From the Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel
Received for publication, March 8, 2001, and in revised form, May 1, 2001
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ABSTRACT |
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FKBP12 is a ubiquitous and a highly conserved
prolyl isomerase that binds the immunosuppressive drugs FK506 and
rapamycin. Members of the FKBP12 family have been implicated in many
processes that include intracellular protein folding, transport, and
assembly. In the budding yeast Saccharomyces cerevisiae and
in human T cells, rapamycin forms a complex with FKBP12 that inhibits
cell cycle progression by inhibition of the TOR kinases. We
reported previously that rapamycin does not inhibit the vegetative
growth of the fission yeast Schizosaccharomyces pombe;
however, it specifically inhibits its sexual development. Here we show
that disruption of the S. pombe FKBP12 homolog,
fkh1+, at its chromosomal locus results in a
mating-deficient phenotype that is highly similar to that obtained by
treatment of wild type cells with rapamycin. A screen for
fkh1 mutants that can confer rapamycin resistance
identified five amino acids in Fkh1 that are critical for the effect of
rapamycin in S. pombe. All five amino acids are located in
the putative rapamycin binding pocket. Together, our findings indicate
that Fkh1 has an important role in sexual development and serves as the
target for rapamycin action in S. pombe.
Cyclosporin A (CsA),1
FK506, and rapamycin are microbial products that exhibit
immunosuppressive activity (1). These three compounds bind with high
affinity to cytoplasmic proteins termed immunophilins (2, 3). CsA binds
an immunophilin called cyclophilin-18, whereas FK506 and rapamycin,
which are structurally related, bind a different immunophilin called
FKBP12. The immunosuppressive drugs form a drug-immunophilin complex,
which binds and inhibits a third component. The complexes
CsA-cyclophilin-18 and FK506-FKBP12 bind and inhibit the activity of
the Ca2+-dependent protein phosphatase,
calcineurin (4-6). The rapamycin-FKBP12 complex binds and inhibits the
activity of the phosphatidylinositol-like kinase, TOR (7-10, 36,
37).
In addition to their immunosuppressive activity, CsA, FK506, and
rapamycin have side effects that may stem, at least in part, from
inhibition of the physiological function of the immunophilins. For
example, in mammals, FKBP12 functions as a subunit of ryanodine calcium
release channels and is thought to modulate intracellular Ca2+ levels in the heart (11-13). Mice deficient in FKBP12
show severe heart defects associated with loss of function of cardiac
ryanodine receptors (14). Similarly, treatment with high doses of FK506 can lead to severe heart failure (15).
Although FKBP12 and cyclophilin-18 are unrelated in primary sequence,
both classes of immunophilins exhibit a peptidyl
prolyl-cis/trans-isomerization (PPIase) activity that
accelerates a rate-limiting step in the folding of peptide and protein
substrates in vitro (3, 16-18). The PPIase activity of the
immunophilins is inhibited upon binding to their specific
immunosuppressive drugs, suggesting an overlap between the
PPIase-active site and the drug-binding site. According to atomic
structure analyses of human cyclophilin-18 and FKBP12, both proteins
contain a deep hydrophobic binding pocket (19-21). These pocket
structures accommodate the specific immunosuppressive-acting ligands
and model tetrapeptides used as pseudosubstrates.
The cellular functions of the immunophilins, as well as the relevance
of the PPIase activity within the cellular environment, is not well
understood. However, some of the important natural substrates of the
immunophilins are now known (reviewed in Ref. 22). For example, the
human cyclophilin-18, CyPA, binds the Gag polyprotein of the human
immunodeficiency virus, type 1, virion (23-25). The human FKBP12
protein is physically associated with calcium release channels (11-13,
26), the type I tumor growth factor, transforming growth factor- Genetic studies in the budding yeast Saccharomyces
cerevisiae have played a critical role in elucidating the mode of
action of the immunosuppressive drugs in higher eukaryotes (reviewed in
Refs. 32 and 33). Similar to the effect of rapamycin in T cells and
certain non-lymphoid cells, rapamycin treatment of S. cerevisiae cells results in a G1 cell cycle arrest
(34). S. cerevisiae cells contain one FKBP12 homolog, named
FPR1 (34), also known as RBP1 (35). Disruption of
FPR1 results in slightly slowly growing but viable cells
that are completely resistant to rapamycin. This phenotype indicated
that FPR1 is a nonessential gene and is the main mediator of
the effect of rapamycin (34, 35). Later it was shown that Fpr1p forms a
complex with rapamycin that binds and inhibits the functions of the
TOR1 and TOR2 gene products in cell cycle
progression (34, 36-39). Several proteins that interact physically
with Fpr1p in the absence of rapamycin have been identified, and it has
been suggested that their activity may be regulated by the interaction
with Fpr1p. These include calcineurin (40), the biosynthetic enzyme
aspartokinase (41), the high mobility group HMG 1/2 proteins (42), and
the transcription factor homolog FAP1 (43).
We reported previously (44) that rapamycin does not affect vegetative
growth in the fission yeast, Schizosaccharomyces pombe, but
severely inhibits its sexual development pathway. S. pombe cells are induced to enter the sexual development pathway under starvation conditions (45). If the sexual development pathway is
chosen, cells of opposite mating type conjugate to form diploid zygotes
that immediately undergo meiosis and sporulation (45). Rapamycin
strongly inhibited sexual development at an early stage, before mating
had occurred, but did not affect entrance into stationary phase (44).
