YEAST VOL. 12: 943-952 (1996) Identification of Two CyP-40-like Cyclophilins in Saccharomyces cerevisiae, One of Which is Required for Normal Growth ANDREA A. DUINA, JAMES A. MARSH AND RICHARD F. CABER* Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University 2153 Sheridan Rd, Evanston, IL 60208, U . S 4. Received 20 March 1996; accepted 2 April 1996 We report the analysis of two Saccharomyces cerevisiae cyclophilins, Cpr6 and Cpr7, identified by their ability to interact in vivo with the transcriptional regulator Rpd3. Both cyclophilins have an extended carboxy-terminal region containing a three-unit tetratricopeptide repeat (TPR) motif and share significant amino acid identity with the mammalian cyclophilin CyP-40. Neither CPR6 nor CPR7 is essential but deletion of CPR7 results in a significant impairment of the rate of cell division. This is the first demonstration that a member of the cyclophilin family is required for normal cell growth,-’The nucleotide sequences encoding CPR6 and CPR7 have been deposited in GenBank (U48867 and U48868). KEY WORDS - Saccharomyces cerevisiae; cyclophilin; transcriptional regulator; budding yeast. INTRODUCTION Cyclophilins constitute a large family of proteins highly conserved throughout evolution. Members of this family are present in a wide variety of tissues and are localized in multiple cellular compartments (Galat, 1993). In vitro, they catalyse the cis-trans isomerization of proline peptide bonds in short polypeptides (Fischer et al., 1989; Takahashi et al., 1989) and can accelerate the slow refolding steps of a number of proteins (Lang et al., 1987; Freskgard et al., 1992). The immunosuppressive drug cyclosporin A (CsA) binds cyclophilins (Handschumacher et al., 1984; Galat, 1993) and inhibits their isomerase activity (Fischer et al., 1989; Takahashi et al., 1989). A complex formed between CsA and its major intracellular receptor, CyP-18 (Handschumacher et al., 1984), mediates the immunosuppressive activity of the drug. The CyP- 18/CsA complex binds and inhibits the Ca2+- and calmodulin-dependent phosphatase calcineurin, a key component of the T-lymphocyte activation pathway (Liu et al., 1991b, 1992; Clipstone and Crabtree, 1992). *Corresponding author. CCC 0749-503x/96/100943-10 0 1996 by John Wiley & Sons Ltd Cyclophilins have been implicated in the proper folding and hence maturation of some proteins through the secretory pathway. For example, the Drosophila ninaA protein, an integral membrane cyclophilin localized in the secretory pathway, has been shown to be required for the proper synthesis of Rhl and Rh2 rhodopsin in photoreceptor cells (Stamnes et al., 1991). Similarly, cyclophilins are involved in the maturation of procollagen (Smith et al., 1999, homo-oligomeric ligand-gated ion channels (Helekar et al., 1994), and transferrin (Lodish and Kong, 1991) within the secretory pathway. Additional studies implicate cyclophilins in collagen triple helix formation (Steinmann et al., 1991), apoptosis (Montague et al., 1994), calciumsignal transduction (Bram and Crabtree, 1994), HIV-1 replication cycle (Luban et al., 1993; Franke et al., 1994; Thali et al., 1994), mitochondrial folding events (Matouschek et al., 1995; Rassow et al., 1995) and steroid receptor activity (Ratajczak et al., 1993). Cyclophilins have also been implicated in transcriptional regulation. Yang et al. (1995) have recently shown that CyP-18 binds the mammalian transcription factor YY 1 and can regulate its activity. In other studies, exposure to CsA resulted in 944 increased expression of the gene encoding laccase (Zac-1) in the filamentous fungus Cryphonectria parasitica (Larson and Nuss, 1993), and stimulated expression of a reporter gene under the control of the glucocorticoid receptor in mouse fibroblasts (Renoir et al., 1995). We report