Molecular Cloning and Characterization of Aspergillus nidulans Cyclophilin B

Fungal Genetics and Biology 27, 55–66 (1999) Article ID fgbi.1999.1131, available online at http://www.idealibrary.com on

Molecular Cloning and Characterization of Aspergillus nidulans Cyclophilin B

James D. Joseph,* Joseph Heitman*,†,‡ and Anthony R. Means*,§ *Department of Pharmacology and Cancer Biology, †Department of Genetics, §Department of Medicine, and the ‡Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

Accepted for publication March 23, 1999

effect sensitivity of A. nidulans to cyclosporin A. cypB mRNA levels were significantly elevated under severe heat shock conditions, indicating a possible role for the A. nidulans cyclophilin B protein during growth in high stress environments. r 1999 Academic Press

James D. Joseph, Joseph Heitman, and Anthony R. Means 1999. Molecular Cloning and Characterization of Aspergillus nidulans Cyclophilin B. Fungal Genetics and Biology 27, 55–66. Cyclophilins are an evolutionarily conserved family of proteins which serve as the intracellular receptors for the immunosuppressive drug cyclosporin A. Here we report the characterization of the first cyclophilin cloned from the filamentous fungus Aspergillus nidulans (CYPB). Sequence analysis of the cypB gene predicts an encoded protein with highest homology to the murine cyclophilin B protein. The sequence similarity includes an N-terminal sequence predicted to target the protein to the endoplasmic reticulum (ER) as well as a C-terminal sequence predicted to retain the mature protein in the ER. The bacterially expressed hexa-histidine tagged protein displays peptidyl-prolyl isomerase activity which is inhibited by cyclosporin A. In the presence of cyclosporin A, the expressed protein also inhibits purified calcineurin. When the endogenous cypB gene was disrupted and placed under the control of the regulatable alcohol dehydrogenase promoter, the strain demonstrated no detectable growth phenotype under conditions which induce or repress cypB transcription. Induction or repression of the cypB gene also did not

Index Descriptors: A. nidulans; cyclophilin b; heat shock. Immunophilins are a class of ubiquitously expressed proteins composed of the cyclophilin and FK506 binding protein (FKBP)1 families. Cyclophilins and FKBPs are the intracellular receptors of the immunosuppressive drugs cyclosporin A and FK506, respectively. Both families possess intrinsic cis-to-trans peptidyl-prolyl isomerase activity which is inhibited by drug binding. However, the immunosuppressive actions of the drugs are not due to the inhibition of isomerase activity. The immunosuppressive activities of cyclosporin A and FK506 result from the drug–protein complexes binding and inhibiting the calcium–calmodulin-dependent protein phosphatase, calcineurin (reviewed in Kunz, 1993; Schreiber and Crabtree, 1992). Since the discovery of the first cyclophilin, many other members of this family have been identified in organisms ranging from bacteria to humans. Many higher eukaryotic systems maintain multiple cyclophilin proteins, produced by independent genes. In Saccharomyces cerevisiae, whose genome has been completely sequenced, eight independent cyclophilin genes have been identified (Dolinski et al., 1997). The four major classes of low-molecular-weight cyclophilins are A through D in mammalian systems,

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Abbreviations used: FKBP, FK506 binding protein; ER, endoplasmic reticulum; cyp, cyclophilin; YG, yeast extract glucose; MMG, minimal medium glycerol; MMD, minimal medium dextrose; MMGT, minimal medium glycerol threonine; DMSO, dimethylsulfoxide; PCR, polymerase chain reaction; DTT, dithiothreitol; CaM, calmodulin; TCA, trichloroacetic acid; bp, base pairs; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis

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corresponding to cpr1, cpr2, cpr4 and cpr3, respectively, in S. cerevisiae. Cyclophilins are classified predominantly on the basis of subcellular localization. Cyclophilin A lacks a targeting sequence and is cytoplasmic, the B and C types possess N-terminal sequences which target the proteins to the ER and secretory pathway, and cyclophilin D is mitochondrial (Dolinski and Heitman, 1997; Marks, 1996). Evidence exists that cyclophilin B participates in protein folding and in calcium signaling events within the ER. In numerous eukaryotic systems exposure to heat shock causes an increase in unfolded proteins within the ER (Kelley and Schlesinger, 1978). In response to the elevated culture temperatures, S. cerevisiae induces the transcription of a number of genes involved in protein folding including that of the cyclophilin B homologue, cpr2. The disruption of the cpr2 gene also leads to a modest twofold decrease in survival following heat shock (Sykes et al., 1993). The phenotype of the cpr2 disrupted strain, together with in vitro results that several cyclophilins can facilitate protein folding, suggests that cpr2 may play a role in protein folding in vivo. Immunolocalization studies of mammalian cells demonstrate that cyclophilin B is colocalized within the ER with calreticulin, calsequestrin, and other markers of calcium storage and stimulated release and is distinct from other ER resident proteins (Arber et al., 1992). Cyclophilin B has also been demonstrated to associate with the ER integral membrane protein CAML. Although the true physiological function of CAML is unknown, it had been demonstrated to induce calcium mobilization and subsequent NFAT activation when overexpressed in Jurkat cells (Bram and Crabtree, 1994). Finally, overexpression of cypb in mammalian cells increases the sensitivity of the cells to cyclosporin A (Bram et al., 1993). In contrast, overexpression of the other ER resident cyclophilin, cypc, has no effect on the sensitivity of the cells to cyclosporin A. Initially these results were interpreted as indicating that cypb is sublocalized with calcineurin and capable of inhibiting calcineurin-dependent NFAT activation from the ER. However, multiple studies suggest that calcineurin activation of NFAT occurs in the cytosol (Flanagan et al., 1991; Shaw et al., 1995), leading to the prediction that either cypb is unable to be transported to the ER when bound to cyclosporin A or when overexpressed cypb leaks from the ER into the cytosol where it is free to bind to cyclosporin A and inhibit calcineurin. These cumulative results suggest that mammalian cypb may play a role in protein folding and calcium signaling within the ER. We set out to investigate the physiological roles of cyclophilins in the filamentous fungus Aspergillus nidulans