More recently, we reported that S. pombe contains two TOR
homologs, tor1+ and tor2+
(46). tor2+ is an essential gene of as yet
unknown function. tor1+ is required under
starvation and a variety of other stress conditions that include
osmotic and oxidative stresses. Interestingly, none of the studied
functions of the S. pombe TOR homologs appear to be
inhibited by rapamycin (46).
To understand further the response of S. pombe to rapamycin,
we isolated and characterized the S. pombe FKBP12 homolog.
We found one FKBP12 homolog and named it fkh1+.
Disruption of fkh1+ results in a mating
deficient phenotype that is highly similar to that of rapamycin-treated
cells. We identified, using a genetic screen, amino acid substitutions
in Fkh1 that confer rapamycin resistance. These substitutions occur in
conserved residues of FKBP12 and are potentially involved in rapamycin
binding. Our analyses of the fkh1 null and
rapamycin-resistant mutants suggest that rapamycin exerts its effect on
sexual development in S. pombe by inhibiting the function of Fkh1.
Yeast Strains, Media, and Yeast Techniques--
Yeast strains
used in this paper are described in Table
I. Media used are based on those
described (47). EMM-N contains no nitrogen; EMM lowG contains 0.1%
glucose. Transformation of S. pombe cells was performed by
electroporation (48). Rapamycin was added to a final concentration of
0.2 µg/ml in liquid or agar-containing media, unless otherwise
indicated. An equal volume of the drug vehicle solution (1:1
Me2SO:methanol) was used as a control in all
experiments. Assays for mating or sporulation efficiency were carried
out as follows. Cells were grown at 30 °C in EMM medium to the
density of ~5 × 106-1 × 107
cell/ml. The cultures were then washed three times with double distilled water and 5 µl containing 5 × 106 cells
were spotted on EMM, EMM-N, EMM-lowG, or ME medium (see Ref. 44 for
detailed description of medium composition). After 3 days of incubation
at 30 °C, a toothpick was used to pick some of the cells from the
center of each patch, and the cells were briefly sonicated and examined
microscopically. The percentage of mating was calculated by dividing
the number of zygotes, asci, and free spores by the number of total
cells. The percentage of sporulation was calculated by dividing the
number of asci and free spores by the number of total cells. One zygote
or one ascus was counted as two cells and one spore was counted as
half-cell. In each experiment 500-1000 cells were counted. Cell
viability after entry into stationary phase was determined as follows.
Cells were grown in minimal medium (EMM) or rich medium (YE) at
30 °C to confluence, and aliquots were sampled every 24 h. Cell
viability was determined by the capacity of cells to form colonies.
FACS Analysis--
Cells were stained with the DNA
fluorochrome propidium iodide and analyzed by a Becton Dickinson
FACSort as described (49). Data were analyzed by Cell Quest software
for Macintosh.
Disruption of S. pombe fkh1+--
A 1.7-kilobase
pair fragment containing the entire fkh1+ gene
was amplified by PCR using a genomic S. pombe DNA
preparation as a template and primers 101 (5'-GCTCAGAATGATCGACATATACAAC) and 102 (5'- CAAACCAGCTACATAGCACAG). The
resulting PCR fragment was cloned into a pGEM-T vector (Promega) to
give pGEMT-fkh1. This plasmid was cut with
HindIII, within the fourth exon of
fkh1+. The HindIII restriction site
lies within the predicted active site and rapamycin binding pocket. The
HindIII cut plasmid was ligated with a HindIII
fragment containing ura4+, resulting in the
plasmid pfkh1::ura4+.
NotI and SacI were used to release the
3.5-kilobase pair
fkh1::ura4+ disruption
fragments that were gel-purified and transformed into the homothallic
strain TA16. Stable Ura4+ haploids were selected and
subjected to PCR analysis with primer 135 (5'-GTTATAAACATTGGTGTTGGAACAG) that is complementary to sequences within the ura4+ gene and primer 136 (5'-GTTCGAATATAT TCGGTGCGCC) that is complementary to sequences of the
fkh1+ locus that are 100 bp downstream of the 3'
end of the disruption construct. The resultant PCR fragment of 1200 bp
confirmed that the disruption cassette integrated into the
fkh1+ locus. In addition, we used primer 136 in
combination with primer 103 that is complementary to sequences that are
100 bp upstream of the 5' end of the disruption construct. The
amplification of a single PCR product of the size of 3700 bp is
consistent with a single site insertion of the disruption cassette. We
analyzed the phenotype of two independently isolated Cloning of fkh1+ cDNA--
The cDNA of
fkh1+ was isolated by PCR amplification from an
S. pombe cDNA library (50) with the primers 92 (5'-GGAATTCCATATGGGTGTCGAAAAGCAAGTTATTTC; underlined
is the NdeI restriction site) and 81 (5'-TGACCAATGGCGAAGAAGTCC). The PCR product of 486 bp was cloned under
the control of the thiamine-repressible nmt1 promoter in
pREP1 (50). In all experiments cells were grown under derepression
conditions (in the absence of thiamine) for full activity of the
nmt1 promoter. The cDNA of fkh1+
was also cloned into the S. cerevisiae expression vector
pCM189 (51) using primers 92 and 81. In addition, the S. cerevisiae FKBP12 homolog, FPR1, was cloned into the
pCM189 vector and pCM189' (a vector that differs from pCM189 only in
that it contains LEU2 as a selective marker and not
URA3) using primers 106 (5'-ATAAGAATGCGGCCGCCGGATCCCGCTCGAGGTCG) and 110 (5'-ATAAGAATGCGGCCGCCAATTAAGGCTCAGATACTTACC). The
NotI sites in both primers are underlined.
