Molecular Biology of the Cell
Vol. 7, 25-42, January 1996
TOR Controls Translation Initiation and Early GI
Progression in Yeast
Nik C. Barbet,* Ulrich Schneider,* Stephen B. Helliwell,* Ian Stansfield,t
Michael F. Tuite,t and Michael N. Hall*t
*Department of Biochemistry, Biozentrum, University of Basel, CH-4056 Basel, Switzerland; and
tResearch School of Biosciences, University of Kent at Canterbury, Canterbury, Kent CT2 7NJ,
Submitted August 11, 1995; Accepted October 26, 1995
Monitoring Editor: Leland Hartwell
Saccharomyces cerevisiae cells treated with the immunosuppressant rapamycin or depleted
for the targets of rapamycin TOR1 and TOR2 arrest growth in the early Gi phase of the
cell cycle. Loss of TOR function also causes an early inhibition of translation initiation
and induces several other physiological changes characteristic of starved cells entering
stationary phase (GO). A Gi cyclin mRNA whose translational control is altered by
substitution of the UBI4 5' leader region (UBI4 is normally translated under starvation
conditions) suppresses the rapamycin-induced Gl arrest and confers starvation sensitiv-
ity. These results suggest that the block in translation initiation is a direct consequence of
loss of TOR function and the cause of the Gl arrest. We propose that the TORs, two
related phosphatidylinositol kinase homologues, are part of a novel signaling pathway
that activates eIF-4E-dependent protein synthesis and, thereby, Gl progression in re-
sponse to nutrient availability. Such a pathway may constitute a checkpoint that prevents
early Gl progression and growth in the absence of nutrients.
The immunosuppressant rapamycin and the related
compound FK506 exert their immunosuppressive ef-
fects by inhibiting intermediate steps in signal trans-
duction that lead to T cell activation and proliferation
(Heitman et al., 1991; Schreiber and Crabtree, 1992;
Sigal and Dumont, 1992; Liu, 1993; Fruman et al.,
1994). FK506 in complex with its intracellular receptor
FKBP inhibits the Ca21 /calmodulin-dependent phos-
phatase calcineurin. As a downstream effector of the T
cell receptor (TCR), calcineurin normally triggers nu-
clear import of a subunit of the transcription factor
NF-AT which, in turn, activates 50-100 genes, includ-
ing the gene encoding the lymphokine interleukin-2
(IL-2) (Weiss and Littman, 1994). Rapamycin also
forms a toxic complex with FKBP, but instead of in-
hibiting the TCR signaling pathway, inhibits a subse-
quent signal transduction cascade that is stimulated
by IL-2 (Bierer et al., 1990; Dumont et al., 1990). The
IL-2 signaling pathway mediates Gl progression (pro-
liferation) of a T cell. Rapamycin prevents the phos-
phorylation and activation of p70 S6 kinase, a down-
stream effector of IL-2 and several other growth
factors, including insulin, EGF, PDGF, IL-3, and eryth-
ropoietin (Calvo et al., 1992, 1994; Chung et al., 1992;
Kuo et al., 1992; Price et al., 1992; Terada et al., 1992;
Ferrari et al., 1993; Lane et al., 1993). Although best
known for its inhibition of IL-2-dependent p70 S6
kinase activation, rapamycin also inhibits p70 S6 ki-
nase activation in response to these other mitogens
(Calvo et al., 1992; Chung et al., 1992, 1994; Price et al.,
1992). The p70 S6 kinase phosphorylates the ribosomal
protein S6 which, in turn, leads to the activation of
translation initiation (Kuo et al., 1992; Thomas, 1992;
Jefferies et al., 1994; Terada et al., 1994). The p70 S6
kinase thus links mitogenic stimulation and the initi-
ation of protein synthesis. A homologue of the yeast
TOR proteins (FRAP/RAFT1/RAPT1/mTOR) (see be-
low) has recently been identified in mammalian cells
as a direct target of the rapamycin-FKBP complex
(Brown et al., 1994; Chiu et al., 1994; Sabatini et al.,
1994; Sabers et al., 1995), suggesting that FRAP/
RAFTfl/RAPT1/mTOR is required for p70 S6 kinase
activation and is part of a general mitogenic signaling
© 1996 by The American Society for Cell Biology
N.C. Barbet et al.
pathway (for a figure that summarizes the pathway,
see Downward, 1994).
In the yeast Saccharomyces cerevisiae, rapamycin-
FKBP inhibits the TOR1 and TOR2 gene products and
arrests cells with an unbudded morphology, indica-
tive of a Gi cell cycle arrest similar to that observed in
T cells (Heitman et al., 1991; Cafferkey et al., 1993;
Kunz and Hall, 1993; Kunz et al., 1993; Helliwell et al.,
1994; Stan et al., 1994; Zheng et al., 1995). A dominant
point mutation in either TOR gene renders a cell re-
sistant to rapamycin, whereas disruption of both
genes results in an unbudded morphology, similar to
that seen when wild-type cells are treated with rapa-
mycin, and a ln DNA content (Heitman et al., 1991;
Cafferkey et al., 1993; Kunz et al., 1993; Helliwell et al.,
1994). TOR1 and TOR2 are large (-280 kDa), function-
ally homologous proteins that are structurally related
to phosphatidylinositol kinases (PI kinases) (Kunz et
al., 1993; Garcia-Bustos et al., 1994; Helliwell et al.,
1994; Yoshida et al., 1994). PI kinases are required for
production of phosphatidylinositol-derived second
messengers (Carpenter and Cantley, 1990). Thus,
TOR1 and TOR2, like FRAP/RAFT1/RAPT1/mTOR,
may be components of a rapamycin-sensitive signal-
ing pathway required for cell cycle progression in Gi.
In S. cerevisiae, the decision to commit to a cell cycle
is made at a point in late Gi termed START. Traversal
of START and entry into S phase is regulated by the
activity of the cyclin-dependent kinase encoded by
CDC28 (Reed, 1992; Nasmyth, 1993). Three Gl-specific
cyclin genes were originally identified and named
CLN1, CLN2, and CLN3 (Cross, 1988; Nash et al., 1988;
Richardson et al., 1989; Wittenberg et al., 1990); addi-
tional candidate Gl cyclin genes have subsequently
been identified and named HCS26, ORFD, CLB5, and
CLB6 (Frohlich et al., 1991; Ogas et al., 1991; Epstein
and Cross, 1992; Kuehne and Linder, 1993; Schwob
and Nasmyth, 1993). All except CLN3 are transcribed
only in late Gl with CLN1, CLN2, and HCS26, and
possibly ORFD, under control of the transcription fac-
tor SBF. CLN3, whose transcript is present throughout
the cell cycle, is regulated post-transcriptionally and
acts as an upstream activator of other Gl cyclins
(Nasmyth and Dirick, 1991; Ogas et al., 1991; Tyers et
al., 1992, 1993; Cvrckova and Nasmyth, 1993).