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Joseph, Heitman, and Means

based on the fact that calcium–calmodulin signaling events and the activation of calcineurin in particular play a critical role in the growth of this organism (Rasmussen et al., 1994; Rasmussen et al., 1990). In this study we present the cloning and characterization of cyclophilin B from A. nidulans. Similar to other cyclophilins the Aspergillus CYPB possesses cyclosporin A-sensitive peptidyl-prolyl isomerase activity and inhibits the activity of purified calcineurin in the presence of cyclosporin A. Repression of A. nidulans cypB expression results in no obvious growth phenotype or change in cyclosporin A sensitivity. However, exposure of wild-type A. nidulans to high temperatures induces an elevation in cypB mRNA, suggesting that cypB may be involved in the heat shock response of A. nidulans.

MATERIALS AND METHODS A. nidulans Strains Used GR5 (A773; pyrG89; wA2; pyroA4) R153 (wA2; pyroA4) AlcCypB (alc:cypB; wA2; pyroA4) pAL5 #6 (A773; wA2; pyroA4)

Medium and Growth Conditions Conidia were grown in minimal media containing 50 mM glycerol (MMG), 50 mM glucose (MMD), or 50 mM glycerol plus 100 mM threonine (MMGT). Minimal media and rich media (YG) were prepared as described by Lu and Means (1993). To grow pyrG89 mutant strains the media was supplemented with 5 mM uridine and 10 mM uracil. When utilized, cyclosporin A, using DMSO as a carrier, was added after autoclaving. All strains were grown at 37°C unless otherwise indicated.

Isolation of A. nidulans cypB Genomic and cDNA Clone The cypB genomic clone was obtained by screening an A. nidulans genomic DNA library. The probe used to screen the A. nidulans genomic library was obtained by PCR using purified GR5 genomic DNA as a template and the degenerate oligonucleotides 58-GTICCIAA(G/ A)ACIG(T/C)IIIIAA(T/C)TT-38 and 58-CC(G/A)AAIACIAC(G/A)TG(T/C)TTICC(G/A)TCIA(G/A)CCA-38, where I indicates inosine. Conditions used in the PCR reaction were as follows: anneal at 25°C for 1.5 min, extend at 72°C

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Cloning and Characterization of A. nidulans Cyclophilin B

for 1.5 min, and denature at 95°C for a total of 30 cycles using Taq DNA polymerase. The final PCR product was subcloned into the pCRII vector (Stratagene) using conditions recommended by the manufacturer and sequenced using the dideoxynucleotide-termination reaction using the Sequenase kit (U.S. Biochemical Corp.). A 0.5-kb EcoRI fragment containing a PCR product predicted to encode a cyclophilin in pCRII was used to screen the ␭pAn A. nidulans genomic DNA library (gift of Dr. G. May). An initial high stringency screen yielded 11 positive plaques. Plasmid DNA containing the library insert was excised from the plaque-purified phage by the method described by Holt and May (1993). From the excised plasmid of clone 10, a 1.3-kb BamHI fragment was subcloned into pUC18 (GIBCO/BRL) and sequenced in both directions. Reverse transcriptase PCR was used to obtain the cypB cDNA clone. The RT-PCR reaction was performed as recommended by the manufacturer (Promega), using Aspergillus GR5 total RNA as a template and oligonucleotides corresponding to the predicted initiating ATG and terminating TAA codons. The RNA was prepared as described by Lu and Means (1993). The reaction products were subcloned into pCRII and sequenced.

Bacterial Expression of A. nidulans CYPB For convenience in subcloning the cypB cDNA, a BamHI restriction site was introduced at Glu26, and a HindIII site at the terminating codon by PCR using the cypB cDNA as a template and the oligonucleotides 58TCTTGTTAATGCCTCGGATCCC-38 and 58-AAGCTTTTAAAGCTCGTTGTGGCTACC-38. The resulting product was subcloned into pCRII and sequenced to ensure the absence of any PCR induced mutations. The BamHI/ HindIII product was then subcloned into pTrcHisB (Invitrogen). The hexa-histidine-tagged protein was expressed and purified as described by Cardenas et al. (1994).