pCM189-fkh1+ and pCM189-FPR1 were
transformed into the S. cerevisiae strains JK9-3d
(MAT
fkh1+ cDNA was also isolated during a screen
for S. pombe genes that could suppress the
rapamycin-sensitive phenotype in S. cerevisiae. The S. cerevisiae strain, RS188N (MATa
leu2-3,2-112 trp1-1 ura3-1 ade2-1 his3-11, 15 can1-100), was
transformed with a S. pombe cDNA library constructed
using a S. cerevisiae high copy number expression vector in
which expression of inserts is regulated by the strong ADH1
promoter (53). Transformants were plated onto minimal medium plates
containing 0.1 µg/ml rapamycin at 30 °C. 25 rapamycin-resistant
colonies were isolated from over 105 transformants. Of
these, 8 exhibited rapamycin resistance upon re-streak on
rapamycin-containing plates, and in 4 the rapamycin resistance
phenotype was dependent on the presence of the
plasmid.2 Sequence analysis
revealed that one of these, pR22, encodes for fkh1+. This clone contained the entire open
reading frame of fkh1+ flanked by 22 and 150 bp
at 5' and 3' ends of the open reading frame, respectively.
Western Blot Analysis of Fkh1--
Total protein extracts were
prepared from mid-log wild type (TA16), Isolation of Rapamycin-resistant fkh1
Mutants--
fkh1 mutants were obtained by PCR-based
mutagenesis. Conditions for PCR-based random mutagenesis of
fkh1+ were essentially as described (54).
Briefly, 5 ng of plasmid pR22 were taken for PCR amplification of
fkh1+ cDNA with 0.2 µg of primers 92 (5'-GGAATTCCATATGGGTGTCGAAAA GCAAGTTATTTC, NdeI
site is underlined) and 81 (5'-TGACCAATGGCGAAGAAGTCC). The PCR buffer
contained 10 mM Tris-HCl (pH 8.7), 50 mM KCl, 5 µg/ml bovine serum albumin, 0.5 mM MnCl2, 4.2 mM MgCl2, 5 units of Taq polymerase,
250 µM each of dNTP, and an excess of 1.5 mM
of one dNTP nucleotide concentration over the others. Four separate
PCRs were performed, and in each reaction a different dNTP was present in excess. 25 cycles of PCR were performed with the following temperature profile: 94 °C, 30 s; 55 °C, 30 s;
72 °C, 30 s. The four PCRs were pooled and fractionated in a
1.5% agarose gel and eluted. The resultant 550-bp DNA fragments were
digested with NdeI and ligated with a
NdeI-SmaI digested pREP1 S. pombe
vector. The ligation product was used for PCR amplification with
primers 189 (5'-GAATAAGTCATCAGCGGTTGTTTC) and 190 (5'-TCATCCATGCGGCCAATCTTGTCG). These DNA fragments containing the
mutated fkh1+ cDNA flanked by pREP1
sequences were co-transformed with pREP1 into the S. pombe
strain TA77 (leu1-32 ura4-D18
fkh1::ura4+ h90).
Transformants were plated on minimal medium and after 4 days of
incubation at 30 °C replica-plated to minimal medium with or without
0.2 µg/ml rapamycin. After an additional 5 days of incubation at
30 °C, the plates were exposed to iodine vapor. Iodine vapor is
routinely used to detect sporulating colonies. Spores are darkly stained by iodine vapor, whereas vegetative cells remain unstained. In
the presence of rapamycin (44) or in Identification of the FKBP12 Homolog in the S. pombe
Genome--
Most of the S. pombe genome has been sequenced
through the coordination of the Sanger Center, UK. Based on sequence
comparisons, we identified one S. pombe FKBP12 homolog on
chromosome II and named it fkh1+ (for
FKBP12 homolog). The open reading frame of
fkh1+ is interrupted by 4 introns of 182, 128, 105, and 48 base pairs. fkh1+ encodes a putative
112-amino acid protein with a predicted mass of 12 kDa. We cloned the
fkh1+ cDNA by PCR amplification using a
fission yeast cDNA library as a template (see "Experimental
Procedures"). Sequence analysis confirmed that the 4 introns
predicted in the genomic sequence are spliced out in the cDNA clone.
Analysis of the predicted amino acid sequence encoded by
fkh1+ reveals that this gene is very similar to
its S. cerevisiae homolog, FPR1 (72% overall
identity). The similarity between fkh1+ and the
human FKBP12 homolog is comparable to the similarity between
FPR1 and the human FKBP12 (55% overall identity).
fkh1+ encodes all the amino acids required for
rapamycin binding as predicted by the high resolution structure of the
human FKBP12-rapamycin complex (Ref. 21 and see Fig. 5).
