoplasmic reticulum (ER). In analysed fibroblasts, we observed dilated ER, higher amount of aberrant mitochondria and reduced mitochondrial mass compared to controls. Respiratory chain complex IV, cytochrome c oxidase (COX), activity and the ratio between COX and citrate synthase (control enzyme) were significantly increased in comparison to controls (P < 0.05). Furthermore, the activity at least from one of other respiratory chain complexes was increased in each studied cell line. Mitochondrial membrane potential as well as reactive oxygen species production were comparable with controls. Based on our results, we hypothesize more profound effect of swelled and damaged mitochondria and ER dilatation on tissues with higher energy demand than fibroblasts have.     

Temperature-sensitive auditory neuropathy associated with an otoferlin mutation: Deafening fever!

Transient deafness associated with an increase in core body temperature is a rare and puzzling disorder. Temperature-dependent deafness has been previously observed in patients suffering from auditory neuropathy. Auditory neuropathy is a clinical entity of sensorineural deafness characterized by absent auditory brainstem response and normal otoacoustic emissions. Mutations in OTOF, which encodes otoferlin, have been previously reported to cause DFNB9, a non-syndromic form of deafness characterized by severe to profound prelingual hearing impairment and auditory neuropathy.Here we report a novel mutation in OTOF gene in a large family affected by temperature-dependent auditory neuropathy. Three siblings aged 10, 9 and 7 years from a consanguineous family were found to be affected by severe or profound hearing impairment that was only present when they were febrile. The non-febrile patients had only mild if any hearing impairment. Electrophysiological tests revealed auditory neuropathy. Mapping with microsatellite markers revealed a compatible linkage in the DFNB9/OTOF region in the family, prompting us to run a molecular analysis of the 48 exons and of the OTOF intron-exon boundaries. This study revealed a novel mutation p.Glu1804del in exon 44 of OTOF. The mutation was found to be homozygous in the three patients and segregated with the hearing impairment within the family. The deletion affects an amino acid that is conserved in mammalian otoferlin sequences and located in the calcium-binding domain C2F of the protein.     

Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones Hsp70 and Hsp60.

We studied the role of mitochondrial cyclophilin 20 (CyP20), a peptidyl-prolyl cis-trans isomerase, in preprotein translocation across the mitochondrial membranes and protein folding inside the organelle. The inhibitory drug cyclosporin A did not impair membrane translocation of preproteins, but it delayed the folding of an imported protein in wild-type mitochondria. Similarly, Neurospora crassa mitochondria lacking CyP20 efficiently imported preproteins into the matrix, but folding of an imported protein was significantly delayed, indicating that CyP20 is involved in protein folding in the matrix. The slow folding in the mutant mitochondria was not inhibited by cyclosporin A. Folding intermediates of precursor molecules reversibly accumulated at the molecular chaperones Hsp70 and Hsp60 in the matrix. We conclude that CyP20 is a component of the mitochondrial protein folding machinery and that it cooperates with Hsp70 and Hsp60. It is speculated that peptidyl-prolyl cis-trans isomerases in other cellular compartments may similarly promote protein folding in cooperation with chaperone proteins.