From the Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences. Tel Aviv University, Tel Aviv 69978, Israel
Received for publication, November 20, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Targets of rapamycin (TORs) are conserved
phosphatidylinositol kinase-related kinases that are involved in the
coordination between nutritional or mitogenic signals and cell growth.
Here we report the initial characterization of two
Schizosaccharomyces pombe TOR homologs,
tor1+ and tor2+.
tor2+ is an essential gene, whereas
tor1+ is required only under starvation and
other stress conditions. Specifically, The target of rapamycin (TOR)1-mediated signaling
pathway in the yeast Saccharomyces cerevisiae activates a
cell growth program in response to nutrient availability (reviewed in
Refs. 1 and 2). S. cerevisiae contains two TOR homologs,
TOR1 and TOR2. TOR1 is a nonessential gene that
shares a common function with TOR2 in controlling
G1 progression (3-6). This common function is sensitive to
the drug rapamycin. Thus, either the loss of function of both
TOR1 and TOR2 or the inhibition of their products
by rapamycin results in a G1 arrest (6). The rapamycin
G1 arrested cells exhibit various characteristics of
starved cells (6-11), suggesting that the rapamycin-sensitive activity
of the two Tor proteins inhibits the program that leads to stationary
phase. Tor2p also controls the polarization of the actin cytoskeleton
(12, 13), an essential function that is not shared by Tor1p and is not
inhibited by rapamycin (4, 5). Similar to the S. cerevisiae
TORs, the mammalian TOR homolog FRAP/RAFT1/mTOR is also involved in signaling pathways that regulate transcription, translation, and G1 progression (reviewed in Ref. 20).
All known TORs contain a conserved domain, the FKBP12-rapamycin binding
(FRB) domain, that lies adjacent to the
phosphatidylinositol kinase domain in the C-terminal region of
the protein (14, 15). A conserved serine residue within this domain is
crucial for the binding of the rapamycin-FKBP12 complex. Studies in
mammals (14-16), S. cerevisiae (3, 5, 17-19), and
Cryptococcus neoformans (20) have shown that a
mutation at the conserved serine residue confers dominant rapamycin
resistance by abolishing the binding to the FKBP12-rapamycin complex.
The importance of the conserved serine for this binding is reinforced
by the atomic structure of the ternary complex FRB-rapamycin-FKBP12
(21). Despite the high conservation of this serine, TOR proteins that
are expressed at normal levels and carry a mutated serine appear to
retain wild type activities other than FKBP12-rapamycin binding
(5, 22-24).
Schizosaccharomyces pombe is genetically tractable yeast
that is highly divergent from S. cerevisiae. These two
yeasts often have distinct differences in carrying out the same
cellular functions, which makes their comparative study especially
revealing (25). Upon starvation, S. pombe cells enter either
the stationary phase or the sexual development pathway (reviewed in
Ref. 26). We previously reported that rapamycin has a different effect
on S. pombe compared with its effect on S. cerevisiae. Rapamycin does not inhibit the growth of S. pombe but specifically inhibits sexual development in response to
starvation (27). As a first step toward understanding the response to
rapamycin in S. pombe, we cloned and initiated a functional
analysis of the S. pombe TOR homologs, named
tor1+ and tor2+. We show
here that at least some of the functions of each of these TORs are
distinct. Thus, tor2+ is essential for growth,
whereas tor1+ is required only under starvation
and other stress conditions. We also demonstrate that the conserved
serine residue within the FRB domain of Tor1 is important for the
protein function and does not play a detectable role in the response of
starved S. pombe to rapamycin.
Yeast Strains, Media, and General Techniques--
Yeast strains
used in this paper are described in Table
I. All media used in this study are based
on those described previously (28). EMM-N contains no nitrogen; EMM
lowG contains 0.1% glucose. Rapamycin was used as described previously
(27). Transformation of S. pombe cells was performed by
electroporation (29). Assays for mating or sporulation efficiency were
carried out as described in (27).
Fluorescence-activated Cell Sorter Analysis--
Cells were
stained with the DNA fluorochrome propidium iodide and analyzed by a
Becton Dickinson FACSort as described by (30). Data were analyzed by
Cell Quest software for Macintosh.
Disruption of S. pombe tor1+--
A fragment
containing 5.84 kbp of the C-terminal region of
tor1+ gene was amplified by PCR using the Expand
Long Template PCR System (Roche Molecular Biochemicals) with a genomic
S. pombe DNA preparation as a template and the primers 104 (5'-TTGAAGAATCTGCAGCAATAAATATTC) and 105 (5'-AAGATTTGATCGGCATTTGGCAC).