When Expressed in S. cerevisiae, fkh1+
Functions Similarly, but Not Identically, to the S. cerevisiae FKBP12
Homolog--
The S. cerevisiae FKBP12 protein, Fpr1p, binds to
rapamycin. FKBP12-rapamycin complexes bind the TOR proteins and thus
inhibit some of their functions (see Introduction). Since the S. pombe fkh1+ gene shows a significant level of homology
with FPR1, we examined whether fkh1+
can replace FPR1 in mediating the effect of rapamycin in
S. cerevisiae. To this goal, we expressed
fkh1+ cDNA in S. cerevisiae using
ADH1 promoter-driven vector, pCM189 (51). Wild type and
Unexpectedly, following a prolonged incubation, cells expressing
pCM189-fkh1+ exhibited slow growth in the
presence of rapamycin, either in the genetic background of wild type or
We also screened an S. pombe cDNA library for genes that
can confer rapamycin resistance in S. cerevisiae cells (see
"Experimental Procedures"). Wild type S. cerevisiae was
transformed with the S. pombe cDNA library, and the
transformants were plated on rapamycin-containing plates. Sequence
analysis of one of the isolated cDNA clones revealed that it
encoded fkh1+. The weak rapamycin resistance
phenotype conferred by overexpression of fkh1+
was observed in several S. cerevisiae strains, including
RS188N and JK9-3d (see "Experimental Procedures" for full
genotypes), indicating that this suppression activity is not
strain-specific.
We examined the ability of fkh1+ to suppress
rapamycin sensitivity at different drug concentrations ranging from 10 to 150 ng/ml. S. cerevisiae wild type cells transformed with
either pCM189-fkh1+ or pCM189-FPR1
were streaked on rapamycin-containing plates, and their growth was
monitored. Cells transformed with
pCM189-fkh1+ grew faster on 10 ng/ml rapamycin
than on 100 ng/ml rapamycin (Fig. 2A) and did not form
colonies on 150 ng/ml rapamycin (data not shown).
pCM189-FPR1 had no significant suppression activity at any
drug concentration.
One possibility to explain the dosage-dependent suppression
of fkh1+ is that the gene product, Fkh1, forms a
complex with rapamycin that does not inhibit the S. cerevisiae TOR proteins as efficiently as Fpr1p-rapamycin
complexes. According to such a model, Fpr1-rapamycin complexes would
compete with Fkh1-rapamycin complexes. Thus overproduction of Fpr1p is
expected to counteract the weak rapamycin resistance associated with
fkh1+. To investigate this, wild type cells were
co-transformed with pCM189'-FPR1 and
pCM189-fkh1+, and the resulting transformants
were streaked on rapamycin-containing plates. The results demonstrate
that increased levels of FPR1 abolished the
fkh1+-dependent rapamycin resistance
(Fig. 2B). These findings support our suggestion that the
slight decrease in rapamycin sensitivity in cells overexpressing Fkh1
results from reduced ability in inhibiting the TOR proteins.
Cells Disrupted for fkh1+ Exhibit a Mating Deficient
Phenotype--
To study the cellular function(s) of
fkh1+ and to examine its possible role in the
response to rapamycin in S. pombe, we used the
ura4+ gene to construct a homothallic strain
that carried a disruption of the fkh1+ gene.
Ura4+ stable haploids were isolated, and PCR analysis
confirmed that a single integration event had occurred at the
fkh1 locus (see "Experimental Procedures" and data
not shown). Consistently, no product of the
fkh1+ gene is observed by Western blot analysis
(see Fig. 3B).
Analysis of two independently isolated
We overexpressed fkh1+ in S. pombe by
placing it downstream of nmt1 promoter (see "Experimental
Procedures"). Overexpression of fkh1+ was
verified by Western analysis (Fig. 3B). Overexpression of fkh1+ in wild type cells (TA16) did not affect
either growth or the efficiency of sexual development (Fig.
3A). Thus, whereas fkh1+ is required
for sexual development, its natural level is not a limiting factor for
this process.
Nitrogen starvation is also a signal for diploid cells to enter
meiosis, a later stage in the sexual development pathway (reviewed in
Ref. 45). To examine whether fkh1+ plays a role
in meiosis/sporulation, we constructed a diploid strain homozygous to
the disruption in fkh1+ (TA94). These diploid
cells were induced to undergo meiosis/sporulation under nitrogen
starvation. Microscopic examination revealed that normal asci
containing four spores were formed with the same efficiency (50%) as
in wild type cells (TA07, 54%). Thus, although
fkh1+ is required for mating it is not required
for meiosis/sporulation.