the identification of two Saccharomyces cerevisiae cyclophilins, Cpr6 and Cpr7, through their interaction with the global transcriptional regulator Rpd3 (Vidal and Gaber, 1991). Rpd3 was identified as a transcriptional repressor of the potassium transporter gene, TRK2 (Vidal et al., 1990) and was subsequently shown to be required for the full activation and repression of numerous S. cerevisiae genes (Vidal and Gaber, 1991). Five cyclophilins have been previously identified in S. cerevisiae (Haendler et ul., 1989; Koser et al., 1990; Franco et a[., 1991; Koser et al., 1991; Tanida et al., 1991; Davis et al., 1992; McLaughlin et al., 1992; Frigerio and Pelham, 1993), none of which is required for normal cell growth. Cpr6 and Cpr7 are most closely related to CyP-40 (Kieffer et al., 1992, 1993), a mammalian cyclophilin associated with specific Hsp90-steroid hormone receptor complexes (Ratajczak et al., 1993; Johnson and Toft, 1994). Disruption of CPR7 leads to a growth defect, demonstrating for the first time the requirement of a cyclophilin for normal cell growth. A. A. DUINA ET AL. EcoRI and BarnHI sites of pGEX-2T. The plasmid encoding the GST-Hsp70PBD (provided by B. Freeman and R. Morimoto) represents a fusion between GST and the peptide-binding domain (amino acids 386-640) of Hsp70. The plasmid encoding Rpd3-3HA (pAAD55) was constructed by inserting a 99-bp fragment encoding three tandem copies of the influenza A haemagglutinin (HA) epitope immediately upstream of the termination codon of RPD3. The RPD3::3HA fusion was then subcloned into pET21-a( +) (Novagen). Plasmid pAAD87, used to delete CPR6 from the S. cerevisiae genome, was constructed as follows. Two PCR-amplified products corresponding to 400 bp immediately upstream and 500 bp immediately downstream from the non-translated region of the CPR6 ORF were cloned into pRS306 (an integrative plasmid harboring the URA3 selectable marker; Sikorski and Hieter, 1989). Plasmids pAAD92 and pAAD95 were used to delete CPR7. pAAD92 was constructed by amplifying two genomic fragments of -480 bp and 3 10 bp corresponding to sequences immediately upstream and immediately downstream from the CPR7 ORF, respectively, and cloned into pRS304 (an integrative plasmid which carries the TRPl selectable marker; Sikorski and Hieter, 1989). pAAD95 was constructed by excising a -790 bp BamHI-XhoI fragment from pAAD92 and inserting it into pRS306 digested with the same enzymes. - - - MATERIALS AND METHODS Media Standard genetic techniques and growth media used were those described in Sherman et al. (1986). Plasmid construe t ions Plasmid pAAD24, expressing the Gal4 DNA binding domain (Gal4BD) fused to Rpd3 was constructed by subcloning the open reading frame (ORF) of RPD3 into the EcoRI and SulI sites of plasmid pGBT9 (Bartel et al., 1993). Plasmid pAAD35, encoding the Gal4BD-Rpd3 construct fused to the Gal4 activation domain (Gal4BDRpd3-Gal4AD) was constructed by subcloning the Gal4AD-encoding segment from pGAD424 (Bartel et al., 1993) into pAAD24. The GST-Cpr6-expressing plasmid (pAAD66) was created by subcloning the CPR6 ORF into the BamHI and EcoRI sites of pGEX-2T (Pharmacia). The plasmid expressing GST-Cpr7 (pJM5) was constructed by cloning the CPR7 ORF into the Two-hybrid screen Transformations of S. cerevisiae cells were accomplished by electroporation (Becker and Guarente, 1991). Strain Y153 (Durfee et al., 1993) containing the Gal4BD-Rpd3-expressing plasmid pAAD24 was transformed to Leu+ with plasmid DNA containing S. cerevisiae genomic DNA fragments fused to the Gal4 activation domain (Chien et al., 1991). To assay for HIS3 reporter expression, transformants were replica-plated to media lacking histidine (His - ) and containing 25 m ~ - 3 aminotriazole (Sigma; Durfee et al., 1993). His+ colonies were assayed for LacZ expression