The resulting PCR fragment was subcloned into a pGEM-T vector (Promega)
to give pGEMtor1. A 3.63-kbp HindIII fragment of pGEMtor1
containing the kinase and FRB-like domains was replaced with an
HindIII fragment containing the entire
ura4+, resulting in the plasmid
ptor1::ura4. NotI and SacI were used to
release the 4-kbp tor1::ura4 disruption fragment, and this was gel purified and transformed into the diploid TA07. Stable Ura4+ diploids were selected, and their DNA was extracted
and subjected to PCR analysis with primers 105 and 137 (5'-TTGTAAATAGGATAGCCAGCACC), which lies 100 bp upstream of the 5' end
of the disruption construct.
Disruption of S. pombe tor2+--
0.4- and 0.5-kbp
fragments containing the very 5' and 3' ends of the
tor2+ open reading frame, respectively, were
amplified by PCR, using genomic DNA as the template. The primers were:
152 (5'-ATAAGAGTCGACTCACAAGTGTTGTGAACTTGGTGG); 154 (5'-GGGGTACCGAGCTCATTCCTGCTTTTCAACCCAGG); 153 (5'-ATAAGAGTCGACAGATACGTGAAGAGGGGTGGTGAC); and 155 (5'-CGGGATCCGTTTCTGGTAGGTGACAGTCCC). The resulting 0.4- and
0.5-kbp PCR fragments were digested with KpnI and
BamHI, respectively (sites are underlined in the sequence of
the primers) and ligated with the 1.8-kbp
kpnI-BamHI fragment containing the entire
ura4+ gene. The fragment containing the
ura4+ gene flanked by
tor2+ sequences was amplified by PCR using 152 and 153 and used to transform the diploid strain TA07. The correct
disruption of tor2+ was verified by PCR analysis
using the primer 165 (5'-CGGGATCCCCATTTAATAGAGAAAGGGATATTAGC) that lies
32 bp upstream of the 5' end of the disruption construct in combination
with primer 135 (5'-GTTATAAACATTGGTGTTGGAACAG) that lies in the
ura4+ gene.
Cloning of S. pombe tor1+ and tor2+ and
Site-directed Mutagenesis--
The tor1+ and
tor2+ gene were obtained using the Expand Long
Template PCR System (Roche Molecular Biochemicals) with a genomic S. pombe DNA preparation as the template. For amplifying
tor2+ we used primers 152 (5'-ATAAGAGTCGACTCACAAGTGTTGTGAACTTGGTGG) and 153 (5'-ATAAGAGTCGACAGATACGTGAAGAGGGGTGGTGAC) (SalI
sites used for subsequent cloning are underlined). For amplifying
tor1+ we used primers 141 (5'-CGGGATCCGCGGCCGCCAATGTGAATGCATATCTTTAGTCC) and 142 (5'-ACGCGTCGACTGCTCTGAAGTCAATTCCGAAGTG) (BamHI,
NotI, and SalI sites used for subsequent
cloning are underlined). The resulting 8.3-kbp
tor1+ and 8 kbp tor2+ PCR
fragments were cloned into the S. pombe pIRT2 expression plasmid (31), resulting in pIRT2-tor1+ and
pIRT2-tor2+, respectively.
To express tor1+ in S. cerevisiae we
used pIRT2-tor1+ as a template in a PCR reaction
with primers 143 (5'-CGGGATCCGTCTATCGTTTCACTCGCTCTC) (BamHI, for subsequent cloning is underlined) and primer
141. This reaction resulted in the amplification of a 7.4-kbp fragment that contains the tor1+ ORF, 30 and 300 bp
upstream and downstream of the open reading frame, respectively. This
fragment was cloned into the pCM190 S. cerevisiae expression
vector (32), resulting in pCM190-tor1+.
The mutation at the conserved serine in the Tor1 FRB domain,
S1843R, was created by site-directed mutagenesis by overlap extension (33). pIRT2-tor1+ served as a template in a PCR
reaction with primers 145 (5'-TGCATCCTCAGGCATTGGTGTATTC) and 146 (5'-GAAAAATAAGCCTGACGAGCTTCCTCTAATC; mutations are in bold
type). The resulting PCR product (220 bp) was gel purified and used as
a primer for a second-round of PCR with primer 144 (5'-AATAGATCTCTCGTTGAGTCCTTCG). The resulting 1.58-kbp PCR product was
cleaved with BglII and Bsu36I, whose restriction
sites reside near the fragment ends. This fragment was used to replace the corresponding fragment in pIRT2-tor1+ and
pCM190-tor1+. We verified that only the desired
mutation had occurred during PCR or cloning of the
BglII/Bsu36I fragment by DNA sequence analysis. In addition, to further ascertain that the only defect of the tor1S1834R resided in Ser1834, the
plasmid carrying tor1S1834R was cut with
BglII/Bsu36I, and the 1.58-kbp fragment
containing the site of the mutation was recovered. This fragment was
used as the template in a PCR reaction performed to replace
Arg1834 back with serine. The fragment containing
Ser1834 was ligated back to the
BglII/Bsu36I cut plasmid to reconstruct a wild
type tor1+. This tor1+,
derived from tor1S1834R, fully restored
tor1+ function.