When S. pombe cells enter stationary phase they become
smaller and round and can maintain their viability over long periods (56). Some of the S. pombe mutants that are impaired in
sexual development are also impaired in their ability to acquire normal stationary phase morphology and physiology. These include mutants of
the cAMP-dependent pathway (57, 58) and mutants of the Spc1-Wis1 stress-activated mitogen-activated protein kinase pathway (59-61). In such mutants, the sterile phenotype may stem from an inability to sense or respond properly to starvation conditions. Recently, we reported (46) that null mutants of the S. pombe TOR homolog, tor1, are defective both in sexual development
and entrance into stationary phase. In contrast, rapamycin-treated cells can enter stationary phase properly (44). Here we found that
Finally, in some sterile mutants, the defect in mating is associated
with an inability to arrest in G1 in response to nitrogen starvation (46, 59). We analyzed the DNA content of starved
Our findings indicate that fkh1+ is required
specifically for an early stage of the sexual development pathway. Like
treatment with rapamycin (44), Isolation of Rapamycin-resistant fkh1 Mutants--
The close
similarity between the phenotypes of
We randomly mutated fkh1+ cDNA using
error-prone PCR (see "Experimental Procedures"). Strain TA77
(leu1 ura4
Alignment of the amino acid sequence of Fkh1 with that of HuFKBP12
reveals that all the fkh1 rapamycin-resistant mutations fall
into or adjacent to the predicted rapamycin binding pocket (21). Four
of the five mutations, F47S, L56F, I92F, and F100L, correspond to amino
acid residues of HuFKBP12 that most closely interact with rapamycin in
the atomic structure of the HuFKBP12-rapamycin complex (see Fig.
5). In particular, Phe-47 and Phe-100
correspond to the HuFKBP12 amino acid residues that surround the
portion of rapamycin that penetrates most deeply into the protein (21). Cys-49 in Fkh1, corresponding to Phe-48 in HuFKBP12 and Cys-55 in
S. cerevisiae Fpr1p, is the only amino acid out of the five identified that is not mapped to the very core of the rapamycin binding
pocket but resides in a close vicinity. The identification of
rapamycin-resistant fkh1 mutants carrying mutations near or at the predicted rapamycin-binding pocket argues that the rapamycin resistance of these mutants is due to impaired rapamycin binding.
We were curious if the fkh1 mutants were also impaired in
rapamycin binding in S. cerevisiae cells. If so, the
fkh1 mutants could not restore rapamycin sensitivity in
S. cerevisiae
Our findings indicate that whereas all the fkh1 mutants
confer complete rapamycin resistance in S. pombe, they are
not identical in their ability to restore rapamycin sensitivity in
The immunophilins, FKBPs and cyclophilins, are highly conserved
from bacteria to human and have been found to be both widely distributed and abundantly expressed. In vitro, these
proteins exhibit PPIase activity that accelerates the refolding of
denatured proteins (16-18). Given these properties, it has been
suggested that immunophilins may play a general role in protein folding (reviewed in Ref. 3). However, more recent studies strongly suggest
that immunophilins play specialized roles, dependent on their ability
to selectively bind to other proteins. For example, the mammalian
FKPB12 specifically interacts with the ryanodine calcium release
channel, altering its sensitivity to Ca2+ and stabilizing
its closed state (11-13). Studies in S. cerevisiae are
consistent with the suggestion that the immunophilins do not carry out
a general, housekeeping, role, since mutants lacking all the
immunophilins are viable (62).
Little is known about the functions of the immunophilins in S. pombe, a yeast that is distantly related to S. cerevisiae. Only two members of the family have been subjected to
detailed analysis as follows: wis2+, a heat
shock-inducible 40-kDa cyclophilin that is involved in cell cycle
regulation (63), and fkb39+, a 39-kDa FKBP
homolog that is localized to the nucleus (64). The isolation and
expression of a cyclophilin-18 homolog has been reported (63, 65). In
the present study we isolated the S. pombe FKBP12 homolog
fkh1+ and demonstrated that it is specifically
required for an early step of the sexual development pathway.
In S. cerevisiae, the FKBP12 homolog, FPR1, has a
critical role in mediating the effect of rapamycin to the TOR proteins
(see Refs. 38 and 39 and reviewed in Ref. 66). The amino acid sequence
encoded by fkh1+ is highly similar to that of
FPR1. Consistently, fkh1+ can replace
FPR1 in mediating the effect of rapamycin in S. cerevisiae (Fig. 1). Slight differences do appear between
fkh1+ and FPR1, since overexpression
of fkh1+ but not of FPR1 can slightly
reduce the sensitivity to rapamycin in S. cerevisiae cells
(Fig. 2A). We suggest that fkh1+
reduces rapamycin sensitivity in S. cerevisiae by
forming Fkh1-rapamycin complexes that are not as efficient in
inhibiting the S. cerevisiae TOR proteins as Fpr1p-rapamycin
complexes (Fig. 2B).
One of the key observations that led to the currently accepted model
for rapamycin mode of action in S. cerevisiae was that cells
disrupted for FPR1 are viable and rapamycin-resistant (34, 35). Unlike this finding, disruption of fkh1+
does not result in rapamycin resistance in S. pombe.