When nutrients are limiting, haploid yeast cells do
not proceed to START in late Gi, but instead exit the
mitotic cell cycle in early Gl and enter a stationary or
GO phase (for review see Werner-Washburne et al.,
1993). Stationary phase enables a cell to maintain via-
bility for long periods when nutrients are not avail-
able, and is characterized by several physiological
properties including ln DNA content, failure to reach
START, reduced protein synthesis, accumulation of
glycogen, acquisition of thermotolerance, and changes
in the pattern of transcription (Werner-Washburne et
al., 1993). Nutrient sensing and the regulation of entry
into stationary phase are poorly understood, but are
generally thought to involve the RAS/cAMP pathway
(Broach, 1991; Thevelein, 1994). However, this is not
the sole nutrient-sensing pathway, as mutants in the
RAS/cAMP pathway have been isolated that exhibit a
normal response to starvation independently of intra-
cellular cAMP levels (Cameron et al., 1988).
Here we report that cells lacking TOR function (cells
treated with rapamycin or depleted of TOR) arrest
growth and rapidly exhibit, by all criteria examined,
properties diagnostic of GO or stationary phase, in-
cluding a reduction in translation initiation. The cell
cycle arrest upon loss of TOR function is suppressed
by altering the translational control of the Gl cyclin
CLN3. Our results and analogy with mammalian cells
suggest that TOR is part of a novel signal transduction
pathway required for translation initiation and Gi
progression, perhaps in response to nutrients.
MATERIALS AND METHODS
Strains, Plasmids, and Media
The parental strain in this study was JK9-3da (MATa leu2-3, 112
ura3-52 trpl his4 rmel HMLa). Isogenic derivatives with only the
changes indicated are shown in Table 1. The composition of rich
medium (YPD), synthetic galactose/glycerol medium (SGal/Gly),
and synthetic glucose medium (SD) supplemented with the appro-
priate nutrients was as described (Sherman, 1991). All cultures were
incubated at 30'C unless otherwise indicated. Rapamycin (provided
by Sandoz Pharma, Basel, Switzerland) was added to the medium to
a final concentration of 0.2 ,ug/ml. Rapamycin was diluted into
media from a stock solution of 1 mg/ml in 10% Tween-20/90%
ethanol (Heitman et al., 1993). Plasmid pJK5 contains the entire
TOR2 gene under control of the GALI promoter (Kunz et al., 1993).
YEplac181::tor2-61ts (ampr 2 ,. LEU2) contains the entire TOR2 gene
and was isolated by hydroxylamine mutagenesis as a temperature-
sensitive TOR2 allele (Barbet and Hall, unpublished data). The
RAS2va119 allele on plasmid YEp213 (ampr 2 ,u URA3) (Broek et al.,
1987) was transformed into JK9-3da. YCplaclll is ampr CEN4
LEU2 (Gietz and Sugino, 1988). The BCY1 gene was disrupted
(bcyl::URA3) as described using the one-step gene replacement
technique (Toda et al., 1987; Rothstein, 1991). Integration of ADH-
Table 1. Strains used in this study
MATa leu2-3,112 ura3-42 trpl his4 rmel HMLa
JK9-3da torl:LEU2-4 tor2::ADE2-3/pJK5
JK9-3da his3 HIS4 torl::HIS3
JK9-3da ura3::[URA3 ADH-CLN2J
JK9-3da ura3::[URA3 CLN3-11
JK9-3da ade2 his3 HIS4 torl::HIS3 tor2::ADE2//
Molecular Biology of the Cell
TOR Signaling Pathway
CLN2 (Nasmyth and Dirick, 1991) at the ura3 locus was achieved by
linearizing the plasmid containing the CLN2 construct with EcoRV.
Disruptions and integrations were confirmed by Southern blot anal-
ysis. All transformations were performed using the lithium acetate
procedure (Ito et al., 1983).
Overnight cultures of yeast in SD complete medium were diluted to
OD600 <0.05 and allowed to grow before the addition of rapamycin
at OD600 = 0.2. Three hundred-microliter samples were taken from
these cultures at hourly intervals, sonicated for 2 min, and imme-
diately fixed by addition of 700 ,ul absolute ethanol. Samples were
incubated overnight at 4°C, washed, and resuspended in 50 mM
sodium citrate, pH 7.4, and treated with RNAse (0.25 mg/ml) for 1
h at 37°C. DNA was stained by the addition of 500 ,ul citrate buffer
containing 16 ,ug/ml propidium iodide. For each timepoint taken,
10,000 events were analyzed for DNA content using a Becton Dick-
inson FACScan (Mountain View, CA) and data was processed using
Lysys II software (Lincoln Park, NJ).
A reciprocal shift experiment was performed with the temperature-
sensitive tor2 strain NB35 and a-factor as described (Hereford and
Hartwell, 1974) and also with the modification of a 1.5-h overlap in
which both blocks were imposed. Because the effects of rapamycin
are irreversible (presumably because the drug cannot be washed
out) we were unable to perform a standard reciprocal shift experi-
ment with a-factor and rapamycin. To circumvent this problem, we
performed a double block experiment and an a-factor to rapamycin
shift experiment. For the double block experiment, logarithmically
growing cultures of JK9-3da in SD medium were treated with
nocodazole (10jig/ml;Sigma, St. Louis, MO) for 2.5 h to arrest the
cells in mitosis. Cells were harvested by filtration and nocodazole
was washed out of the cells with 10 volumes of sterile water fol-
lowed by 10 volumes of SD medium. Cells were then incubated in
fresh SD medium either with no addition, with 10jig/mlmating
pheromone (a-factor), with 0.2 jtLg/ml rapamycin, or with both
mating pheromone and rapamycin. Samples were taken at 30-min
intervals, sonicated for 2 min to separate cells, and scored for
emergence of buds and appearance of the shmoo phenotype (Spra-
gue, 1991). For the a-factor to rapamycin shift experiment, logarith-
mically growing cultures of JK9-3da in SD medium were treated
with 10 ,ug/ml a-factor for 2.5 h. Cells were harvested by filtration
and washed with 10 volumes of sterile water followed by 10 vol-
umes of SD medium. The culture was then split; one half of the
culture received 0.2 ,tg/ml rapamycin and the other half received
drug vehicle alone. Samples were removed at 30 min intervals,
sonicated for 2 min, and scored for the emergence of buds.