using the colony lift assay described by Bartel et al. (1993). A full-length CPR6 cDNA was cloned from an S. cerevisiae DNA library (Liu et al., 1991a) by colony hybridization using a 32P-labeledsynthetic oligonucleotide corresponding to a 39-bp sequence within the CPR6 ORF. A clone encompassing the CPR7 gene was obtained from a genomic library S. CEREVISIAE CYCLOPHILIN REQUIRED FOR NORMAL GROWTH (provided by C. Connelly and P. Hieter) by colony hybridization using the CPR7 insert obtained from the two-hybrid screen as the probe. D N A sequence analysis DNA sequence analysis was performed by the dideoxy method (Sanger et al., 1977). Database searches were performed using BLAST (National Center for Biotechnology Information). Sequence alignments and analysis were performed using the Geneworks software (IntelliGenetics, Inc.) and the DNA Inspector IIe (Textco, Inc.). The DNA sequences for CPR6 (U48867) and CPR7 (U48868) have been deposited in GenBank. In vitro binding assay GST fusion proteins were expressed from pGEX vectors (Pharmacia). Rpd3-3HA was expressed from plasmid pET-21a(+) (Novagen). All pGEX plasmids were transformed into Escherichia coli strain DH5a. The PET-Rpd3-3HA vector (pAAD55) was transformed into hDE3 lysogens of strain BL21. GST proteins and Rpd3-3HA were induced using isopropyl-p-thiogalactopyranoside. Samples of fusion protein-expressing bacterial extracts were diluted in PBS and incubated in the presence of glutathione-Sepharose 4B beads (Pharmacia) for 1 h at 4°C. Following incubation, the beads were collected by centrifugation at 500 g for 5 min and washed twice with PBS. Equal amounts of Sepharose bead-bound fusion proteins ( - 5 pg) were added to reaction mixtures containing 100 pl of 2 x binding buffer (1 x binding buffer: 20 mM-Tris-HC1 [pH 7.51, 150 mM-KCl, 2 mM-CaCl,, 2 mM-MgCl,, 5 mM-dithiothreitol, 0.5% IGEPAL, 0.5 mM-phenylmethyl-sulfonyl fluoride, 5% glycerol), 40 p1 of lysate containing Rpd3-3HA (except for the Rpd3-minus control reactions), and ddH,O to a final volume of 200 pl. The binding reactions were incubated for 1 h at 4°C; the Sepharose beads were then harvested and washed four times with binding buffer. The bound proteins were eluted from the beads with 0.5 x glutathione elution buffer (1 x elution buffer: 20 mM reduced glutathione [Sigma] in 50 mM-TrisHCl [pH 8.01). The Sepharose beads were removed by centrifugation and aliquots of each supernatant (15 pl per sample) were subjected to SDS-PAGE. The gels were stained with Coomassie blue or processed for immunoblot analysis. Rpd3-3HA was detected on immunoblots with 12CA5 mouse monoclonal antibody (BabCO) 945 which binds to the HA epitope. Antibody detection was performed using the ECL Western blotting detection reagents (Amersham) following the manufacturer’s protocols. Construction of deletion strains Strains in which the entire ORFs of CPR6 andlor CPR7 were deleted were constructed by the ‘gamma’ deletion technique (Sikorski and Hieter, 1989) using linearized plasmids described above. Diploid cells harboring deletion mutations of CPR6 and/or CPR7 were generated by transformation with plasmids pAAD87 and pAAD92 (or pAAD95), respectively. Haploid cells containing the cpr deletion mutations were obtained as meiotic segregants from these heterozygous diploids or by direct transformation of the isogenic haploid strains (listed in Table 1). All deletion mutations were confirmed by Southern blot analysis (data not shown). RESULTS Cpr6 and Cpr7 interact with Rpd3 and are related to CyP-40 The yeast two-hybrid system (Chien et al., 1991) was used to identify proteins that interact with the global transcriptional regulator Rpd3 (Vidal and Gaber, 1991). Because Rpd3 can function as a repressor of transcription (Vidal and Gaber, 1991), we first performed an experiment to ensure