Epitope Tagging and Western Blot Analysis of Tor1 and
Tor1S1834R--
The wild type and mutant tor1
genes were amplified from the plasmids
pIRT2-tor1+ and
pIRT2-tor1S1834R using the primers 141 (containing a BamHI site, see above) and 199 (ATAAGAATGCGGCCGCATGGAGTATTTTAGTGATCTAAAAAAC) (the
NotI site is underlined). PCR products were digested with
BamHI/NotI and cloned into the
BglII/NotI sites of the thiamine-repressible
vector pSLF273 (34), downstream and in frame with the plasmid sequences encoding the triple hemagglutinin (HA) epitope domain. The resulting plasmids carrying the N-terminal HA-tagged wild type and mutated proteins were transformed into a Identification of TOR Homologs in the S. pombe Genome--
The DNA
sequence of most of the S. pombe genome has been determined
through the coordination of the Sanger Center. Based on sequence
comparisons, we identified two TOR homologs in chromosome II. We named
one of these, on cosmid SPBC30D10, tor1+ and the
second, contained on two overlapping cosmids, SPBC216 and SPBC646,
tor2+. The open reading frames of
tor1+ and tor2+ encode
for 2335- and 2337-amino acid proteins, respectively. No introns are
found in either of the open reading frames. The two S. pombe
TOR homologs share 52% overall identity. A slightly lower level of
overall identity (42-44%) is revealed when the amino acids encoded by
tor1+ or tor2+ are
aligned with the human TOR, the S. cerevisiae Tor1p or
Tor2p, or the C. neoformans Tor1. The C-terminal regions of
the S. pombe Tor1 and Tor2 proteins contain the FRB and the
phosphatidylinositol kinase-like domains and are the most conserved
regions of the proteins. The C-terminal regions of Tor1 and Tor2,
residues 1814-2171 and 1817-2174, respectively, show 64-65%
identity with the corresponding amino acids of other TOR homologs in
the data bases. Amino acid comparisons of these C-terminal regions
indicate that both Tor1 and Tor2 in S. pombe contain
the conserved structural features that characterize the TOR family of
proteins. This includes the conserved serine at position 1834 or 1837 in Tor1 or Tor2, respectively (Fig.
1).
The S. pombe tor2+, but Not
tor1+, Is Required for Growth--
As a first
step toward understanding the physiological function(s) of the TORs in
S. pombe, we disrupted the chromosomal copies of
tor1+ or tor2+ by
one-step gene replacement ("Experimental Procedures"). Fig. 2 shows a tetrad analysis of a
heterozygous diploid strain (TA137) in which one of the copies of
tor2+ was replaced with the selective marker
ura4+. A total of 25 tetrads were dissected, and
all yielded only two or one viable spores, none of which cosegregated
with the ura4+ marker, indicating that the
tor2 disruption was lethal. Microscopic examination revealed
that in the nonviable segregants, the spores produced single cells that
did not undergo cell divisions (data not shown). This lethal phenotype
was rescued by reintroduction of tor2+. Strain
TA137 transformed with plasmid pIRT2-tor2+
(tor2+ expressed under the regulation of its
endogenous promoter) was sporulated and dissected. Progeny disrupted in
the chromosomal copy of tor2+ were obtained, but
in all cases they carried the plasmid-borne wild type
tor2+ gene.
We also used the ura4+ selective marker to
disrupt tor1+ ("Experimental Procedures").
In contrast to tor2 disruption, sporulation of diploid
heterozygous for tor1+ disruption (TA82) yielded
viable Ura4+ haploids. PCR analysis confirmed that the
Ura4+ haploids carried a disputed allele of tor1
(data not shown). Thus, whereas tor2+ is an
essential gene, tor1+ is a nonessential gene.
tor1+ Is Required for Entrance into Stationary Phase
and for Sexual Development--
We further analyzed two independently
isolated
We also noted that
Nitrogen or carbon starvation is also a signal for diploid cells to
enter meiosis (reviewed in Ref. 26). Under these conditions most of the
wild type diploid cells, >60%, underwent sporulation, whereas <1%
of homozygous
Analysis of the DNA content of growing and starved tor1+ Is Required for Growth under Osmotic or Oxidative
Stress Conditions--
We noted that the phenotype of
Ser1834 in the FRB Domain of Tor1 Is Required for Tor1
Activity--
We previously reported that rapamycin specifically
inhibits sexual development in S. pombe (27). As indicated
above,
The tor1+and tor1S1834R
genes, cloned into the S. pombe expression vector pIRT2
("Experimental Procedures") were transformed into
Our finding that Ser1834 is critical for the function of
Tor1 is surprising given that equivalent mutations did not affect TOR function in S. cerevisiae. To ascertain that the only defect
of the tor1S1834R resided in S1834, the plasmid
carrying tor1S1834R was used as the template in
a PCR reaction performed to replace Arg1834 back with
serine (see "Experimental Procedures"). This
tor1+, derived from
tor1S1834R, fully restored
tor1+, demonstrating that
tor1S1834R carries no mutations other than
S1834R. We also examined whether the mutation at S1834 affected protein
stability. To this aim, the wild type and mutated Tor1 proteins were
tagged with the HA epitope (see "Experimental Procedures").