Enzymatic Reactions Proline isomerization assays were performed by monitoring the chymotrypsin-mediated cleavage of the synthetic peptide N-succinyl-Ala-Xaa-Pro-Phe-p-nitroanilide spectrophotometrically, as described by Heitman et al. (1993). Purified hexa-histidine-tagged S. cerevisiae FKBP12 was used as a control. Calcineurin inhibition assays were performed using purified bovine calcineurin (Sigma) and purified inhibi-

tor-1 (gift of Dr. S. Shenolikar) as a substrate. Inhibitor-1 was phosphorylated with [␥-32P]ATP by purified bovine protein kinase A as described by Ingebritsen and Cohen, (1983). The assays were performed in 60-µl final reaction volume using the following conditions: 40 mM Tris, pH 7.5, 5 mM MgCl2, 0.1 mM MnCl2, 0.2 mM CaCl2, 0.1 mM DTT, 0.1 mg/ml BSA, 5 µM 32P-labeled Inhibitor-1, 20 ng calcineurin, 1.9 µM purified CYPB, and identical volumes of cyclosporin A in DMSO were added for final concentrations of 0 to 1000 nM. Reactions were initiated by addition of 100 nM CaM and incubated for 5 min at 30°C. Reactions were terminated by the addition 0.1 ml of 6 mg/ml BSA and 0.1 ml of 20% ice-cold TCA. Relative calcineurin activity was determined by measuring the amount of 32P in 200 µl of supernatant by liquid scintillation counting following centrifugation for 10 min at 4°C and 15000g.

Southern and Northern Analyses Genomic DNA and total RNA were isolated from washed, lyophilized mycelia as described by Lu and Means (1993). Ten micrograms of genomic DNA was digested overnight at 37°C with the appropriate restriction enzyme, phenol–chloroform extracted, ethanol precipitated, and separated by 0.8% agarose electrophoresis. Ten micrograms of total RNA was separated by 0.8% denaturing agarose gel electrophoresis. Both Southern and Northern blots were transferred to nitrocellulose membrane (zeta-probe, Bio-Rad) in 10⫻ SSC and were probed with either the full-length BamHI/HindIII cypB cDNA or a 1.4-kb BstEII fragment of the benA33 genomic clone of Aspergillus ␤-tubulin (gift of Dr. G. May) random labeled with [␣-32P]dCTP (Amersham) as described by Church and Gilbert (1984).

Generation and Analysis of AlcCypB To generate the pALCypB plasmid, the 58 end of the cypB gene was amplified by PCR using the oligonucleotides 58-CTCATCTTTGAACTTCGCAC-38 and 58-GGTCGGAAAGCTTTGGACGATG-38 and subcloned into the pCRII vector (Stratagene). The correct product was then subcloned into the SmaI site of pAL3 as a blunt-ended HindIII fragment (Waring and May, 1989). The GR5 strain of Aspergillus was transformed with pALCypB according to the protocol described by Lu and Means (1993). Positive transformants were selected for growth in the absence of uridine and uracil. To generate a nutritionally complemented control strain (pAL5#6), GR5 protoplasts were also

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transformed with the empty pAL5 vector (Doonan et al., 1991). Homologous recombination of the pALCypB plasmid was determined by Southern analysis of purified genomic DNA. Positive strains were purified by passing them three times on selective media, after which recombination was reconfirmed by repeating the Southern analysis. Proper carbon source-dependent regulation of the alcAcontrolled cypB gene was determined by Northern analysis of strains grown in minimal media with dextrose, glycerol, or glycerol plus threonine as the sole carbon source. Sensitivity of AlcCypB to cyclosporin A was determined by growth on minimal media plates with differing carbon sources. The diameters of the AlcCypB, pAL5#6, and the R153 strains were measured after 48 h of growth at 37°C.

Expression in Response to Heat Shock GR5 spores were germinated for 12 h at 37°C and then shifted to 47°C, and 50-ml aliquots were removed at 0, 30, 60, 90, 120 min. Samples were immediately filtered, washed with cold water, and lyophilized. RNA was isolated and analyzed by Northern blot. Following densometric analysis of the cypB autoradiogram, the expression level of cypB mRNA was determined by normalizing the data relative to ␤-tubulin expression and represented as fold induction compared to levels at t ⫽ 0.

RESULTS Cloning of A. nidulans Cyclophilin B To characterize the function of immunophilins in A. nidulans we sought to identify and clone cyclophilins from this organism. To this end, we generated degenerate oligonucleotides corresponding to highly conserved regions of cyclophilins for use in PCR. Using the oligonucleotides and A. nidulans genomic DNA as a template, we performed PCR utilizing low annealing temperatures. At an annealing temperature of 25°C, several PCR products were identified and subcloned. Of the subcloned and sequenced reaction products, one 465-bp product was predicted to encode a cyclophilin-related protein. Using this cyclophilin gene fragment as a probe we screened the A. nidulans ␭AN genomic DNA library using stringent conditions. Of 10 positive clones identified, 1 contained what was predicted to be a full-length cyclophilin gene. The nucleotide sequence, shown in Fig. 1, consists of 932 bp containing five predicted introns and an