that it would not interfere with transcriptional activation in the two-hybrid screen. A fusion in which Rpd3 was inserted between the Gal4 DNA-binding domain (Gal4BD) and activation domain (Gal4AD) was constructed and tested for activation in strain Y 153. Whereas Gal4BD-Rpd3 did not activate transcription of either reporter gene, Gal4BDRpd3-Gal4AD stimulated expression of both LacZ and HZS3 at levels comparable to the native Gal4 protein (data not shown). Thus, the inherent repression activity of Rpd3 did not prevent transcriptional activation in the two-hybrid screen. A fusion between Gal4BD and full-length Rpd3 was screened for interaction with hybrid proteins derived from a library of S. cerevisiae genomic fragments fused to a sequence encoding the Gal4 activation domain (Gal4AD; Chien et al., 1991). From approximately 200,000 library clones screened in strain Y 153, three activated transcription in a manner dependent on the presence of Gal4BD-Rpd3. 946 A. A. DUINA ET AL. Table 1. Strains used in this study. Strains Genotype Source W303 MATaIMATa 1eu2-3, 112lku2-3, 112 ura3-llura3-1 trpl-lltrpl-1 his3-11, lSlhis3-11, 15 ude2-llade2-1 canl-100Icunl-100 GALIGAL SUC2ISUC2 J. Thevelein W303-1A MATa leu2-3, 112 ura3-1 trpl-1 his3-11, 15 ade2-1 canl-100 GAL SUC2 J. Thevelein AAD128 MATa leu2-3, 112 ura3-1 trpl-1 his3-11, 15 ade2-1 canl-100 GAL SUC2 cpr6A:: URA3 This study AAD 129 MATaIMATa leu2-3, 112lleu2-3, 112 ura3-llura3-1 trpl-lltrpl-1 his3-11, 15lhis3-1I, 15 ade2-llade2-1 canl-100Icanl-100 GALIGAL SUC2ISUC2 cpr7A:: TRPlICPR7 This study AAD131 MATa leu2-3, 112 ura3-1 trpl-1 his3-11, 15 ade2-I canl-100 GAL SUCZ cpr7A::TRPl AAD 129 AAD132 M A T a leu2-3, 112 ura3-1 trpl-1 his3-11, 15 ade2-1 canl-100 GAL SUCZ cpr7A:: TRPl AAD 129 AAD135 Diploid strain derived from mating AADB128 and AAD132 AAD136 MATa leu2-3, 112 ura3-1 trpl-1 his3-11, 15 ade2-1 canl-100 GAL SUC2 cpr6A:: URA3 cpr7A::TRPl AAD135 M517 MATaIMATa ura3-53lura3-52 trpl Alltrpl A1 his3-200lhis3-200 leu2-lIleu2-1 trkl A1 trkl A Vidal and Gaber (1991) AAD120 M A TaIMA Ta ura3-53lura3-52 trpl A1 ltrpl A1 his 3-200lhis3-200 leu2-lIleu2-1 trkl Al trkl A cpr6A:: URA3ICPR6 This study AADll8 AAD138 M A T a uru3-52 trpl A1 his3-200 leu2-I trkl A cpr6A:: URA3 M A TaIMA Ta ura3-53lura3-52 trpl A1 ltrpl A1 his3-200lhis3-200 leu2-1lleu2-1 trkl A1 trklA cpr7A:: URA3ICPR7 AAD 120 This study AAD139 AAD141 M A T a ura3-52 trplAl his3-200 leu2-1 trkl Acpr7A:: URA3 Diploid strain derived from mating AADll8 and AAD139 AAD138 This study AAD 142 MATa ura3-52 trplA1 his3-200 leu2-I trkl A cpr6A:: URA3 cpr7A:: URA3 DNA sequence analysis of the positive clones revealed the presence of two different inserts. Two clones contained identical in-frame fusions between GAL4AD and an ORF of 918 nucleotides; the third plasmid contained a different in-frame O RF of 969 nucleotides. These ORFs represent portions of two new members of the cyclophilin family and will be referred to as Cpr6 and Cpr7. Clones representing the entire CPR6 and C P R 7 genes were obtained from separate cDNA and genomic libraries as described in Materials and Methods. The sequences for both CPR6 and C P R 7 have been identified recently through the This study AAD141 genomic sequencing project and were recognized to encode putative members of the cyclophilin family. The CPR6 OR F is identical to a sequence on S. cerevisiae chromosome XI1 (Johnston et al., 1994). A sequence nearly identical to CPR7 has been reported by Zagulski et al. (1995) as an OR F present in a 24.3-kb fragment of the S. cerevisiae chromosome X. CPR6 and C P R 7 encode proteins with predicted molecular weights of 42 kDa and 45 kDa, respectively. The two share 38% sequence identity (Figure 1A) and contain two distinct domains (Figure 1B). One domain is similar in sequence and in length to 947 