Analysis of
Because the S. pombe tor1+ shows a significant
level of homology with the S. cerevisiae TOR genes (about
43% identity), we examined the functions of the S. pombe
tor1+ and tor1S1834R in
S. cerevisiae. Plasmids carrying
tor1+ or tor1S1834R,
expressed under the control of the S. cerevisiae ADH1
promoter, were introduced into wild type S. cerevisiae
cells. Fig. 7 shows that
tor1S1834R but not tor1+
conferred dominant rapamycin resistance in S. cerevisiae.
This is similar to the effect of equivalent point mutations in the S. cerevisiae TOR proteins (3, 5, 19), except that
tor1S1834R did not confer rapamycin resistance
at a high concentration of rapamycin (100 ng/ml, results not shown).
The dominant resistance exhibited by the S. pombe
tor1S1834R indicates that the S. pombe TOR
homolog can complement the function of the S. cerevisiae
TORs. It also implies that the conserved serine in
tor1+ is critical for the binding of
FKBP12-rapamycin when expressed in S. cerevisiae cells. In
conclusion, it appears that although S1834 is required for Tor1
function in S. pombe, it is not required for the
rapamycin-sensitive TOR function of S. cerevisiae.
We report here the identification and initial characterization of
the S. pombe homologs of the TOR genes. We found the
S. pombe tor2+ gene is essential for growth,
whereas tor1+ is required only under starvation,
osmotic stress, and oxidative stress.
The inability of Mutational analysis of tor1+ revealed that the
conserved serine residue within the FRB domain of Tor1 plays a critical
role in the protein cellular function. Thus, although the mutation at
Ser1834 did not affect the level of protein expression
(Fig. 6), tor1S1834R can only partially
complement the defects observed in None of the functions of the S. pombe TORs described in this
work appear to be inhibited by rapamycin. Rapamycin specifically inhibits sexual development in S. pombe, at an early stage,
before mating (27). Cells disrupted for tor1+
are deficient in their ability to undergo mating. However, the effects
of Tor1 and rapamycin on sexual development appear to be unrelated. The
inability of If neither Tor1 nor Tor2 is the protein target for rapamycin, than what
is the target for the action of rapamycin in S. pombe? Recent findings in our lab show that cells disrupted for the S. pombe FKBP12 homolog exhibit a phenotype highly similar to
treatment with rapamycin. Thus, it is most probable that rapamycin
inhibits sexual development by inhibiting the cellular function of
FKBP12 in sexual development3
and not by inhibiting TOR-related function.
Why rapamycin does not inhibit neither the function of Tor1 or Tor2?
Our functional analysis of tor1+ and
tor1S1834R in S. cerevisiae cells
(Fig. 5A) suggests that Tor1 can bind the FKBP12-rapamycin
complex, at least in S. cerevisiae cells. It is possible
that Tor1 interacts with FKBP12-rapamycin complexes in S. pombe, but this interaction does not inhibit the studied functions
of Tor1 or Tor2. By analogy, the function of the S. cerevisiae Tor2p in the control of the actin cytoskeleton
organization is not inhibited by rapamycin (5).
The features of S. pombe TOR proteins, together with other
studies of TOR functions, indicate that these proteins are involved in
many distinct cellular functions. Given that the C-terminal region
containing the FRB and the kinase domains of the TORs is highly
conserved, the differences between the TORs might reside in the less
conserved N-terminal region. This is an intriguing possibility yet to
be explored.
tor1 cells fail
to enter stationary phase or undergo sexual development and are
sensitive to cold, osmotic stress, and oxidative stress. In complex
with the prolyl isomerase FKBP12, the drug rapamycin binds a conserved
domain in TORs, FRB, thus inhibiting some of the functions of TORs.
Mutations at a conserved serine within the FRB domain of
Saccharomyces cerevisiae TOR proteins led to rapamycin
resistance but did not otherwise affect the functions of the proteins.
The S. pombe tor1+ exhibits different features;
substitution of the conserved serine residue, Ser1834, with
arginine compromises its functions and has no effect on the inhibition
that rapamycin exerts on sexual development in S. pombe.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Strains used in this study
tor1 strain, TA163.
Total protein extracts were prepared from cells growing under
conditions that allow protein expression (in the absence of thiamine)
following the method described by (28). Aliquots of whole cell extracts containing 60 µg of protein were fractionated by SDS-polyacrylamide gels and transferred to membrane filters. The immobilized proteins were
detected using the PerkinElmer Life Sciences enhanced chemiluminescence system. The HA-tagged proteins were detected with monoclonal antibody HA.11 (Berkley Antibody Co.). Polyclonal antibodies raised against S. cerevisiae FKBP12 (a kind gift of J. Heitman, Duke
University Medical Center) were used to detect the S. pombe
FKBP12 proteins as an internal loading control.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (58K):
[in a new window]
Fig. 1.