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Joseph, Heitman, and Means

open reading frame of 642 bp predicted to encode a protein of 214 amino acids.2 The nucleic acid sequence of the predicted mRNA was confirmed by sequencing the cDNA isolated by reverse transcriptase PCR. The five introns of the novel cyclophilin gene all follow the basic 58 GT donor and 38 AG acceptor sites found in higher eukaryotes (Mount, 1982). As shown in Fig. 2, the amino acid sequence shares high sequence identity with B type cyclophilins and maintains conservation in the residues identified in crystal structures to be involved in peptide and cyclosporin A binding (Kallen and Walkinshaw, 1992; Theriault et al., 1993). The A. nidulans cyclophilin is most closely related to mouse and human cyclophilin B, sharing 61 and 57% identity, respectively. Two elements of the amino acid sequence suggest that the A. nidulans cyclophilin B homologue resides within the ER as is the case for the mammalian cyclophilin B counterparts. First, the N-terminal 24 amino acids of the protein possess the characteristics of an ER targeting sequence, similar to B type cyclophilins from other organisms. N-terminal targeting sequences generally consist of three components, a basic N-terminal region, a central hydrophobic core, and a polar C-terminus. The sequence serves to target the protein to translocate into the ER where it is cleaved following entry. Following the rules set forth by von Heijne (1986), the predicted site of cleavage is following Ala24. Second, the C-terminus of the protein ends with the short ER retention sequence HNEL. Although divergent from the conventional KDEL ER retention sequence, HNEL has been demonstrated to function similarly to the KDEL sequence in the receptormediated ER retention system (Bu et al., 1995; Medda and Proia, 1992). The presence of both ER determinants is rare in B type cyclophilins from mammals, but has been observed in a second ER-targeted cyclophilin of S. cerevisiae, cpr5 (Pelham and Frigerio, 1993), which is homologous to the yeast cyclophilin B homologue cpr2. On the basis of the predicted localization and sequence homology this gene is identified as the A. nidulans homologue of cyclophilin b, cypB.

Expression and Characterization of CYPB The cypB cDNA was bacterially expressed as an Nterminal hexa-histidine-tagged fusion protein to allow rapid purification. The protein was expressed without the N-terminal ER targeting sequence so that the expressed 2

Sequence data from this article have been deposited in the GenBank Database under Accession No. AF107254.

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Cloning and Characterization of A. nidulans Cyclophilin B

FIG. 1. Genomic sequence of A. nidulans cypB. Nucleic acid and predicted protein sequences were identified as described under Materials and Methods and Results. The sequence of the predicted cDNA and resulting protein was confirmed by cloning of the cDNA. The bold sequence identifies the predicted N-terminal ER targeting sequence, and the underlined sequence identifies the ER retention sequence.

tagged protein would be fully processed. Expressed in E. coli, purified by Ni2⫹ affinity column, the purified tagged protein migrates at the predicted molecular weight of 25 kda on SDS–PAGE. Following purification, CYPB was tested for intrinsic peptidyl-prolyl cis–trans isomerase activity. Isomerase activity was followed using the standard spectrophotometric assay based upon the chymotrypsin-mediated cleavage of the trans isomer of the peptide N-succinyl-Ala-Xaa-Pro-Phe-pnitroanilide (Heitman et al., 1993). When the Xaa-Pro bond is isomerized from cis to trans, chymotrypsin cleavage liberates the p-nitroanilide chromophore allowing the rate of Xaa-Pro isomerization to be measured spectrophotometrically at 395 nm. CYPB is able to catalyze the cis to trans isomerization of the Xaa-Pro bond as shown in Fig. 3. Also demonstrated in Fig. 3 is the ability of cyclosporin A to inhibit the isomerase activity of CYPB. In the presence of cyclosporin A the isomerization rate of the peptide is reduced to the rate observed without any added cyclophilin, representing the intrinsic isomerization rate of the synthetic peptide.

Because cyclosporin A inhibited the isomerase activity of CYPB, we tested whether CYPB could inhibit the activity of calcineurin in the presence of cyclosporin A. Since A. nidulans calcineurin has not yet been purified, we used purified bovine calcineurin and measured the ability of the cyclophilin–cyclosporin A complex to inhibit the dephosphorylation of phosphorylated inhibitor-1. As demonstrated in Fig. 4, the addition of A. nidulans CYPB and cyclosporin A inhibits the in vitro phosphatase activity of bovine calcineurin. Inhibition is dose-dependent with respect to cyclosporin A with a KI of approximately 3 nM (Fig. 4). This value is similar to that determined for cyclophilins from various organisms.

cypB Is Dispensable for A. nidulans Growth and Sensitivity to Cyclosporin A To determine if cypB is required for the survival of A. nidulans, a strain was generated in which the expression of cypB is regulatable. Using a promoter replacement strat-

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Joseph, Heitman, and Means

FIG. 2. Amino acid comparison of A. nidulans cyclophilin B with the highly homologous sequences of mouse cypb (M60456) (Hasel et al., 1991), human cypb (M60857) (Price et al., 1991), chicken cypb (M63553) (Caroni et al., 1991), S. cerevisiae CPR2 (X51497) (Koser et al., 1990), and S. cerevisiae CPR5 (X73142) (Pelham and Frigerio, 1993). Residues identical in greater than half of the sequence are shaded black, while homologous residues are shaded gray. The alignment was performed using FASTA and shading by BOXSHADE at Baylor College of Medicine Molecular Computational Biology Resources homepage.

egy, the endogenous cypB gene of the strain GR5 was placed under the control of the alcA promoter. In A. nidulans, the alcA gene is regulated transcriptionally in a carbon source-dependent manner. Growth of Aspergillus in glucose represses alcA transcription, glycerol allows a

FIG. 3. Cis–trans peptidyl isomerization activity of CYPB. Spectrophotometric proline isomerization assay performed as described by Heitman et al. (1993) using either 300 ng hexa-histidine-tagged CYPB (small dashed line), 300 ng tagged CYPB ⫹ 1 µM cyclosporin A (large dashed line), 300 ng hexa-histidine-tagged FKBP12 (doted line), or the absence of any protein (solid line). The graph is representative of several independent experiments.