S. CEREVISIAE CYCLOPHILIN REQUIRED FOR NORMAL GROWTH A ~ p r 7 50 Cpr6 CyP-40 37 49 Cpr7 Cpr6 cyp-40 100 79 90 Cpr7 Cpr6 cyp-40 126 137 Cpr7 Cpr6 cyp-40 198 176 185 Cpr7 Cpr6 cyp-40 276 149 247 230 Cpr7 297 275 278 Cpr6 cyp-40 Cpr6 cyp-40 347 325 324 Cpr7 CRr6 cyp-40 393 371 370 ~pr7 B TPR Units CYP-40 CyP-18-like domain Figure 1. Amino acid and schematic comparisons between Cpr7, Cpr6 and CyP-40. (A) Amino acid alignment between Cpr7, Cpr6 and human CyP-40. Identical residues are shaded. Underlined sequences correspond to the TPR domains; boxed amino acids correspond to residues that fit the TPR motif *--*G-*Y/F-----*--A*--Y/F--A*-*P----- (where - is any amino acid and * is any large hydrophobic amino acid; Hirano et al., 1990). The Trp to His substitution within the CyP-18-like domain (refer to text) is shown in negative. Dashed underline indicates sequences contained in putative calmodulinbinding site. (B) Schematic comparison between Cpr7, Cpr6 and CyP-40. The TPR units are hatched. Location of putative calmodulinbinding site is indicated by solid boxes. Numbers under the diagrams refer to amino acid positions. cyclophilin-18 (CyP-18), which has been shown to mediate isomerase and CsA-binding activities (Handschumacher et al., 1984: Harding et ul., 1986: Fischer et al., 1989: Takahashi et al., 1989). The second domain, absent from the other five S. cerevisiue cyclophilins, is reminiscent of the carboxy-terminal extension present in CyP-40 (Kieffer et al., 1992, 1993), a mammalian cyclophilin found in association with some Hsp90- steroid hormone receptor complexes (Ratajczak et al., 1993; Johnson and Toft, 1994). The similarity between Cpr6, Cpr7 and CyP-40 extends throughout the entire proteins. Cpr6 and CyP-40 are the most closely related (44% identity). Their CyP- 18-like domains are 58% identical, whereas their carboxyl extensions share 31% identity. Ratajczak, et al. (1993) observed that a highly conserved tryptophan thought to interact with 948 A. A. DUINA ET AL. + + - - - - + + - _ _ - - + - _ _ - _ + + - + - + + A- GST-Cpr6 GST-Cpr7 GST-Hsp70PBD GST Rpd3-3HA 83.0 kD50.6 kD20.9 kD- B 83.0rkD- 4 50.6 kD20.9 kD- 1 2 3 4 5 6 7 8 Figure 2. In vitvo binding assay. CST-Cpr6 (lanes 3 and 4), GST-Cpr7 (lanes 5 and 6), GST-HSP70PBD (lane 7), and GST alone (lane 8) were expressed in bacteria and lysates incubated in the presence of either bacterially expressed Rpd3-3HA (lanes 3, 5, 7 and 8) or buffer alone (lanes 4 and 6 ) . Following affinity purification using glutathione-Sepharose beads, the binding reactions were subjected to SDS--PAGE analysis and Coomassie blue staining (A) or Western blot analysis using anti-HA antibodies to detect the presence of Rpd3-3HA (B). Lane 1 shows 1 p1 of total Rpd3-3HA lysate. Lane 2 shows molecular weight standards. A protein species (*) that crossreacted with the anti-HA antibody was also detected in nonRpd3-3HA expressing lysates (data not shown). CsA (residue 121 in CyP-18) is replaced by histidine in CyP-40; Cpr6 and Cpr7 have the same substitution (Figure I). The three proteins also share significant sequence identity within a region containing a three-unit tetratricopeptide repeat (TPR) motif which is believed to be involved in protein-protein interactions (Lamb et al., 1995). Finally, a putative calmodulin-binding domain, similar to that discovered in the immunophilin FKBP-59 (Lebeau et al., 1992), is present at the extreme carboxy terminus of each of the three cyclophilins. Cpr6 and Cpr7 bind Rpd3 in vitro To provide further evidence for the authenticity of the Cpr61Rpd3 and Cpr71Rpd3 interactions, the proteins were expressed in bacteria and their binding activities were tested in vitro. Both cyclophilins were expressed in E. coli as GST fusions and, following affinity chromatography on glutathioneSepharose beads, yielded