The FRB domains of the S. pombe
Tor1 and Tor2 are highly conserved. The predicted amino acid
sequences of the S. pombe Tor1 (SpTOR1) and S. pombe Tor2 (SpTOR2) FRB domains are aligned with the analogous
regions from S. cerevisiae Tor1p (ScTOR1), S. cerevisiae Tor2p (ScTOR2), C. neoformans (CnTOR1), and
mammalian mTOR. Asterisks denote the positions of the amino
acid residues that closely interact with rapamycin in the ternary
structure of the mammalian FRB domain with FKBP12-rapamycin (21).
View larger version (43K):
[in a new window]
Fig. 2.
tor2+ is an essential
gene. A diploid
tor2+/ tor2 was
sporulated and dissected on a nonselective medium. All viable spores
were Ura
, indicating that tor2+ is
an essential gene.
tor1 haploid clones. Under optimal growth
conditions, the growth rate and cell morphology of
tor1 cells were indistinguishable from that of
wild type cells (Fig. 3A).
However,
tor1 cells exited the logarithmic phase at a
lower cell density, were abnormally long, and lost viability rapidly
(Fig. 3). The loss of viability of
tor1
depends on the growth medium. Whereas
tor1
cells died when they reached saturation in rich medium, they maintained
viability comparable with wild type cells when grown to saturation in
minimal medium (Fig. 3B). This suggests that
tor1 cells are defective in the response to
particular sets of conditions rather than in the actual cellular
processes that allow cells to acquire the stationary phase
physiology.
View larger version (58K):
[in a new window]
Fig. 3.
tor1+ is required for
entrance into stationary phase. A, growth curves of
wild type (WT, TA100) and tor1 (TA99) cells in
a rich (YE) medium. B, viability in stationary
phase. Cells were grown to stationary phase in rich or minimal
(EMM) medium. After the cells entered the stationary phase,
at the indicated time points, cell viability was determined by plating
efficiency on a complete medium. C, the morphology of
starved wild type and
tor1 cells. Photographs were taken
48 h after cells exited the logarithmic phase in rich
medium.
tor1 cells failed to cross with wild
type strains. Microscopic examination suggested a defect at an early stage of sexual development, before conjugation had occurred. Media
limiting for either nitrogen or carbon sources are conventionally used
for a quantitative analysis of conjugation (mating). Under these
conditions, haploid cells of the opposite mating types can conjugate to
form a diploid zygote, which rapidly undergoes meiosis and sporulation
and produces an ascus (reviewed in Ref. 26). Under either nitrogen or
carbon starvation, ~60% of wild type cells underwent mating compared
with less than 1% of
tor1 cells. The sterile phenotype
of
tor1 was efficiently suppressed when we reintroduced
tor1+ (Fig.
4A).
View larger version (41K):
[in a new window]
Fig. 4.
Phenotypes of
tor1 cells under starvation, osmotic,
and oxidative stress. A, heterothallic
tor1 strain (TA99) was transformed with the S. pombe vector pIRT2 or pIRT2-tor1+. The
transformants were mixed with wild type (WT) cells of the
opposite mating type (TA02) and induced to undergo sexual development
in EMM-N medium. Wild type is TA16 induced to undergo sexual
development in EMM-N. B, exponentially growing wild type
(TA100) and
tor1 (TA99) cells in minimal medium (log)
were collected, washed and resuspended in nitrogen free
(EMM-N), low glucose (EMMlowG), or EMM
(Stationary) medium, and incubated at 25 °C for 3 days.
Aliquots were removed from growing and starved cells, and the DNA
content of individual cells was measured by fluorescence-activated cell
sorter. C,
tor1 cells transformed with vector
only or plasmid carrying tor1+ were streaked
onto YE agar plates supplemented with 1 M KCl or 0.5 M NaCl and incubated for 5 days at 32 °C. D,
tor1 cells transformed with vector only or plasmid
carrying tor1+ were streaked onto YE agar plates
supplemented with 0 or 5 mM H2O2
and incubated for 5 days at 32 °C.
tor1 diploids sporulated (results not shown). Thus, in addition to its role in mating,
tor1+ is also required for
meiosis/sporulation.
tor1
cells also indicates that these cells are defective in their response to starvation. In the absence of a mating partner, starved S. pombe cells become arrested in either G1 or
G2, depending on the growth medium; nitrogen starvation
arrests cells mainly at the G1 phase, and carbon starvation
arrests cells mainly at the G2 phase (35). The DNA profile
of
tor1 cells under optimal growth conditions shows a
major 2n DNA peak, characteristic of growing wild type cells
(see Refs. 36 and 37 and Fig. 4B). However, under nitrogen
starvation,
tor1 cells show an abnormal DNA profile as
cells failed to arrest in G1 (Fig. 4B). Because
G1 arrest is a prerequisite for mating, the failure of
tor1 cells to arrest their growth in G1 may
be associated with their inability to undergo sexual development.