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low basal level of transcription, and glycerol plus threonine induces transcription (Pateman et al., 1983). The GR5 strain was transformed with the pAL3 plasmid in which the 58 end of the cypB gene was cloned adjacent to the alcA promoter (pALCypB). The pAL3 plasmid contains the N. crassa pyr4 gene, which complements the pyrG89 mutation in the GR5 strain, allowing selection of positive transformants by the ability to grow in the absence of uridine and uracil (Waring and May, 1989). Analysis of a homologous integration of the pALCypB plasmid into cypB by Southern blot reveals the loss of the 2.6-kb BamHI fragment representing the endogenous cypB and the appearance of 1.85- and 1.7-kb hybridizing bands representing the disrupted locus. Following selection of approximately 200 positive ura⫹ transformants, one strain (AlcCypB) in which the pALCypB plasmid had integrated at the cypB locus was identified by Southern analysis (Fig. 5A). The positive strain was plated to a single colony three times to ensure strain purity, and Southern analysis repeated to confirm that the isolated strain maintained the pALCypB plasmid homologously integrated at the genomic cypB locus. The GR5 parental strain was transformed with the pAL5 vector (pAL5#6) containing no insert for use as a control in growth experiments. The AlcCypB strain displays carbon source-dependent

Cloning and Characterization of A. nidulans Cyclophilin B

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regulation of cypB mRNA expression. As seen in Fig. 5B, no cypB mRNA was detectable by Northern blot when AlcCypB was grown in the presence of glucose, low levels were detectable in glycerol, and high expression levels were detected when grown in glycerol and threonine. When AlcCypB was germinated at 37°C, no difference was observed in either the rate of nuclear division or the time of bud emergence regardless of the carbon source. No differences in growth rates over a 3-day period in any carbon source were observed between the AlcCypB and nutritionally complemented control strains as determined by comparing colony diameter (data not shown). Finally, growth of the AlcCypB strain was not detectably impaired when grown at either elevated or reduced culture temperatures of 32° and 42°C in any carbon source (data not shown). Thus on the basis of these observations we conclude that cypB is not essential for vegetative growth in A. nidulans. To determine whether cypB plays a role in cyclosporin A-mediated growth inhibition of A. nidulans, the ability of cyclosporin A to inhibit growth of the AlcCypB strain was compared to control strains. Growth was measured by colony diameter on minimal media plates containing glycerol, glucose, or glycerol and threonine as the sole carbon source and either untreated or treated with cyclosporin A at 0.01 to 100 µg/ml. Figure 6 shows the growth after 2 days at 32°C of the different A. nidulans strains in response to increasing concentrations of cyclosporin A.

FIG. 4. Calcineurin is inhibited in the presence of CYPB and cyclosporin A. Purified bovine calcineurin activity measured in the presence of CYPB and increasing concentrations of cyclosporin A as described under Materials and Methods. The graph is representative of three experiments performed in duplicate.

FIG. 5. Southern and Northern analyses of AlcCypB. (A) Southern analysis of the AlcCypB strain, several strains in which the pALCypB plasmid integrated randomly, and the parental GR5 strain. Performed as described under Materials and Methods. (B) Northern analysis of cypB mRNA expression in GR5 grown in MMD, and AlcCypB grown in MMG (basal), MMGT (inducing), or MMD (repressing) media. Ethidium bromide-stained gel is also shown to verify equal sample loading.

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Joseph, Heitman, and Means

FIG. 6. Sensitivity of AlcCypB to cyclosporin A. AlcCypB (open squares), pAL5#6 nutritionally complemented control strain (shaded circles), and wild-type R153 strain (shaded squares) were inoculated onto minimum media plus glycerol, glucose, or glycerol plus threonine including various concentrations of cyclosporin A; the colony diameter was measured after 3 days of growth at 37°C. The data are represented as a percentage of the colony diameter of each strain grown in the absence of any addition.

The data are presented as percentage growth of R153 grown in the absence of cyclosporin A. As can be seen, the AlcCypB strain showed no difference in growth after 48 h in inducing, repressing, or derepressing medium at any concentration of cyclosporin A when compared to wildtype A. nidulans. Although the absolute levels of growth

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inhibition vary slightly in the three different carbon sources, the IC50 of cyclosporin A for all the strains tested was approximately 500 ng/ml. Thus, increasing or decreasing expression of cypB does not confer resistance or hypersensitivity to growth inhibition by cyclosporin A in A. nidulans.

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FIG. 7. Induction of cypB mRNA in response to heat shock. A. nidulans GR5 was grown for 12 h at 37°C followed by heat shock at 47°C, and samples were collected for the indicated times, RNA was isolated, and Northern blots were performed. The fold induction of cypB is relative to the ␤-tubulin mRNA levels.