Extraction of total cellular RNA was performed as previously de-
scribed (Jensen et al., 1983). For Northern analysis, 10 ,ug of total
RNA was separated on 1% agarose gels containing 6% formalde-
hyde, and transferred overnight to Hybond-N+ nylon membrane
(Amersham, Arlington Heights, IL) in 20X SSC. The HCS26, ORFD,
CTT1, SSA3, UBI4, CLB5, and CLN2 DNA probes were amplified
from genomic DNA by the polymerase chain reaction (PCR). The
primers used for PCR were as follows, with the 5' primer listed first
and the fragment size generated given in parentheses: HCS26, 5'-
CAT-3' (963 bp); ORFD, 5'-ATGTCAAACTACGAAGCC-3' and 5'-
(998 bp); CTT1, 5'-ATGAACGTG-
TTCGGTAAA-3' and 5'-TGGCACTTGCAATGGACC-3' (1686 bp);
SSA3, 5'-ATGTCTAGAGCAGTTGGT-3' and 5'-ATCAACCTCTTC-
CACTGT-3' (1947 bp); UBI4, 5'-ATGCAGATTTTCGTCAAG-3' and
(1278 bp); and CLN2, 5'-ATGGCTAGTGCTGAACCA-3' and 5'-
TATTACTTGGGTATTGCC-3' (1634 bp). The SWI4 probe was a
2.2-kbp BamHI fragment from the plasmid YCplac33::SWI4 (gift of
K. Nasmyth). The SWI6 probe was a 2.2-kbp XhoI/CiaI fragment
from 1941 (gift of K. Nasmyth). The CLN3 probe was a 500-bp
HindIII/EcoRI fragment from pBF30 (Nash et al., 1988). The probe
for CLNI was a 2-kbp HindIll fragment from pclnl::URA3 (Had-
wiger et al., 1989). The probe for CDC28 was a 1.2-kbp XhoI/XbaI
fragment from YEpl3::CDC28 (gift of K. Nasmyth). The probe for
TORI was a 4.3-kbp HindIll fragment from pPW20 (Helliwell et al.,
1994). The TOR2 probe was a 5.3-kbp BglII fragment from pJK3-3
(Kunz et al., 1993). The HSP26 probe was a 800-bp BglII/NdeI
fragment from pHSP26 (gift of S. Lindquist). The probe for SSB1
was a 2.2-kbp HindIll fragment from pFKR15. The probe for SSA1
was a 2-kbp Sall fragment from EC551 (gift of E. Craig). The ACTi
probe was a 1-kbp EcoRI/PstI fragment from pUC18::ACT1 (gift of
P. Linder). SSA1 and SSA2 transcripts are indistinguishable, as are
SSB1 and SSB2 transcripts, because the DNA sequences of these
pairs of genes are 97% and 94% identical, respectively (Werner-
Washburne et al., 1989). Probes were labeled with [32P]dATP using
the random-primed DNA labeling kit (United States Biochemical,
Cleveland, OH).Filters were exposed
X-OMAT) AR at -70°C with intensifying screens (Dupont Cronex).
Signals were quantitated by scanning appropriately exposed films
using a Molecular Dynamics densitometer (Sunnyvale, CA). In the
experiment shown in Figure 7B, the total cellular RNAs of strains
NB36 and NB38 were prepared identically, run on the same gel,
transferred to the same filter, and hybridized to the same probe at
the same time.
Incorporation of [35SIMethionine into Total
For analysis of gross protein synthesis, tricholoracetic acid (TCA)-
precipitable counts were quantitated from pulse-labeled cultures at
the indicated times after treatment. For rapamycin treatment, expo-
nentially growing cultures of JK9-3da in SD medium minus methi-
onine were treated with 0.2 ,ug/ml rapamycin, 100 ,Lg/ml cyclohex-
imide, or with drug vehicle alone (10% Tween-20/90% ethanol). For
TOR depletion, exponentially growing cultures of the torts strain
NB35 and the control strain NB17-3d in SD minus methionine were
resuspended in prewarmed medium at 37°C. For each timepoint,
0.01OD600equivalents were removed and labeled at 30°C for 7 min
with 2 ,uCi [35Slmethionine (Amersham). Aliquots of the pulse-
labeled cells were lysed on Whatman filters presoaked in 50% TCA,
and deacylated by boiling for 10 min in 5% TCA. Filters were
washed in acetone, air dried, and TCA-precipitable counts were
quantitated by scintillation counting using a Canberra Packard
1900TR liquid scintillation analyzer.
Polysome Gradient Analysis
Strains JK9-3da and NB35 were grown in YPD to a cell density of
107 cells m-1'. Following harvesting, polysomes were prepared as
described (Stansfield et al., 1992), except that polysomes were re-
solved on a 15-50% w/v sucrose gradient by centrifuging for 2.1 h
at 17,000 x g using a Beckman SW40 Ti rotor. Cycloheximide (200
jig/ml)and rapamycin (0.2 ,ug/ml) were added to cultures at the
indicated times before harvest. Drugs used in this way to inhibit
yeast cultures were also included at the same concentration in the
lysis buffer (Stansfield et al., 1992).
Logarithmically growing cultures in SD medium were treated with
0.2 ,Lg/ml rapamycin and incubated at 30°C in the presence of the
drug. At hourly intervals up to 5 h after rapamycin addition, 5 OD
equivalents of cells were harvested onto Millipore HA filters (Bed-
Vol. 7, January 1996
N.C. Barbet et al.
ford, MA), placed upon a solid agar matrix, and exposed to iodine
vapor for 1 min.
Construction and Analysis of the UBI4-CLN3
The 5' region (containing the untranslated leader and promoter
sequences) of the UBI4 polyubiquitin gene and a sequence contain-
ing the open reading frame of the CLN3 gene were amplified from
S. cerevisiae genomic DNA using the polymerase chain reaction.
Oligonucleotides were designed to produce a 752-bp UBI4 5' region
fragment flanked by a 5' HindIll and a 3' SalI restriction site, and a
1821-bp CLN3 fragment flanked by 5' SalI and 3' SmaI sites. The
oligonucleotides were as follows: UBI4 5' end, 5'-GCAAAGCTTC-
CCACCACCAGCACTAGCTTAGAT-3'; UBI4 3' end, 5'-AATGTC-
TACGTCGACTGTACGATGGCCATATTGAAGGAT-3'; and CLN3 3'
UBI4-CLN3 construct was obtained by first introducing the HindlIlI
Sall-cut UBI4 5' region fragment into a HindIII/SaiI cut YCplaclll
vector (CEN4 LEU2). Following transformation and amplification in
E. coli, this "parent plasmid" was digested with Sall and SmaI and
the Sail/SmaI-cut CLN3 fragment was introduced. The UBI4 5'
region was fused 7 bp upstream of the CLN3 start codon. The
resultant plasmid (YCplaclll::UBI45'-CLN3) and its parent plasmid
(YCplaclll::UBI45`) were transformed into the wild-type haploid
yeast strain JK9-3da to yield strains NB36 and NB37, respectively.