tor1 cells was particularly similar to that of cells
disrupted for atf1+, a gene that encodes a bZIP
(basic leucine zipper) transcription factor (37). Under starvation
conditions, both
tor1 and
atf1 cannot
arrest in G1, exhibit an abnormal elongated morphology, lose viability in rich but not minimal medium, and are sterile. atf1+ has also been implicated in regulating the
cellular response to a variety of stress conditions, such as cold,
osmotic stress, and oxidative stress (37-39). We found that
tor1+ is also required under these stress
conditions; unlike wild type cells,
tor1 could not form
colonies on medium containing 0.5 M NaCl or 1 M
KCl (Fig. 4C) or on medium containing 5 mM
H2O2 (Fig. 4D) or below 20 °C
(results not shown). Taken together, our findings reveal a striking
similarity between the phenotypes of
tor1 and
atf1 cells. It remains to be determined whether tor1+ and atf1+ are
involved in the same signaling pathway.
tor1 cells are unable to undergo sexual
development. Because rapamycin is known to inhibit the TOR proteins in
mammals, S. cerevisiae, and C. neoformans, we
considered the possibility that rapamycin exerts its effect by
inhibiting the function of the S. pombe Tor1 during sexual
development. A conserved serine residue in TORs has been identified as
the site for missense mutations (serine substituted with arginine,
isoleucine, or glutamic acid) conferring dominant rapamycin resistance
(see the Introduction). We mutated the equivalent serine residue in
S. pombe Tor1, Ser1834, into arginine
("Experimental Procedures").
tor1 strains TA99 or TA157. TA99 and TA157 are isogenic
except that TA157 is a homothallic strain (cells can switch their
mating types between h+ and
h
every other generation), whereas TA99 is a
heterothallic strain composed of h
cells only.
Surprisingly, we found that the mutation at Ser1834
diminished the activity of Tor1; the mating efficiency was extremely low when TA157 cells carrying tor1S1834R were
induced to undergo mating (0.9%, Fig.
5A). In crosses between wild
type and
tor1 cells (TA99) carrying
tor1S1834R, we observed that the wild type cells
partially suppressed the sterility of
tor1 cells carrying
tor1S1834R (Fig. 5A). The mutation
S1834R also diminished the activity of Tor1 under osmotic stress
conditions (Fig. 5C) or in acquisition of stationary phase
physiology (data not shown). Because mating is very inefficient in
tor1S1834R mutants, it was no surprise that this
mutated allele did not confer dominant resistance to rapamycin in wild
type TA16 transformants (Fig. 5B).
View larger version (37K):
[in a new window]
Fig. 5.
Expression of
tor1S1834R gene in S. pombe
cells. A, heterothallic tor1
strain (TA99) or homothallic
tor1 strain (TA157) were
transformed with the S. pombe tor1+ or
tor1S1834R. TA99 transformants were mixed with
wild type (WT) cells of the opposite mating type (TA02) and
induced to undergo sexual development in EMM-N medium. TA157
transformants were also induced to undergo sexual development in EMM-N
medium. B, wild type homothallic cells (TA16) transformed
with the S. pombe tor1+ or
tor1S1834R were induced to undergo sexual
development in EMM-N in the presence or absence of rapamycin.
C,
tor1 cells (TA99) transformed with vector
only, plasmid carrying tor1+, or
tor1S1834R were streaked onto YE agar plates
supplemented with 0.8 M KCl and incubated at 32 °C.
Photographs were taken after 4 days of incubation.
tor1 cells expressing these proteins
demonstrated that the HA tagging did not affect the activity of the
proteins in vivo (data not shown). Western blot analysis
using antibody raised against the HA epitope demonstrated that the
level of Tor1 and Tor1S1834R are comparable (Fig.
6). Therefore, we conclude that the
S1834R mutation did not affect protein stability in vivo,
but rather the S1834 residue is required for the function of Tor1 under
starvation or osmotic stress conditions.
View larger version (52K):
[in a new window]
Fig. 6.
Wild type and mutant HA-TOR1 fusion proteins
are stably expressed. Top panel, HA-Tor1 and
HA-Tor1S1834R fusion proteins were expressed in strain
TA163 and detected by Western blot with antibodies against the HA
epitope. Bottom panel, the same extracts as in
the top panel were detected using FKBP12 antiserum
raised against the S. cerevisiae FKBP12 homolog. This
antibody cross-reacts with the S. pombe FKBP12 protein and
is used here as a control for protein loading.
View larger version (42K):
[in a new window]
Fig. 7.