Induction of cypB mRNA in Response to Heat Shock Because cyclophilins have been implicated in mediating protein folding, we sought to determine if the A. nidulans cypB mRNA was induced under conditions which lead to increased levels of unfolded proteins in the ER. Elevation of growth temperature has been shown to increase the cellular levels of unfolded proteins (Kelley and Schlesinger, 1978). In A. nidulans the response to heat shock has not been thoroughly studied, but there is evidence that increasing the temperature of an exponentially growing culture induces the expression of several proteins whose identities are, as yet, unknown. Stephanou and Demopoulos (1986) identified the optimal A. nidulans heat shock temperature, relative to protein synthesis, to be 43°C, with little to no protein induction at 47°C. Therefore we examined the induction of mRNA following heat shock at 43° and 47°C. Under mild heat shock conditions of 43°C, cypB mRNA was slightly induced (data not shown). However, as shown in Fig. 7, when A. nidulans is subjected to the severe heat stress of 47°C, cypB mRNA is induced approximately 19-fold. Maximal cypB induction occurs at 60 min post heat shock and began to decline by 180 min post heat shock.

DISCUSSION We have explored the physiological functions of cyclophilins in A. nidulans based on previous studies that implicate cyclophilins in calcium-mediated signaling events. The requirements of a number of calcium/calmodulin-depen-

dent processes for the growth and nuclear division cycle of A. nidulans make this an ideal genetic system for study (reviewed in Nanthakumar et al., 1996). To this end, through the use of degenerate PCR, we cloned the first cyclophilin from A. nidulans. Two elements of the predicted sequence suggest this protein resides in the ER/ secretory pathway. The predicted N-terminal endoplasmic reticulum signaling sequence serves to target the protein for import into the ER where it is cleaved following import, resulting in the mature, fully processed protein. The presence of the C-terminal, HNEL retention sequence also ensures that the protein remains localized to the ER via receptor-mediated translocation. On the basis of the predicted ER localization and the high homology with B type cyclophilins from other species, we have identified this gene as the A. nidulans cyclophilin B homologue, cypB. While the degenerate PCR screen identified only cypB, multiple lines of evidence lead to the prediction that A. nidulans will express multiple cyclophilin genes. In all eukaryotic systems studied multiple cyclophilin genes have been identified, including eight in the budding yeast. The extreme sensitivity of A. nidulans to the immunosuppressive drug cyclosporin A even in the absence of CYPB also argues for the existence of an additional cyclophilin gene. Finally a recent search of the A. nidulans expressed sequence-tagged database3 identified what is predicted to be the A. nidulans cyclophilin A homologue. When expression was either induced or repressed to nondetectable levels we observed no discernible growth

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Aspergillus nidulans and the Neurospora crassa cDNA Sequencing Project, B.A. Roe, D. Kupfer, S. Clifton, R. Prade, and J. Dunlap.

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phenotype. Although the repression of cypB mRNA in the presence of glucose cannot be interpreted as an absolute absence of protein, we have previously used the alcA promoter to successfully repress the expression of the endogenous calmodulin (Lu et al., 1992), calcineurin (Nanthakumar et al., 1996), and CaM kinase (Dayton and Means, 1996) genes, all of which are essential genes, and thus growth is arrested under repressing conditions. Thus, the lack of any growth phenotype when AlcCypB is grown in glucose leads us to conclude that cypB does not play an essential role in the growth of the fungus. However, the absence of a change in the cyclosporin A sensitivity under conditions which either overexpress or repress expression was surprising. We demonstrate that in the presence of cyclosporin A, CYPB is capable of inhibiting bovine calcineurin activity in vitro, and one might have expected to see a change in cyclosporin A sensitivity in vivo. The most obvious conclusion from these results is that similar to ER resident human cyclophilin C, cypB plays no role in the cyclosporin A sensitivity of A. nidulans. Bram and colleagues (1993) identified the C-terminal 35 amino acids to be required for overexpressed cyclophilin B to affect the cyclosporin A sensitivity of Jurkat cells. Interestingly, Arber et al. (1992) first identified the evolutionarily conserved C-terminal 10 amino acids, VEKPFAIAKE, as being responsible for sublocalizing mammalian cyclophilin B within the ER. They found that when glia-derived nexin was C-terminally tagged with this peptide it was then colocalized with endogenous cyclophilin B. The ability of overexpressed cypb but not cypc to effect the cyclosporin A sensitivity of NFAT-dependent transcriptional activation in Jurkat cells was initially thought to be due to the specific sublocalization of cypb with calcineurin within the ER. However, NFAT dephosphorylation occurs in the cytoplasm, after which it is translocated to the nucleus (Flanagan et al., 1991; Shaw et al., 1995), thus it is unlikely that the inhibition of calcineurin within the ER would play a role in the inhibition of NFAT-dependent transcription. A more plausible explanation may be that either the binding of cyclosporin A to cypb inhibits its ER translocation in a manner similar to the methotrexate inhibition of tagged DHFR transport into the mitochondria (Eilers and Schatz, 1986) or the sublocalization of cypb within the ER may make it more susceptible to leaking into the cytosol when overexpressed. Ultimately, the localization of CYPB to the ER by two localization sequences could explain the inability of cypB to affect cyclosporin A sensitivity in Aspergillus. The ability of A. nidulans cypB mRNA to be induced by elevated temperatures indicates that one physiological role may be during the heat shock response. Although little is