Unexpectedly, the phenotype of Does Fkh1 form a toxic complex with rapamycin that inhibits the
functions of the S. pombe TOR proteins? We have recently
determined that the S. pombe tor1+ gene is
required under various stress conditions, including starvation, whereas
tor2+ is an essential gene (46). Since rapamycin
does not affect entrance into stationary phase or response to stress
conditions, it appears that most, if not all, of the S. pombe TOR functions are not inhibited by the drug. Sterility is
the sole phenotype common to rapamycin treatment and loss of function
of TOR activity. However, the sterile phenotypes of tor1
mutants and rapamycin-treated cells seem to be unrelated. In
tor1 mutants the sterile phenotype is likely to be
associated with the inability to respond to starvation conditions,
whereas rapamycin-treated cells are specifically defective in the
sexual development pathway (46). Are the phenotypes of tor1
and fkh1 mutants related? Thus far we have failed to show any genetic link between tor1 and fkh1 mutants;
overexpression of tor1+ does not alleviate the
sterility of The rapamycin-binding site of FKBP12, revealed through structure
analyses of HuFKBP12 (21), is composed of aromatic side chains that
form a hydrophobic pocket. In this work we have exploited the yeast
genetic system to identify residues in Fkh1 involved in the response to
rapamycin. We identified 5 amino acid residues in Fkh1 that are
critical for the effect of rapamycin in S. pombe. Four of
the five amino acids identified, Phe-47, Leu-56, Ile-92, and Phe-100,
correspond to amino acid residues of HuFKBP12 that most closely
interact with rapamycin. The fifth amino acid, Cys-49, corresponding to
Phe-48 in HuFKBP12, is not mapped to the very core of the
ligand-binding pocket but in a close vicinity. The C49R mutation,
however, conferred complete rapamycin resistance in S. pombe, and our studies in S. cerevisiae suggested that
it diminished rapamycin binding (Fig. 6). Since all the
rapamycin-resistant fkh1 mutants are mutated at amino acid
residues potentially important for rapamycin binding, it is likely that
the rapamycin resistance phenotype stems from an inability of the
mutant proteins to bind the drug. This suggestion awaits further
support from binding experiments of rapamycin to the wild type and
mutant Fkh1 proteins.
Notably, all the rapamycin-resistant fkh1 mutants completely
restored mating in The finding that fkh1+ is required for sexual
development provides a novel, genetically amenable system to study the
possible role of this immunophilin in cellular function. In the future we intend to exploit this system to identify the substrate(s) for Fkh1
cellular function in vivo. We have demonstrated in this work
the ability to isolate fkh1 mutants that are
rapamycin-resistant. In the future, a similar approach will be utilized
to isolate fkh1 mutants that are defective in their ability
to support sexual development, thus determining the amino acid residues
critical for Fkh1 activity in this pathway.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
receptor (27-30), and the transcription factor YY1 (31).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S. pombe strains used in this study
fkh1
clones and demonstrated that re-introduction of the wild type
fkh1+ gene rescued the defects observed in these clones.
leu2-3,2-112 trp1-1 ura3-52 his4 rme1
HMLa) and JK9-3d
ade2 fpr1::ADE2 (52), the
kind gift of J. Heitman, Duke University Medical Center.
fkh1 (TA59), and
wild type (TA16) cells transformed with pREP1-fkh1+, following the method described
(47). Aliquots of whole cell extracts containing 40 µg of protein
were fractionated by SDS-polyacrylamide gels and transferred to
membrane filters. The immobilized proteins were detected using the
PerkinElmer Life Sciences ECL system. The Fkh1 proteins were detected
with polyclonal antibodies raised against S. cerevisiae
FKBP12, the kind gift of J. Heitman, Duke University Medical Center.
fkh1 colonies (this study), no dark staining is observed since the sexual development pathway is blocked prior to conjugation. Plasmid DNA was isolated from
fkh1 transformants that stained dark in the presence of rapamycin and used for re-transformation of TA77 and transformation of
bacterial cells for plasmid amplification. Plasmids that conferred rapamycin resistance phenotype upon re-transformation were further subjected to DNA sequence analysis.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
fpr1 S. cerevisiae cells were transformed with
pCM189-fkh1+, and the transformants were
streaked onto plates containing 0.08 µg/ml rapamycin (Fig.
1). As described previously, the wild
type S. cerevisiae cells did not form colonies in the
presence of rapamycin, whereas
fpr1 cells were completely
resistant to the lethal effect of the drug (34) (Fig. 1). Expression of
fkh1+ in
fpr1 cells restored
rapamycin sensitivity (Fig. 1, plate 2), indicating that
fkh1+, like FPR1, is capable of
mediating the effect of rapamycin in S. cerevisiae cells. It
is therefore most likely that the gene product of
fkh1+ forms a toxic complex with rapamycin that
binds and inhibits the S. cerevisiae TOR proteins.
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Fig. 1.
Expression of
fkh1+ in S. cerevisiae
cells. S. cerevisiae wild type cells (WT)
or cells disrupted for the S. cerevisiae FKBP12 homolog
( fpr1) were transformed with vector only or vector
containing the S. pombe fkh1+ gene expressed
from the S. cerevisiae ADH1 promoter. Cells were streaked on
0.08 µg/ml rapamycin-containing plates and incubated at 30 °C.
Photographs were taken after 3 or 8 days of incubation.
fpr1 cells (Fig. 1, plates 3 and
4). Thus, the expression of fkh1+
under the strong ADH1 promoter can slightly increase
rapamycin resistance in S. cerevisiae cells. Overexpression
of FPR1 from the same expression vector did not exhibit such
an effect (see Fig. 2A),
consistent with previous findings (55).
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Fig. 2.
Overexpression of
fkh1+ in S. cerevisiae
cells confers rapamycin resistance that is abrogated by
simultaneous overexpression of FPR1. A, S. cerevisiae cells disrupted for the FKBP12 homolog
( fpr1) were transformed with vector only or vector in
which the S. pombe fkh1+ gene or the S. cerevisiae FPR1 gene are expressed from the ADH1
promoter. Cells were streaked on rapamycin-containing plates as
indicated and incubated at 30 °C. Photographs were taken after 4 days of incubation. B, S. cerevisiae wild type
cells were transformed with overexpression plasmids carrying the
indicated genes. Cells were streaked on plates containing 10 ng/ml
rapamycin and incubated at 30 °C. Photographs were taken after 4 days of incubation.