The S. pombe
tor1S1834R can confer rapamycin resistance in
S. cerevisiae. S. cerevisiae wild type cells,
JK9-3d (19) transformed with plasmids containing the S. pombe
tor1+ or tor1S1834R genes, were
streaked on minimal plates containing 0 or 10 ng/ml rapamycin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
tor1 cells to respond appropriately to
starvation conditions may suggest that the S. pombe
tor1+, like its S. cerevisiae TOR homologs
(2, 40), participates in signal transduction pathways that are involved
in nutrient sensing. However, there are significant differences between
the functions of the S. cerevisiae and S. pombe
TORs. First, the S. pombe tor1+ has a positive
role in the sexual development pathway and entry into stationary phase,
whereas the activity of the S. cerevisiae TORs is required
to repress meiosis (41) and entry into stationary phase (6-10, 42).
Second, the S. pombe tor1+ is required for the
appropriate response to a variety of stress conditions, whereas there
is no evidence that the S. cerevisiae TOR homologs are
involved in the response to stresses other than starvation. Third, each
of the two S. pombe TOR homologs carries out a distinct
function that is not shared by the other homolog. In contrast, the
S. cerevisiae TOR1 is a nonessential gene, and its function
in regulating growth in response to nutrient availability is shared
with TOR2 (3-5).
tor1 cells (Fig. 5).
Although Ser1834 is required for Tor1 function, its
equivalent serine residues in the S. cerevisiae TOR proteins
(3, 5, 18) and possibly the human TOR protein (22-24) do not appear to
play an essential role in the studied functions of the proteins.
However, given the conserved nature of this serine residue, its role in
other TOR homologs may become evident under yet unidentified conditions.
tor1 cells to enter sexual development seems
to be part of a general defect in responding to nutritional deprivation. Thus,
tor1 cells fail to enter stationary
phase, arrest in G1 in response to starvation, or undergo
meiosis/sporulation. In contrast, rapamycin specifically inhibits the
sexual development pathway and does not interfere with other responses
to starvation. Thus, cells treated with rapamycin can enter stationary
phase properly, are only slightly defective in meiosis/sporulation
(27), and can arrest their growth in G1 under starvation
conditions.2 Our finding that
tor1S1834R could not alleviate the inhibitory
effect of rapamycin on sexual development (Fig. 5B) is
consistent with our suggestion that rapamycin does not exert its effect
in S. pombe by forming a toxic FKBP12-rapamycin complex that
inhibits the Tor1 function.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Heitman for S. cerevisiae strains and antibodies against S. cerevisiae FKBP12. We thank M. Varon for helpful comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a Israel Cancer Research Found fellowship (to R. W.) and by a Ela Kodesz Institute for Research on Cancer Development and Prevention grant (to M. C.).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.: 972-3-0407532;
Fax: 972-3-6409407; E-mail: ronitt@post.tau.ac.il.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M010446200
2 R. Weisman and M. Choder, unpublished results.
3 R. Weisman, S. Finkelshtein, and M. Choder, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TOR, target of rapamycin; FRB, FKBP12-rapamycin binding; kbp, kilobase pair(s); bp, base pairs; PCR, polymerase chain reaction; HA, hemagglutinin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hall, M. N. (1996) Biochem. Soc. Trans. 24, 234-239[Medline] [Order article via Infotrieve] |
2. |
Cardenas, M. E.,
Cruz, M. C.,
Del Poeta, M.,
Chung, N.,
Perfect, J. R.,
and Heitman, J.
(1999)
Clin. Microbiol. Rev.
12,
583-611 |
3. | Cafferkey, R., Young, P. R., McLaughlin, M. M., Bergsma, D. J., Koltin, Y., Sathe, G. M., Faucette, L., Eng, W. K., Johnson, R. K., and Livi, G. P. (1993) Mol. Cell. Biol. 13, 6012-6023[Abstract] |
4. | Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M., Movva, N. R., and Hall, M. N. (1993) Cell 73, 585-596[Medline] [Order article via Infotrieve] |
5. | Zheng, X. F., Florentino, D., Chen, J., Crabtree, G. R., and Schreiber, S. L. (1995) Cell 82, 121-130[Medline] [Order article via Infotrieve] |
6. | Barbet, N. C., Schneider, U., Helliwell, S. B., Stansfield, I., Tuite, M. F., and Hall, M. N. (1996) Mol. Biol. Cell 7, 25-42[Abstract] |
7. | Di Como, C. J., and Arndt, K. T. (1996) Genes Dev. 10, 1904-1916[Abstract] |
8. |
Zaragoza, D.,
Ghavidel, A.,
Heitman, J.,
and Schultz, M. C.
(1998)
Mol. Cell. Biol.
18,
4463-4470 |
9. | Beck, T., and Hall, M. N. (1999) Nature 402, 689-692[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Schmidt, A.,
Beck, T.,
Koller, A.,
Kunz, J.,
and Hall, M. N.
(1998)
EMBO J.
17,
6924-6931 |
11. |
Noda, T.,
and Ohsumi, Y.