Strain NB38 is JK9-3da containing the plasmid YEpURA::CLN3
(gift of K. Nasmyth), which consists of a 7-kb genomic BglII frag-
ment containing the CLN3 gene inserted into YEp352. For the asyn-
chronous flow cytometry experiments, strains were grown in SD
medium minus leucine to early log phase, and treated with 0.2
,ug/ml rapamycin. Cell number and DNA content were analyzed
hourly for 5 h following rapamycin treatment. For the synchrony
experiments, NB36 and NB37 were grown to early log phase, then
treated with 10 ,Lg/ml a-factor for 2.5 h to arrest cells at start.
a-Factor was removed by filtration and washing with water, fol-
lowed by SD medium minus leucine, and cells were resuspended in
fresh SD medium minus leucine. Samples were removed at 20-min
intervals, washed, sonicated to separate cells, and assessed for
emergence of buds and DNA content (flow cytometry). At maximal
budding (generally 60 min after release from a-factor), the cultures
were split; half received 0.2 ,ug/ml rapamycin, the remaining half
received drug vehicle alone. Flow cytometry was performed as
Assay of Starvation Sensitivity
Strain NB36 containing the UBI4-CLN3 fusion and control strain
NB37 containing the UBI4 5' region without the CLN3 open reading
frame, on a LEU2 plasmid, were grown in SD medium minus
leucine for 6 days. Samples were removed daily and assessed for
cell number/milliliter of culture, cell viability, and percentage of
budded cells. For viability determination, 103 cells were plated on
rich medium (YPD) in duplicate, and the number of cells able to
form colonies was determined as a percentage of total number of
cells plated. Replica plating to SD medium minus leucine showed
that over 80% of the cells retained their respective plasmid, even
after prolonged incubation.
Rapamycin Blocks Gl Progression
We have shown previously that rapamycin treatment
causes yeast cells to arrest with an unbudded mor-
phology (Heitman et al., 1991; Kunz et al., 1993). Such
a phenotype, although suggestive of, is not necessarily
indicative of a Gi arrest, as mutants have been iso-
lated that are perturbed in budding but not in the
onset of DNA synthesis (Adams et al., 1990; Johnson
and Pringle, 1990; Bender and Pringle, 1991; Cvrckova
and Nasmyth, 1993). We therefore examined whether
yeast cells treated with rapamycin arrest with a ln
DNA content, and are thus indeed impaired in Gl
progression. An exponentially growing asynchronous
culture of the haploid strain JK9-3da was treated with
0.2 ,ug/ml rapamycin, and at hourly intervals samples
were removed for flow cytometry. As shown in Figure
1, a shift to a ln DNA content was observed after 1 h
of rapamycin treatment, and after 2-3 h, -85% of the
cells contained a ln DNA complement (Figure 1D).
The shift to ln DNA content paralleled growth arrest;
rapamycin-treated cells never completed more than
one doubling, as determined by direct counting of the
cells in the treated culture at the different time inter-
vals. A control culture treated with the drug vehicle
alone (10% Tween/90% ethanol) continued to grow
normally, doubling in cell number every 125 min for
the duration of the experiment. Thus, rapamycin
causes a Gi arrest within one generation. As shown*
previously, TOR depletion also causes cells to arrest
growth with a ln DNA content (Helliwell et al., 1994).
When the size distribution of cells was analyzed, we
observed two subpopulations
treated cells (Figure 1E). The major subpopulation of
cells increased in size throughout the experiment,
whereas the minor subpopulation of cells appeared to
remain as small cells. Although the two subpopula-
tions became more evident at later time points as the
larger cells continued to increase in volume, two dis-
crete populations could already be discerned after 2 h.
The small cells most likely represent newly formed,
starved daughter cells (see below) (Johnston et al.,
1977). The increased size of the larger cells can be
accounted for by the observation that they contain an
exceptionally large vacuole (Heitman et al., 1991). Be-
cause an enlarged vacuole is also symptomatic of star-
vation (Granot and Snyder, 1991), these cells might
also be starved (in GO) despite the presence of nutri-
ents. The reason for the biphasic size distribution is
in the rapamycin-
The TOR Restriction Point Is in Early Gl
To determine the TOR restriction point within Gl, we
performed an order-of-function (reciprocal shift) anal-
ysis using a temperature-sensitive tor mutant and the
mating pheromone a-factor (Hereford and Hartwell,
1974). This maps the TOR restriction point relative to
START, the a-factor arrest point. The mutant strain
(NB35) used in this experiment contained a tempera-
ture-sensitive tor2 allele on a plasmid and chromo-
somal disruptions of both TOR1 and TOR2. NB35
Molecular Biology of the Cell
TOR Signaling Pathway
0.2 ,ug/ml rapamycin and sampled for flow cytometry at hourly intervals up to 5 h. (A and D) DNA content, (B and E) cell size, and (C and
F) a two-dimensional plot of cell size distribution (x-axis) versus DNA content (y-axis) for rapamycin-untreated (A-C) and -treated cells
(D-F). The two-dimensional plot corresponds to the 5-h time point. ln and 2n refer to DNA content.
Rapamycin causes wild-type yeast cells (JK9-3da) to arrest with a ln DNA content. Exponentially growing cells were treated with
(torts) arrests growth with a ln DNA content after shift
to the nonpermissive
growth upon return to the permissive temperature.
The growth arrest of NB35 (torts) occurs within one
generation; this strain fails to complete more than one
doubling after shift to the nonpermissive temperature,
as determined by cell counting. Following release
from a mating pheromone block and a simultaneous
shift from the permissive temperature (24°C) to the
nously entered S phase as determined by emergence
of new buds (Figure 2A); cells maintained at 24°C
behaved similarly. In contrast, when cells were ar-
rested at the TOR restriction point, then released by
resuspending in fresh medium at 24°C and treated
with mating pheromone, they formed shmoos and did
not initiate a new round ofbudding for the duration of
the experiment (Figure 2B). Budding after shift from
a-factor to the restrictive temperature was not due to
a slow inactivation of temperature-sensitive TOR.
(37°C), cells synchro-
First, NB35 (torts) arrests within one generation. Sec-
ond, shifting cells to the nonpermissive temperature
1.5 h before release from the a-factor block did not
prevent budding (Figure 2C). Third, wild-type cells
released from an a-factor block into medium con-
taining rapamycin also resumed budding (Figure
2D). The results of a double block experiment per-
formed with a-factor and rapamycin (see MATERI-
ALS AND METHODS) were also consistent with a
TOR restriction point in early Gl; rapamycin pre-
vented nocodazole-synchronized cells from forming
shmoos in response to a-factor (our unpublished
results). Thus, the TOR restriction point is in early
Gl before START.