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Fig. 3.
Phenotype of fkh1-disrupted
cells. A, cell patches of wild type (WT)
strain (TA16) and fkh1 strain (TA59) carrying no plasmids
or transformed with pREP1-fkh1+,
pREP1-FPR1, or vector only, were replica-plated onto ME
plates (conditions that induce mating) in the presence or absence of
rapamycin. After 3 days of incubation at 25 °C, the efficiency of
mating was determined microscopically. B, detection of the
Fkh1 protein was performed by immunoblotting with polyclonal antibodies
raised against the S. cerevisiae Fpr1 protein. Cells
transformed with pREP1-fkh1+ (OP)
exhibited increased expression of a 12-kDa protein compared with wild
type (WT), consistent with the predicted molecular weight of
Fkh1. In protein extracts of
fkh1 cells (
) no 12-kDa
band was observed. C, exponentially growing wild type (TA06)
and
fkh1 (TA96) cells in minimal medium were collected,
washed, and resuspended in nitrogen-free (EMM-N) medium. After 3 days
of incubation at 25 °C, aliquots were removed, and the DNA content
of individual cells was measured by FACS.
fkh1 clones
(fkh1::ura4+ leu1-32
ura4-D18 ade6-M216 h90) showed that the growth rate and
cell morphology of
fkh1 strains were indistinguishable
from the parental wild type strain (data not shown). However, the
ability of
fkh1 strains to enter the sexual development
pathway was greatly diminished. S. pombe cells are induced
to undergo sexual development under starvation conditions, the first
stage being the mating of two haploid cells to form the diploid zygote.
Conventionally, either low nutrient medium such as ME medium, low
nitrogen medium, or low glucose medium are used. We found that under
any of these conditions, the mating efficiency of
fkh1
strains was reduced by 10-40-fold compared with wild type isogenic
strains (Fig. 3A and data not shown). The sterile phenotype
of
fkh1 was suppressed when we re-introduced fkh1+ (Fig. 3A). Interestingly,
introduction of FPR1 on an S. pombe expression
vector also suppressed the mating defect of
fkh1 (Fig. 3A). Thus, although FPR1 does not seem to have a
role in the sexual development pathway in S. cerevisiae, it
can fulfill the function of fkh1+ when expressed
in S. pombe.
fkh1 cells arrested growth as relatively small cells and remained viable over a long period (see "Experimental Procedures" and data not shown). We therefore conclude that
fkh1+ is not required for entrance into
stationary phase.
fkh1 cells and rapamycin-treated cells. The results shown
in Fig. 3C demonstrate that neither
fkh1 nor
rapamycin-treated cells are defective in their ability to arrest in
G1 under nitrogen starvation conditions.
fkh1 cells are
specifically defective in their ability to undergo mating but can
undergo meiosis/sporulation. As in rapamycin-treated cells,
fkh1 cells do not show other defects associated with
abnormal response to starvation, such as entrance into stationary phase
or arrest in G1 in response to nitrogen deprivation. Taken
together, the phenotype of
fkh1 cells is extremely similar to that of rapamycin-treated cells.
fkh1 cells and
rapamycin-treated cells suggests that the direct target of rapamycin in
S. pombe cells is Fkh1. We hypothesized that if Fkh1 is the
target for rapamycin action, then we might isolate fkh1 mutants that can confer rapamycin resistance. Such mutants are expected
to be impaired in rapamycin binding but to retain activity necessary
for the sexual development pathway.
fkh1 h90) was transformed
with a mixture of plasmids bearing mutated fkh1 cDNAs,
plated on minimal medium in the absence of rapamycin. After colonies
had developed they were replica-plated onto 0.2 µg/ml rapamycin-containing plates. Colonies that underwent sexual development despite the presence of rapamycin were identified by exposure to iodine
vapor (see "Experimental Procedures"). Of 25,000 transformants, 28 clones showed plasmid-dependent sporulation on
rapamycin-containing plates. Sequence analysis revealed that 16 of
these 28 clones carried single missense mutations at one of the
following positions: Phe-47, Cys-49, Leu-56, Ile-92, or Phe-100.
The remaining 12 mutants carried 2-4 missense mutations. In each case
at least one mutation occurred at one of the critical positions Phe-47,
Cys-49, Leu-56, Ile-92, or Phe-100 (see Table
II). Since iodine vapor analysis suggested that all mutations conferred similar rapamycin resistance phenotype, representatives of mutants of each of the five critical mutations were chosen for further analysis as follows: F47S, C49R, L56F, I92F, and F100L. Quantitative assessment of the mating
efficiencies of
fkh1 cells expressing these
fkh1 mutants demonstrated that all fully complemented the
mating deficiency phenotype and conferred a complete rapamycin
resistance phenotype (Fig. 4).
Isolation of fkh1 mutants
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Fig. 4.
fkh1 rapamycin-resistant mutants.
fkh1 cells (TA77) transformed with vector only,
fkh1+, or fkh1 bearing the indicated
mutations, were streaked on EMM medium with (+R) or without
(
R) 0.2 µg/ml rapamycin and incubated at 30 °C for 4 days. The cells were then exposed to iodine vapor. Since iodine stains
only spores, only cells that underwent sexual development are stained
with dark color. The mating frequency of each strain was scored under
the microscope, and the averages of the scores obtained in two
independent experiments are shown.