relatively pure proteins of the expected molecular weights (Figure 2A, lanes 3-6). An epitope-tagged version of Rpd3 (Rpd3-3HA) was also expressed in E. coli. The epitope tag, corresponding to three tandem copies of the 9-amino-acid epitope derived from the influenza A virus HA protein, was inserted at the carboxyl terminus of Rpd3 between amino acids 432 and 433. Rpd3-3HA remains fully functional as it conferred Rpd+ phenotypes to cells deleted for RPD3 (data not shown). To test the ability of Cpr6 and Cpr7 to interact with Rpd3 in vitro, bacterial lysates containing different GST fusions (GST-Cpr6, GST-Cpr7, GST fused to the Hsp70 peptide binding domain [GST-HS~~OPBD] and GST alone) were incubated in the presence of Rpd3-3HA. The ability of a GST fusion protein to bind Rpd3-3HA was assessed by analysing the material removed from solution following incubation with glutathioneSepharose beads. Resolution of the proteins bound to the beads by SDS-PAGE showed that the concentration of GST fusion proteins retained by the Sepharose beads was approximately the same in each case (Figure 2A). Immunoblot analysis of the same samples using anti-HA antibody revealed that Rpd3-3HA is specifically retained from solution by either GST-Cpr6 or GST-Cpr7 (- 1 to 5% of Rpd3 removed from solution), but not by GST alone nor by GST-Hsp70PBD (Figure 2B). CPR6 and CPR7 are non-essential but Cpr7 is required for normal cell growth Strains deleted for CPR6 (cpr6A), CPR7 (cpr7A) and CPR6 CPR7 (cpr6A cpr7A) were created by ‘gamma’ deletion (Sikorski and Hieter, 1989). cpr6A and cpr7A mutations were generated in diploid cells and haploid meiotic segregants recovered as spore colonies. Analysis of the tetrads derived from the different diploid cells revealed that c p 7 A cells display a slow growth phenotype. Figure 3A shows tetrads derived from CPR7I cpr7A::TRPl diploid cells. In each case the slow growth phenotype co-segregated with the Trp+ phenotype. Introduction of a plasmid containing the wild-type CPR7 gene fully suppressed the slow growth phenotype (data not shown), confirming that the growth defect was due to the loss of Cpr7. The small colony size of cpr7A cells was not the result of a defect in spore germination. During logarithmic growth the doubling time for wild-type cells was 1.5 h, whereas the doubling time for cpr7A cells was 2.6 h. In contrast, cells containing cpr6A mutation exhibited growth rates indistinguishable from wild-type CPR6 cells. Cells deleted for both CPR6 and CPR7 exhibit a slow growth phenotype indistinguishable from cpr7A cells (Figure 3B). S. CEREVISIAE CYCLOPHILIN REQUIRED FOR NORMAL GROWTH A B Figure 3. Effects of cpr6A and cpr7A mutations on growth. (A) Spore colonies dissected from diploid AAD129 (CPR7I cpr7A::TRPI) onto medium. Each column represents the four meiotic products derived from the same tetrad. The slowgrowing colonies co-segregated with the Trp' phenotype. (B). CPR6 CPR7 (W303-1A), cpr6A (AAD128) cpr7A (AAD131) and cpr6A cpr7A (AAD136) haploid strains grown on YPD medium at 30°C (photographed 48 h after inoculation). To determine whether a functional relationship between Cpr6 and Cpr7 and Rpd3 could be detected in vivo, we tested cpr6A, cpr7A and cpr6A cpr7A cells for Rpd3-related phenotypes. Previous studies showed that disruption of RPD3 results in numerous phenotypes due to the aberrant transcriptional regulation of target genes (Vidal and Gaber, 1991). Four such phenotypes (see Vidal and Gaber, 1991) were examined in haploid cells containing the cpr deletion mutations: (1) ability to sporulate as homozygous diploids; (2) derepression of P H 0 5 in cells grown in media containing high concentrations of phosphate; (3) hypersensitivity to cycloheximide (Cyhhs); and (4) derepression of TRKZ resulting in the ability of t r k l A TRIG! cells to grow on low K' medium. Deletion of CPR6 and/or CPR7 had no obvious effect on the first three phenotypes: cprlcpr diploids sporulated; cpr mutants exhibited normal repression of P H 0 5 , and were not more resistant or more sensitive to cycloheximide (data not shown). However, t r k l A cpr6A, t r k l A cpr7A, and t r k l A cpr6A cpr7A cells did show slightly increased rates of growth on low K' medium. Whether or not this phenotype involves decreased repression of TRK2 by RPD3 will require thorough genetic analysis. 