As further evidence that loss of TOR function
causes an early Gl arrest, we observed that rapa-
mycin-treated cells lack START-specific transcripts
encoding the Gl cyclins (Figure 3) (see below), and
that providing CLN2 under control of the rapamy-
Vol. 7, January 1996
N.C. Barbet et al.
Figure 2. TOR depletion arrests cells in early Gl before START. (A-C) Order-of-function determination by an a-factor (af) and torts
reciprocal shift. The percentage of budded cells was monitored at the indicated time points after release from the first block. (A) Strain NB35
(torts) was arrested by pretreatment with a-factor at the permissive temperature, washed, and resuspended in fresh medium without a-factor
at 24°C (open squares) or 37'C (closed circles). (B) Strain NB35 (torts) was arrested by preincubation for 5 h at the nonpermissive temperature
(37°C), washed, and resuspended in fresh medium at the permissive temperature (24°C) containing vehicle alone (open squares) or a-factor
(closed circles). (C) Strain NB35 (torts) was treated as in panel A with the modification that cells were shifted to the nonpermissive temperature
1.5 h before release from the a-factor block. (D) Order-of-function determination by an a-factor to rapamycin shift. Wild-type strain JK9-3da
was arrested at START by a-factor treatment, washed, and resuspended in medium containing vehicle alone (open squares) or 0.2/ig/ml
rapamycin (closed circles). The percentage of budded cells was determined at the indicated times after release from a-factor.
pombe ADH promoter (Nasmyth and Dirick, 1991)
does not abrogate the rapamycin-induced cell cycle
arrest (our unpublished results). Thus, TOR is not
directly (or solely) required for CLN gene transcrip-
tion, and the loss of START-specific transcripts is a
downstream effect rather than the direct cause of the
cell cycle arrest. A constitutively expressed CLN2
transcript does not suppress the rapamycin-induced
cell cycle arrest presumably because it is not trans-
lated (see below).
The effects of rapamycin treatment on START-spe-
cific transcripts were as follows. The mRNAs for CLN1
Molecular Biology of the Cell
TOR Signaling Pathway
and CLN2 (Figure 3 and our unpublished results for
CLN2) were no longer detectable after 2 h of rapamy-
cin treatment. Surprisingly, the normally constitu-
tively expressed CLN3 transcript was also reduced
with similar kinetics as seen for CLN1 and CLN2, but
was not completely eliminated. As determined by
densitometry of appropriately exposed autoradio-
graphs and normalization to ACTI transcript levels,
the CLN3 mRNA level was maximally reduced by
-60%. The mRNAs for the three additional genes,
HCS26, ORFD, and CLB5, which bear limited homol-
ogy to the CLN genes and are also expressed only in
late Gi also disappeared upon rapamycin treatment,
with kinetics identical to those seen for the CLN1 and
CLN2 transcripts (Figure 3 for HCS26 and ORFD).
Expression of the CLN1, CLN2, and HCS26 genes
(and possibly ORFD) is under control of the transcrip-
tion factor SBF, which is composed of the DNA bind-
ing moiety SWI4 and its regulatory subunit SWI6
(Nasmyth and Dirick, 1991; Ogas et al., 1991). We
therefore assessed the levels of SW14 and SWI6 tran-
mRNA for SWI6 is constitutively expressed whereas
the mRNA for SWI4 oscillates, peaking in late Gl and
falling to a low but detectable basal level elsewhere in
the cell cycle (Breeden and Mikesell, 1991). Like the
CLN3 transcript, the mRNA for SW16 was depleted by
-60% (Figure 3). The transcript for SWI4 fell to basal
levels 2 h after rapamycin treatment, thus behaving
like other START-specific mRNAs.
cells. Normally, the
treatment. Northern blot analysis of RNA isolated from cells (JK9-
3da) treated with 0.2 ,tg/ml rapamycin for 0, 1, 2, 3, 4, and 5 h
(indicated in minutes). The mRNAs for CLN1, ORFD, and HCS26
are abolished, and mRNAs for CLN3 and SWI6 are reduced by
-60% relative to ACTI levels. ACTI encodes actin and is a control
for a message that is not START specific. The level of ACTI message
is not affected by rapamycin. See text for additional information.
START-specific transcripts are depleted upon rapamycin
These observations are not due to a global repres-
sion of transcription as the transcripts for actin (ACTI)
and CDC28 and also the previously identified targets
of rapamycin TOR1 and TOR2 were not depleted
throughout the time course of these experiments (Fig-
ure 3 and our unpublished results). Furthermore,
some transcripts are actually induced upon rapamycin
treatment (see below). As mentioned above, the ab-
sence of START-specific transcripts upon rapamycin
treatment is presumably an indirect consequence of a
cell cycle arrest before START (Hubler et al., 1993). The
reduction in the normally constitutive messages could
reflect the inherent instability of untranslated (see be-
TOR Is Required for Translation Initiation
Because rapamycin blocks activation of protein syn-
thesis in mammalian cells (Jefferies et al., 1994; Terada
et al., 1994) and because inhibition of protein synthesis
in yeast causes an early Gl arrest (Hartwell and Un-
ger, 1977; Pringle and Hartwell, 1981; Brenner et al.,
1988), we investigated whether rapamycin blocks pro-
tein synthesis in yeast by assaying incorporation of
[35S]methionine at intervals after addition of rapamy-
cin. We observed an early decrease in incorporation
upon rapamycin treatment (Figure 4A). Protein syn-
thesis fell to a low (-10% of normal levels) but detect-
able level after 120 min, and remained at this low level
throughout the course of the experiment. The low
level of protein synthesis was greater than that ob-
served in cells treated with cycloheximide (100 ,tg/
ml), which reduced protein synthesis to undetectable
levels. Up to 100-fold higher concentrations of rapa-
mycin did not have a more severe effect on incorpo-
ration. Protein synthesis was not affected in a rapamy-
cin-resistant TORI-i (JH11-lc) or TOR2-1 (JH12-17b)
mutant, as assayed by [35S]methionine incorporation
in the presence of rapamycin. Thus, rapamycin is an
effective inhibitor of protein synthesis acting through
To confirm that TOR is required for protein synthe-
sis, as suggested by the above observation, we exam-
ined the effect of TOR depletion on protein synthesis.
The torts strain NB35 was shifted to the nonpermissive
temperature and levels of protein synthesis were de-
termined at time intervals after the temperature shift.