(1998)
J. Biol. Chem.
273,
3963-3966 |
12. |
Schmidt, A.,
Kunz, J.,
and Hall, M. N.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
13780-13785 |
13. | Schmidt, A., Bickle, M., Beck, T., and Hall, M. N. (1997) Cell 88, 531-542[Medline] [Order article via Infotrieve] |
14. |
Chiu, M. I.,
Katz, H.,
and Berlin, V.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12574-12578 |
15. | Chen, J., Zheng, X. F., Brown, E. J., and Schreiber, S. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4947-4951[Abstract] |
16. | Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S., and Schreiber, S. L. (1994) Nature 369, 756-758[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Stan, R.,
McLaughlin, M. M.,
Cafferkey, R.,
Johnson, R. K.,
Rosenberg, M.,
and Livi, G. P.
(1994)
J. Biol. Chem.
269,
32027-32030 |
18. | Helliwell, S. B., Wagner, P., Kunz, J., Deuter-Reinhard, M., Henriquez, R., and Hall, M. N. (1994) Mol. Biol. Cell 5, 105-118[Abstract] |
19. |
Lorenz, M. C.,
and Heitman, J.
(1995)
J. Biol. Chem.
270,
27531-27537 |
20. |
Cruz, M. C.,
Cavallo, L. M.,
Gorlach, J. M.,
Cox, G.,
Perfect, J. R.,
Cardenas, M. E.,
and Heitman, J.
(1999)
Mol. Cell. Biol.
19,
4101-4112 |
21. | Choi, J., Chen, J., Schreiber, S. L., and Clardy, J. (1996) Science 273, 239-242[Abstract] |
22. | Brown, E. J., Beal, P. A., Keith, C. T., Chen, J., Shin, T. B., and Schreiber, S. L. (1995) Nature 377, 441-446[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Brunn, G. J.,
Hudson, C. C.,
Sekulic, A.,
Williams, J. M.,
Hosoi, H.,
Houghton, P. J.,
Lawrence, J. C., Jr.,
and Abraham, R. T.
(1997)
Science
277,
99-101 |
24. |
Hara, K.,
Yonezawa, K.,
Kozlowski, M. T.,
Sugimoto, T.,
Andrabi, K.,
Weng, Q. P.,
Kasuga, M.,
Nishimoto, I.,
and Avruch, J.
(1997)
J. Biol. Chem.
272,
26457-26463 |
25. | Forsburg, S. L. (1999) Trends Genet 15, 340-344[CrossRef][Medline] [Order article via Infotrieve] |
26. | Davey, J. (1998) Yeast 14, 1529-1566[CrossRef][Medline] [Order article via Infotrieve] |
27. | Weisman, R., Choder, M., and Koltin, Y. (1997) J Bacteriol 179, 6325-6334[Abstract] |
28. | Moreno, S., Klar, A., and Nurse, P. (1991) Methods Enzymol. 194, 795-823[Medline] [Order article via Infotrieve] |
29. | Prentice, H. L. (1992) Nucleic Acids Res. 20, 621[Medline] [Order article via Infotrieve] |
30. |
Snaith, H. A.,
and Forsburg, S. L.
(1999)
Genetics
152,
839-851 |
31. | Booher, R., and Beach, D. (1986) Mol. Cell. Biol. 6, 3523-3530[Medline] [Order article via Infotrieve] |
32. | Gari, E., Piedrafita, L., Aldea, M., and Herrero, E. (1997) Yeast 13, 837-848[CrossRef][Medline] [Order article via Infotrieve] |
33. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
34. | Forsburg, S. L., and Sherman, D. A. (1997) Gene (Amst.) 191, 191-195[CrossRef][Medline] [Order article via Infotrieve] |
35. | Costello, G. F., Rodegers, L., and Beach, D. (1986) Curr. Genet. 11, 119-125 |
36. | Moreno, S., and Nurse, P. (1994) Nature 367, 236-242[CrossRef][Medline] [Order article via Infotrieve] |
37. | Takeda, T., Toda, T., Kominami, K., Kohnosu, A., Yanagida, M., and Jones, N. (1995) EMBO J. 14, 6193-6208[Abstract] |
38. | Shiozaki, K., and Russell, P. (1996) Genes Dev. 10, 2276-2288[Abstract] |
39. | Wilkinson, M. G., Samuels, M., Takeda, T., Toone, W. M., Shieh, J. C., Toda, T., Millar, J. B., and Jones, N. (1996) Genes Dev. 10, 2289-2301[Abstract] |
40. | Cutler, N. S., Heitman, J., and Cardenas, M. E. (1999) Mol. Cell. Endocrinol. 155, 135-142[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Zheng, X. F.,
and Schreiber, S. L.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3070-3075 |
42. |
Hardwick, J. S.,
Kuruvilla, F. G.,
Tong, J. K.,
Shamji, A. F.,
and Schreiber, S. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14866-14870 |