949 DISCUSSION We have identified two new cyclophilins in S. cerevisiae through their interaction with the global transcriptional regulator Rpd3. Cpr6 and Cpr7 share significant sequence identity and domain organization with the human CyP-40 (Kieffer et a/., 1993; Ratajczak et a/., 1993). Deletion of CPR7 leads to a significant decrease in the growth rate. This is the first time that a member of the cyclophilin family has been shown to be required for normal growth. In fact, only one other cyclophilin in S. cerevisiue, Cpr3, has been shown to be associated with any phenotype: cpr3 mutants fail to grow on lactate medium at 37°C (David et al., 1991). It is not known why cpr7A cells are defective in growth but it does not appear to be due to the loss of Rpd3-Cpr7 interaction since deletion of RPD3 does not result in slow growth (Vidal and Gaber, 1991). Therefore, Cpr7 may be involved in additional cellular functions. Genetic identification of suppressors that restore normal growth to cpr7A should provide insights into these functions. Several studies have reported that some cyclophilins are involved in transcriptional regulation (Larson and Nuss, 1993; Renoir et a/., 1995). In particular, Yang et al. (1995) have reported that the mammalian global transcriptional regulator YY1 interacts with and is regulated by CyP-18. Rpd3, also a global transcriptional regulator, may be functionally modified by cyclophilins. Several possibilities may explain why changes in Rpd3related phenotypes were not observed in cells deleted for CPR6 and CPR7: (i) Cpr6 and Cpr7 may be negative regulators of Rpd3 or they may not be required for Rpd3 function; (ii) Cpr6 and Cpr7 may be required for a subset of Rpd3mediated functions not tested in our assays; (iii) other proteins (perhaps cyclophilins) may have functions that overlap with Cpr6 and/or Cpr7; (iv) the sensitivity of the assays performed may have been insufficient to detect effects on Rpd3 functions. The sequence and structural similarity between Cpr6, Cpr7 and CyP-40, as well as the common amino acid substitution at an otherwise conserved residue (Trp to His within the putative CsAbinding domain), raises the possibility that all three cyclophilins may have evolved to perform related functions. Another member of this class of cyclophilins, wis2, has recently been identified as a multi-copy suppressor of a cell cycle defect in Schizosaccharomyces pombe (Weisman et al., 950 1996). CyP-40 has been identified as a component of Hsp90 complexes (Ratajczak et ul., 1993; Johnson and Toft, 1994) that regulate the activity of some steroid receptors (Picard et ul., 1990). Chang and Lindquist (1994) have shown that the components of the Hsp90 complexes are conserved between S. cerevisiue and mammalian systems. In addition, they have identified a protein within an S. cerevisiae Hsp90 complex that is likely to be Cpr6 based on amino-terminal sequence data and molecular weight determinations (Chang and Lindquist, 1994). It will therefore be of interest to determine whether Cpr6 and Cpr7 are indeed part of some Hsp90 complexes and to assess their roles in Hsp90-mediated processes through genetic analysis. ACKNOWLEDGEMENTS We thank Anna Dobrzycka for excellent technical assistance. 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