At the nonpermissive temperature, we observed a
progressive decrease in the levels of [35S]methionine
incorporation (Figure 4B). Incorporation levels fell to a
minimum of -10% after 6 h of incubation at the non-
permissive temperature. Levels of incorporation in
NB35 (torts) at the permissive temperature were less
than those in wild type, indicating that there is a
protein synthesis defect in this mutant even at the
permissive temperature. Thus, TOR is required for
protein synthesis. Furthermore, because an inhibi-
Vol. 7, January 1996
N.C. Barbet et al.
120 180 240 300 360
Cells were assessed for incorporation of [35Slmethionine by labeling
for 7 min at intervals (0, 15, 30, 60, 120, 180, and 240 min) after
addition of 0.2 ,ug/ml rapamycin. Cells treated were wild-type
JK9-3da (open squares) and rapamycin-resistant TORI-i mutant
JH11-lc (closed squares). Also plotted is JK9-3da treated with 100
,tg/ml cycloheximide (closed circles). Incorporation (relative incor-
poration) is plotted as a percentage of the control, wild-type strain
JK9-3da treated with drug vehicle alone. (B) Inhibition of protein
synthesis upon TOR depletion. Strain NB35 (torts) was incubated at
the restrictive temperature, and samples were removed at the indi-
cated time intervals for determination of [35S]methionine incorpo-
ration. Values are plotted as a percentage of [35S]methionine incor-
poration in NB17-3d at the restrictive temperature. An early time
point is not included because a reliable value could not be obtained
for either the temperature-sensitive mutant NB35 or NB17-3d im-
mediately after shift to the nonpermissive temperature. Shown (A
and B) are representative curves of three or more independent
Rapamycin treatment inhibits protein synthesis. (A)
tion of protein synthesis causes an early Gl arrest
(Hartwell and Unger, 1977; Pringle and Hartwell,
1981; Brenner et al., 1988), the protein synthesis
defect may be the cause of the cell cycle arrest; the
relatively slow inhibition of incorporation in NB35
(torts) at the nonpermissive temperature, compared
with rapamycin-treated cells at the permissive tem-
perature, is not necessarily inconsistent with the
first cycle arrest of NB35 (torts) because this strain
has a translation defect even at the permissive tem-
perature and because cells have a longer cell cycle at
the higher temperature.
To determine whether the inhibition of protein syn-
thesis was at the level of initiation or elongation, poly-
some profiles of wild-type cells treated with rapamy-
cin for 1 and 2 h were analyzed. This experiment was
performed in the absence of the translation elongation
inhibitor cycloheximide so that a block in elongation,
if imposed, could be observed. Such a block is char-
acterized by an accumulation of polysomes. No poly-
somes were present in either extract, only a single
peak corresponding to 80S monosomes and ribosomes
(our unpublished results). Rapamycin does not, there-
fore, cause a translation elongation block; however, a
mild defect in the rate of elongation that is not suffi-
ciently stringent to prevent ribosome "run-off" during
the time needed to harvest, wash, and lyse cells in
preparation for sucrose gradients would not be de-
tected. To investigate whether rapamycin causes a
block in translation initiation, wild-type cells were
treated with drug vehicle alone or with rapamycin for
1 and 2 h followed by a 10-min treatment with cyclo-
heximide to prevent run-off of any polysomes present
(Figure 5, A and B; our unpublished result for 1-h
timepoint). Rapamycin treatment caused a progres-
sive decay of polysomes with a coincident increase in
the 80S peak, indicating an initiation block. The ap-
parent discrepancy between the observed inhibition of
[35S]methionine incorporation (-90%) and the inhibi-
tion of polysomes (-60%) after 2 h of rapamycin treat-
ment may reflect a difference in the sensitivities of the
two assays or a mild elongation defect in addition to a
block in initiation.
We next examined the polysome profiles of TOR-
depleted cells using the torts strain NB35. Again, a
severe reduction in the number of polysomes and a
coincident increase in the 80S peak were evident after
incubation for 5 h at the nonpermissive temperature
(Figure 5C). A similar but less pronounced effect was
observed after 3 h at the nonpermissive temperature.
Thus, TOR is required for translation initiation.
Loss of TOR Causes a Starvation Response, but
TOR Is Not Part of the RAS/cAMP Pathway
Starved yeast cells exit the cell cycle (stop dividing)
and enter GO. Cells entering GO are characterized by
Molecular Biology of the Cell
TOR Signaling Pathway
several distinct properties (Werner-Washburne et al.,
1993) including ln DNA content, failure to reach
START (Pringle and Hartwell, 1981), a reduction in
protein synthesis to -10% of normal levels, down-
regulation of CLN3 message (Hubler et al., 1993), and
enlargement of the vacuole (Granot and Snyder, 1991).
As described above, rapamycin-treated or TOR-de-
pleted cells display all these characteristics. Addition-
ally, rapamycin-treated or TOR-depleted cells are still
alive (metabolically active) despite the observed re-
duction in protein synthesis; rapamycin-treated cells
exclude the vital dye phloxin B even 24 h after treat-
ment, and all temperature-sensitive tor2 alleles iso-
lated to date are reversible (Barbet and Hall, unpub-
lished data). This led us to consider that rapamycin
might be causing a starvation response despite the
presence of nutrients, and inducing cells to enter GO.
To test this, we examined by Northern analysis the
effect of rapamycin on the transcription of genes
whose mRNA levels are known to change upon entry
into GO. The heat shock genes SSA3 and HSP26 and
the ubiquitin gene UBI4 are transcriptionally induced
upon entry into GO (Werner-Washburne et al., 1993).
The catalase T gene CTT1 is also transcriptionally
induced upon entry into GO, with enzymatic activity
peaking and then declining 3 h after cells enter sta-
tionary phase (Werner-Washburne et al., 1993). In con-
trast, the mRNA level of the heat shock genes SSA1
and SSA2 (SSA1/2) fluctuates in different ways de-
pending on the starvation regimen but can remain
largely unchanged, and transcription of the cold-in-
ducible "heat shock" genes SSB1 and SSB2 (SSB1/2) is
severely repressed upon entry into GO (Werner-Wash-
burne et al., 1993). As shown in Figure 6A, we ob-
served these same changes in transcription upon ra-
pamycin treatment. The mRNAs for SSA3, HSP26, and
UBI4 were induced upon rapamycin treatment; max-
imal induction occurred 2 h after rapamycin addition
for SSA3 and HSP26, and after 30 min for UBI4. The
CTT1 transcript was also induced upon rapamycin
treatment, and transcript levels remained high for 2 h
before falling. In contrast, the SSB1/2 transcripts de-
creased to almost undetectable levels within 1 h of
treatment. The level of SSA1/2 transcripts fluctuated
but remained largely unchanged. Thus, it appears that
rapamycin causes a starvation response and induces
entry into GO.
tion initiation. (A and B) Polysome profiles of wild-type cells (JK9-
3da) treated with (A) vehicle alone, and (B) 0.2 ,ug/ml rapamycin for
2 h. (C) Polysome profile of the torts strain NB35 after 5 h at the
nonpermissive temperature (37°C). In all the above cases, cyclohex-
imide was added 10 min before harvest, to prevent "run off."