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Joseph, Heitman, and Means

known about the heat shock response in A. nidulans, [35S]-methionine labeling experiments indicate that the levels of several proteins are elevated in response to increased temperatures. In contrast to the induction of cypB mRNA, Stephanou and Demopoulos (1986) demonstrated that the ideal temperature for protein induction is 43°C, with decreased levels observed at higher temperatures including 47°C. In our studies, however, a temperature shock of 43°C caused only a modest increase in cypB mRNA levels and a much greater response was seen at 47°C. The discrepancy in the optimal temperature for induction may lie in the fact that we are measuring either an increase in cypB transcription or mRNA stability, while Stephanou and Demopoulos measured increased protein synthesis, which may have different heat shock sensitivity. The identity of the [35S]-methionine-labeled proteins identified as heat inducible are unknown and the temperature dependence of the transcriptional activation of these genes has not yet been determined. On the basis of the similar heat shock inducibility of S. cerevisiae CPR2 and A. nidulans cypB genes, as well as the predicted similarity in subcellular localization between the two proteins, we predict that cypB plays a physiological role similar to that of CPR2. Analogous to cypB the mRNA levels of CPR2 are induced at elevated culture temperatures and CPR2 is predicted to localize to the ER. Disruption of CPR2 yields no discernible phenotype when the strain is grown under normal culture conditions; however, disruption leads to a twofold reduced ability to survive exposure to extreme temperatures (Sykes et al., 1993). The sensitivity of the ⌬CPR2 strain to elevated temperatures, coupled with the in vitro protein folding activities of cyclophilins (reviewed by Marks, 1996), led to the conclusion that CPR2 plays a role in the heat shock response of S. cerevisiae. Exposure to heat shock leads to a number of adverse affects, including increased levels of unfolded proteins within the cell (reviewed by Parcell and Lindquist, 1994). In response to heat shock virtually all organisms induce a number of proteins, including chaperones and isomerases, which are thought to promote protein folding (reviewed by Lindquist and Craig, 1988). It is our prediction that cypB functions in facilitating the proper folding of proteins within the ER, most importantly under heat shock conditions.

ACKNOWLEDGMENTS We thank Raylene Means, Eric Lim, and Maria Cardenas for technical assistance. We thank Greg May and Shirish Shenolikar for reagents. We

Cloning and Characterization of A. nidulans Cyclophilin B

also thank Scott Daigle and Sara Hook for critical reading of the manuscript. The work was supported by NIH Research Grant GM33976 to A.R.M. J.H is an investigator of the Howard Hughes Medical Institute.

REFERENCES Arber, S., Krause, K. H., and Caroni, P. 1992. s-Cyclophilin is retained intracellularly via a unique COOH-terminal sequence and colocalizes with the calcium storage protein calreticulin. J. Cell Biol. 116: 113–125. Bram, R. J., and Crabtree, G. R. 1994. Calcium signalling in T cells stimulated by a cyclophilin B-binding protein. Nature. 371: 355–358. Bram, R. J., Hung, D. T., Martin, P. K., Schreiber, S. L., and Crabtree, G. R. 1993. Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: Roles of calcineurin binding and cellular location. Mol. Cell. Biol. 13: 4760– 4769. Bu, G., Geuze, H. J., Strous, G. J., and Schwartz, A. L. 1995. 39 kDa receptor-associated protein is an ER resident protein and molecular chaperone for LDL receptor-related protein. EMBO J 14: 2269–2280. Cardenas, M. E., Hemenway, C., Muir, R. S., Ye, R., Fiorentino, D., and Heitman, J. 1994. Immunophilins interact with calcineurin in the absence of exogenous immunosuppressive ligands. EMBO J. 13: 5944–5957. Caroni, P., Rothenfluh, A., McGlynn, E., and Schneider, C. 1991. S-cyclophilin: New member of the cyclophilin family associated with secretory pathway. J. Biol. Chem. 266: 10739–10742. Church, G. M., and Gilbert, W. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81: 1991–1995. Dayton, J. S., and Means, A. R. 1996. Ca(2⫹)/calmodulin-dependent kinase is essential for both growth and nuclear division in Aspergillus nidulans. Mol. Biol. Cell. 7: 1511–1519. Dolinski, K., and Heitman, J. 1997. Peptidyl-prolyl isomerases—An overview of the cyclophilin, FKBP and parvulin families. In Guidebook to Molecular Chaperones and Protein-Folding Catalysts. (M. J. Gething, Ed.) pp. 359–369. Oxford University Press, Oxford, UK. Dolinski, K., Muir, S., Cardenas, M., and Heitman, J. 1997. All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 94: 13093–13098. Doonan, J. H., MacKintosh, C., Osmani, S., Cohen, P., Bai, G., Lee, E. Y. C., and Morris, N. R. 1991. A cDNA encoding rabbit muscle protein phosphatase 1␣ complements the Aspergillus cell cycle mutation, bimG11. J. Biol. Chem. 266: 18889–18894. Eilers, M., and Schatz, G. 1986. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature 322: 228–232. Flanagan, W. M., Corthesy, B., Bram, R. J., and Crabtree, G. R. 1991. Nuclear association of a T-cell transcription factor blocked by FK506 and cyclosporin A. Nature 352: 803–807. Hasel, K. W., Glass, J. R., Godbout, M., and Sutcliffe, J. G. 1991. An endoplasmic reticulum-specific cyclophilin. Mol. Cell. Biol. 11: 3484– 3491.