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Fig. 5.
A comparison of FKBP12 sequences and
positions of rapamycin-resistant fkh1 mutants.
The predicted amino acid sequence of fkh1+
(SpFKBP) is aligned with the S. cerevisiae Fpr1p (ScFKBP)
and the human FKBP12 homolog (HuFKBP). Asterisks denote the
positions in SpFKBP12 in which single point mutations confer rapamycin
resistance phenotype. Boxes indicate the positions that are
predicted to interact most closely with rapamycin according to
structural studies of the HuFKBP12-rapamycin complex (21).
fpr1 mutants. We cloned each of
the fkh1 mutants into an S. cerevisiae expression
vector and transformed it into
fpr1 strain (Fig.
6). Although the wild type
fkh1+ gene could efficiently restore rapamycin
sensitivity in
fpr1 mutants, the F47S mutant completely
failed to restore rapamycin sensitivity, suggesting that this mutation
is strongly impaired in rapamycin binding.
fpr1 cells
transformed with F100L and C49R grew well on plates containing 25 ng/ml
rapamycin but poorly on plates containing 100 ng/ml rapamycin,
suggesting that the F100L and C49R mutants are partially impaired in
rapamycin binding in S. cerevisiae cells. Somewhat
surprisingly, the mutant L56F efficiently restored rapamycin
sensitivity, suggesting that it can efficiently form a toxic complex
with rapamycin in S. cerevisiae.
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Fig. 6.
Expression of fkh1 mutants
in fpr1 S. cerevisiae cells.
S. cerevisiae
fpr1 cells transformed with
vector only (
), fkh1+, or fkh1
bearing the indicated mutations were streaked on plates containing
rapamycin as indicated. Plates were photographed after 3 days of
incubation at 30 °C.
fpr1 S. cerevisiae cells. Of all the mutations only the
F47S mutation completely abolished the ability of fkh1 to
restore rapamycin resistance in
fpr1. The differences in
the behavior of the fkh1 mutants in the two yeast systems
may not be surprising since in S. pombe rapamycin seems to
inhibit directly the FKBP12 function, whereas in S. cerevisiae rapamycin exerts its effect by forming a complex with
FKBP12 that inhibits the TOR proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
fkh1 mutants highly
resembled that of cells treated with rapamycin;
fkh1
cells are defective in an early step of the sexual development pathway,
before mating occurs, but show no defects in later steps such as
meiosis or sporulation. In S. pombe, sexual development is a
process induced only under starvation conditions (see Introduction).
However, neither the sterility of rapamycin-treated cells (44) nor the
sterility of
fkh1 mutants is associated with defects in
response to starvation. The strong similarity between the phenotype of
fkh1 mutants and that of rapamycin-treated wild type
cells suggests that rapamycin inhibits sexual development directly by
inhibiting the cellular function of fkh1+.
fkh1 cells, and overexpression of
fkh1+ does not alleviate the sterility of
tor1
cells.3
fkh1 mutants (Fig. 4). This finding
suggests that the Fkh1 rapamycin-binding site and the putative active
site do not completely overlap. In particular, mutation at the Phe-100 amino acid residue did not impair the cellular activity of Fkh1 in
sexual development, despite its being one of the most conserved amino
acid residues in FKBP12 sequences (21). The effects of mutations at
positions corresponding to Phe-100 in human FKBP12 (F99Y) and in
S. cerevisiae FPR1 (F106Y) have been studied previously (67,
68). Like the F100L mutation in Fkh1, neither the F99Y mutation nor the
F106Y mutation affected the protein function in vivo. The
F99Y mutant supported the ryanodine channel function (67), and the
F106Y mutant complemented the slow growth phenotype observed in
fpr1 (68). It is also interesting to note that the
HuFKBP12 F99Y mutant and the S. cerevisiae F106Y mutant show reduced PPIase activity in the in vitro peptide cleavage
assay (67, 68). More recently, however, it was demonstrated that the
S. cerevisiae Phe-106 mutant retained PPIase activity in a different assay that uses ribonuclease T1 as a substrate (68). It has
thus been suggested by Dolinski et al. (68) that the Phe-106
mutant may retain PPIase activity toward large substrates, leaving it
an open question whether the PPIase activity observed in
vitro is relevant for the cellular activity in
vivo.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Heitman for S. cerevisiae strains and for antibodies raised against the S. cerevisiae FKBP12 homolog. We also thank Y. Aylon for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This research was supported by The Israel Science Foundation Grant 526/00=16.0 and by an Israel Cancer Research Fund (ICRF) fellowship (to R. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be
addressed. Tel.: 972-3-6407532; Fax: 972-3-6409407; E-mail:
ronitt@post.tau.ac.il.
Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M102090200
2 R. Weisman, S. Finkelstein, and M. Choder, manuscript in preparation.
3 R. Weisman, S. Finkelstein, and M. Choder, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CsA, cyclosporin A; PPIase, peptidylprolyl-cis/trans-isomerization; PCR, polymerase chain reaction; bp, base pair; FACS, fluorescence-activated cell sorter.
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