Wild-type strain JK9-3da grown at the nonpermissive temperature
is slightly stimulated for polysome accumulation. The positions of
40S ribosomal subunits (s), 60S ribosomal subunits (1), 80S mono-
somes (m), and polysomal ribosomes (p) are indicated.
Rapamycin treatment or TOR depletion blocks transla-
Vol. 7, January 1996
N.C. Barbet et al.
We next examined whether TOR depletion elicits
the same starvation-induced changes in transcript lev-
els. We could not utilize the torts strain for these ex-
periments, because many of the same changes in tran-
script levels occur normally at high temperature (the
nonpermissive temperature of our tortS mutant) inde-
pendently of starvation. Therefore, we used a strain
containing TOR2 under control of the regulatable
GALl promoter and chromosomal disruptions of both
TOR1 and TOR2 (JK350-21a) to deplete the cells of
TOR (Kunz et al., 1993; Helliwell et al., 1994). After
shifting from galactose- (SGal/Gly) to glucose-con-
taining (SD) medium (TOR-depletion conditions), we
observed changes in the pattern of transcription sim-
ilar to those seen when wild-type cells are treated with
rapamycin (Figure 6B). Thus, TOR depletion also in-
duces a starvation response.
Additional indicators of stationary phase are the
accumulation of the storage carbohydrate glycogen
and acquisition of thermotolerance. We examined
whether cells treated with rapamycin accumulate
glycogen. Cultures were treated for 5 h with rapa-
mycin, harvested by filtration at hourly intervals,
and stained for glycogen using iodine vapor, which
stains glycogen-containing cells dark brown (Ches-
ter, 1968). As shown in Figure 6C for the 5-h time
point, cells treated with rapamycin did indeed stain
darkly when exposed to iodine. Accumulation of
glycogen was weakly detectable after 1 h of treatment.
Also confirming that loss of TOR function induces a
starvation response, we observed that cells depleted
for TOR exhibit increased resistance to the killing
effects of high temperatures when compared with
wild-type cells (our unpublished results).
The RAS/cAMP signal transduction pathway acts in
early Gl (before the mating pheromone arrest point)
and may be involved in the controlled entry into
GO (Broach, 1991; Thevelein, 1994). To investigate
vation response. (A) RNA was isolated from cells (JK9-3da) treated
with 0.2 ,ug/ml rapamycin for 0, 15, 30, 60, 120, 180, 240, and 300
min, and probed by Northern analysis with the indicated genes (see
MATERIALS AND METHODS). SSA1/2 refers to SSAI and SSA2.
SSB1/2 refers to SSBI and SSB2. The observed changes in transcript
levels are characteristic of cells entering GO. (B) Histogram showing
level changes for the indicated transcripts upon depletion ofTOR by
galactose to glucose shift. Conditions of the galactose to glucose
shift were as described (Helliwell et al., 1994). Hatched bars corre-
spond to a wild-type (JK9-3da) strain; solid bars correspond to a
TOR-depleted strain (JK350-21a). Transcript levels were normal-
ized to ACTI mRNA levels. Transcript level values in relative units
are given above each bar. A value of 0 indicates an undetectable
mRNA level. (C) Rapamycin treatment causes accumulation of gly-
cogen. (Top left) Rapamycin-resistant strain JH12-17b treated with
drug vehicle alone. (Top right) JH12-17b treated for 5 h with 0.2
,tg/ml rapamycin. (Bottom left) Wild-type strain JK9-3da treated
with drug vehicle alone. (Bottom right) JK9-3da treated for 5 h with
0.2 ,Lg/ml rapamycin. Filters were exposed to iodine vapor for 1
min to stain for glycogen.
Rapamycin treatment or TOR depletion induces a star-
Molecular Biology of the Cell
TOR Signaling Pathway
whether loss of TOR function induces entry into GO
by inhibiting the RAS/cAMP cascade, we constitu-
tively activated this pathway, and then tested for
abrogation of the rapamycin-induced cell cycle ar-
rest. Two methods were used to constitutively acti-
vate the pathway. First, we disrupted the BCY1 gene
(Toda et al., 1987). A BCY1 disruption activates the
RAS/cAMP pathway by eliminating the negative
regulatory subunit of the cAMP-dependent protein
kinase A (Cannon and Tatchell, 1987; Toda et al.,
1987). Second, we introduced the dominant, acti-
vated RAS2 allele RAS2valI9. The RAS2vall9 mutation
hyperactivates the RAS/cAMP pathway by main-
RAS2ValI9 (NB34) cells were as sensitive as wild-type
cells to rapamycin, based upon growth arrest in the
presence of drug. Flow cytometry on these strains
indicated that greater than 85% of the cells arrested
with a in DNA content after 3 h of rapamycin
treatment, as observed with wild-type cells (see Fig-
ure 1 for wild-type cells). Rapamycin-treated bcyl
and RAS2ValI9 cells also accumulated glycogen, as
determined by iodine staining. Therefore, activation
of the RAS/cAMP pathway does not abrogate the
rapamycin-induced cell cycle arrest, indicating that
TOR is not part of the RAS/cAMP pathway.
Our data do not rule out the possibility that TOR
lies in the RAS/cAMP pathway downstream of
BCY1, but we consider this very unlikely. First,
subcellular localization studies (Kunz, Stevenson,
Schneider, and Hall, unpublished data) and their
homology to lipid kinases indicate that the TORs are
membrane-associated proteins, whereas BCY1 is a
membrane-distal component of the RAS/cAMP
pathway. Second, diploid cells lacking TOR func-
tion arrest in Gl (2n DNA content) but do not sporu-
late, whereas diploids compromised in the RAS/
cAMP pathway do sporulate. Third, activation of
p70 S6 kinase, a presumed downstream component
of TOR in mammalian cells, is independent ofp2lras
(Downward, 1994; Ming et al., 1994). Fourth, there is
no example of, or need for, a lipid kinase in a
signaling pathway that utilizes cAMP as a second
messenger; the lipid kinases mediate production of
the fundamentally different, phosphatidylinositol-
derived second messengers. Thus, TOR1 and TOR2
appear to define a novel nutrient-related process
would be in agreement with the observations of
Cameron et al. (1988), who described mutants that
protein kinase A activity but that still respond ap-
propriately to nutrient conditions, even in the ab-
sence of essential upstream components of the
Expression of CLN3 under Altered Translational
Control Confers TOR-independent GI Progression
The finding that loss of TOR function causes an early
reduction in protein synthesis and a Gi arrest within
one generation suggested that TOR might be control-
ling translation of an unstable protein(s) required for
Gi progression. Good candidates for such proteins
were the Gi cyclins, as these proteins are unstable and
limiting for Gi progression (Cross, 1988; Nash et al.,
1988; Hubler et al., 1993; Tyers et al., 1993). To test
whether cells lacking TOR function arrest in early Gl
(GO) because they do not synthesize Gi cyclins, we
devised a situation in which one of these, CLN3,
would be synthesized upon rapamycin treatment, and
asked whether this would be sufficient to drive rapa-
mycin-treated cells through Gi. CLN3 was chosen
because the transcript for this cyclin is normally
present under conditions of rapamycin treatment (Fig-
ure 3). We fused the CLN3 open reading frame to the
5' region (untranslated leader and promoter) of the
UBI4 gene. The UBI4 5' region was chosen because it
is both transcriptionally and translationally active in
GO and would therefore express CLN3 upon rapamy-
cin treatment (Finley et al., 1987; Brenner et al., 1988;
Werner-Washburne et al., 1993) (Figure 6A). We then
examined whether the UBI4-CLN3 fusion suppresses
the rapamycin-induced cell cycle arrest.