65 Heitman, J., Koller, A., Cardenas, M. E., and Hall, M. N. 1993. Identification of immunosuppressive drug targets in yeast. Methods: a Comp. Meth. Enzymol. 5: 176–187. Holt, C. L., and May, G. S. 1993. A novel phage ␭ replacement cre-lox vector that has automatic subcloning capabilities. Gene 133: 95–97. Ingebritsen, T. S., and Cohen, P. 1983. The protein phosphatases involved in cellular regulation. Eur. J. Biochem. 132: 255–261. Kallen, J., and Walkinshaw, M. D. 1992. The X-ray structure of a tetrapeptide bound to the active site of human cyclophilin A. FEBS Lett. 300: 286–290. Kelley, P. M., and Schlesinger, M. J. 1978. The effect of amino acid analogues and heat shock on gene expression in chicken embryo fibroblasts. Cell 15: 1277–1286. Koser, P. L., Sylvester, D., Livi, G. P., and Bergsma, D. J. 1990. A second cyclophilin-related gene in Saccharomyces cerevisiae. Nucleic Acids Res. 18: 1643. Kunz, J., Hall, M. N. 1993. Cyclosporin A, FK506 and rapamycin: More than just immunosuppression. TIBS 18: 334–338. Lindquist, S., and Craig, E. A. 1988. The heat shock proteins. Annu. Rev. Genet. 22: 631–677. Lu, K. P., and Means, A. R. 1993. Conditional mutants for studying functions of calmodulin in Aspergillus nidulans. Meth. Mol. Genet. 2: 255–275. Lu, K. P., Rasmussen, C. D., May, G. S., and Means, A. R. 1992. Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans. Mol. Endocrinol. 6: 365–374. Marks, A. R. 1996. Cellular functions of immunophilins. Phys. Rev. 76: 631–649. Medda, S., and Proia, R. L. 1992. The carbocylesterase family exhibits C-terminal sequence diversity reflecting the presence or absence of endoplasmic-reticulum-retention sequences. Eur. J. Biochem. 203: 801–806. Mount, S. M. 1982. A catalogue of splice junction sequences. Nucleic Acids Res. 10: 459–472. Nanthakumar, N. N., Dayton, J. S., and Means, A. R. 1996. Role of Ca⫹⫹/calmodulin binding proteins in Aspergillus nidulans cell cycle regulation. Prog. Cell Cycle Res. 2: 217–228. Parcell, D. A., and Lindquist, S. 1994. Heat shock proteins and stress tolerance. In The Biology of Heat Shock Proteins and Molecular Chaperones. (R. I. Morimoto, A. Tissieres, and C. Georgopoulos, Eds.), pp. 457–494. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Pateman, J. A., Doy, C. H., Olsen, J. E., Norris, U., Creaser, E. H., and Hynes, M. 1983. Regulation of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) in Aspergillus nidulans. Proc. R. Soc. Lond. 217: 243–264. Pelham, H. R. B., and Frigerio. 1993. A Saccharomyces cerevisiae cyclophilin resident in the endoplasmic reticulum. J. Mol. Biol. 233: 183–188. Price, E. R., Zydowsky, L. D., Jin, M. J., Baker, C. H., McKeon, F. D., and Walsh, C. T. 1991. Human cyclophilin B: A second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence. Proc. Natl. Acad. Sci. USA 88: 1903–1907. Rasmussen, C., Garen, C., Brining, S., Kincaid, R. L., Means, R. L., and Means, A. R. 1994. The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans. EMBO J. 13: 3917–3924.

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66 Rasmussen, C. D., Means, R. L., Lu, K. P., May, G. S., and Means, A. R. 1990. Characterization and expression of the unique calmodulin gene of Aspergillus nidulans. J. Biol. Chem. 265: 13767–13775. Schreiber, S. L., and Crabtree, G. R. 1992. The mechanism of action of cyclosporin A and FK506. Immun. Today 13: 136–142. Shaw, K. T.-Y., Ho, A. M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P. G. 1995. Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells. Proc. Natl. Acad. Sci. USA 92: 11205–11209. Stephanou, G., and Demopoulos, N. A. 1986. Heat shock phenomena in Aspergillus nidulans. I. The effect of heat on mycelial protein synthesis. Cur. Genet. 10: 791–796.

Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

Joseph, Heitman, and Means

Sykes, K., Gething, M.-J., and Sambrook, J. 1993. Proline isomerases function during heat shock. Proc. Natl. Acad. Sci. USA 90: 5853– 5857. Theriault, Y., Logan, T. M., Meadows, R., Yu, L., Olejniczak, E. T., Holzman, T. F., Simmer, R. L., Fesik, S. W. 1993. Solution structure of the cyclosporin A/cyclophilin complex by NMR. Nature 361: 88–91. von Heijne, G. 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14: 4683–4690. Waring, R. B., and May, G. S. 1989. Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulincoding genes. Gene 79: 119–130.