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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. This work was supported by a grant
from the National Institutes of Health
(GM45739).
NOTE ADDED IN PROOF
A search of the genome sequence indicated that
CPR6 and CPR7 are the only CyP-40-type cyclophilins in S. cerevisiue
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