An asynchronously growing wild-type yeast strain
containing the UBI4-CLN3 fusion on a centromeric
plasmid (NB36) was treated with rapamycin, and at
hourly intervals the DNA content of the cells was
analyzed by flow cytometry. Like a control strain
(NB37) containing a plasmid-borne UBI4 5' region
without the CLN3 open reading frame, NB36 cells
arrested growth after approximately 2 h of rapamycin
treatment. This was expected because rapamycin
causes a general inhibition of protein synthesis (Figure
4), and TOR has an essential non-cell cycle function in
addition to its essential role in Gi (Kunz et al., 1993).
Analysis of DNA content of the arrested cells, how-
indicated that NB36 (UBI4-CLN3)
throughout the cell cycle, whereas the control strain
arrested in Gi (Figure 7). Thus, cells containing the
UBI4-CLN3 fusion no longer arrest in Gi upon rapa-
Northern analysis of the strain (NB36) containing
the UBI4-CLN3 fusion indicated that it produces ap-
proximately 20-fold more CLN3 mRNA upon rapamy-
cin treatment than an isogenic strain lacking the fu-
sion. To determine whether the suppression of the cell
cycle arrest in strain NB36 was due to altered control
of CLN3 translation or merely to the increased dosage
of the CLN3 transcript, we examined whether cells
containing the wild-type CLN3 gene on a high-copy-
number plasmid (NB38) also arrested outside of GI
upon rapamycin treatment. After 2 h of treatment,
Vol. 7, January 1996
N.C. Barbet et al.
growing cells containing the UBI4-CLN3 fusion (NB36) were treated with 0.2 ,ug/ml rapamycin and sampled for flow cytometry at hourly
intervals up to 5 h. Rapamycin-treated NB36 arrested throughout the cell cycle, as indicated by a roughly even distribution of cells with a
ln and 2n DNA content. Results of flow cytometry on control strain NB37 treated with rapamycin were indistinguishable from the results
in Figure 1D. (B) Northern analysis of strains treated with rapamycin and assessed for levels of CLN3 transcript. Shown are the levels of CLN3
transcript in strain NB38 (hatched bars) containing the wild-type CLN3 gene in a high copy number plasmid and strain NB36 (solid bars)
containing the UBI4-CLN3 fusion, at the indicated times (in minutes) following rapamycin treatment. All values were normalized to the levels
of actin transcript. At 120 min following treatment, cells had arrested growth. (C and D) A UBI4-CLN3 strain treated with rapamycin is able
to traverse GI. (C) Percentage of budded cells of rapamycin-treated (closed squares) or -untreated (open squares) NB36 (UBI4-CLN3)
compared with rapamycin-treated (closed circles) or -untreated (open circles) control strain NB37. Percentage ofbudded cells was determined
at 20-min intervals following release from the a-factor block at START. Rapamycin (rap) was added 60 min following release from a-factor.
(D) Flow cytometry of NB36 (UBI4-CLN3) and NB37 control cells released from an a-factor block at time 0 and treated with rapamycin 60
min following release from a-factor. Shown is the DNA content of the rapamycin-treated cells at the end of the experiment (240 min). The
rapamycin-treated cells arrested growth approximately 150 min after rapamycin addition. The left panel shows the DNA content for strain
NB36, the right panel shows the DNA content for NB37 (Gl arrest). DNA content of untreated cells at 240 min was indistinguishable from
that shown in the left panel. Data shown is representative of three independently performed experiments.
Altered translational control of CLN3 suppresses the cell cycle-specific arrest of rapamycin-treated cells. (A) Exponentially
NB38 cells arrested growth, with -85% of cells con-
taining a ln DNA content. Northern analysis of the
rapamycin-treated NB36 (UBI4-CLN3) and NB38 (high
copy CLN3) cells indicated that the level of CLN3
transcripts in NB38 was greater than that in NB36
(Figure 7B). In addition, high level overexpression of
CLN3 from the inducible GALl promoter was also
unable to overcome a rapamycin-induced Gi arrest,
and plasmid-borne UBI4-CLN3 still caused a random
arrest despite disruption of the chromosomal copy of
Molecular Biology of the Cell
TOR Signaling Pathway
CLN3 (our unpublished results). Furthermore, an in-
tegrated copy of the CLN3-1 allele (strain NB33),
which bears a mutation that stabilizes the CLN3 pro-
tein but does not otherwise affect its cyclin function
(Cross, 1988; Nash et al., 1988), had the same effect as
UBI4-CLN3 in causing a random arrest upon rapamy-
cin treatment. This confirms that the UBI4-CLN3 fu-
sion does not promote Gl progression simply because
of an elevated level of CLN3 transcripts.
To determine more directly whether rapamycin-
treated cells containing the UBI4-CLN3 fusion are able
to traverse the Gl phase of the cell cycle, we examined
the effect of rapamycin on synchronized cells. Strain
NB36 (UBI4-CLN3) and the control strain NB37 were
synchronized at START by addition of a-factor. Fol-
lowing release from the pheromone block, the cultures
were split into two and rapamycin was added to one
half, the remaining halves receiving drug vehicle
alone. As shown in Figure 7C, NB37 control cells
treated with rapamycin entered Gl and arrested as
CLN3), however, entered Gl but then began to pro-
duce new buds before arresting growth, indicating
that cells were traversing Gl and beginning a new
cycle. Analysis of the arrested cells by flow cytometry
confirmed that the NB36 (UBI4-CLN3) cells had tra-
versed Gl whereas the NB37 control cells had not
(Figure 7D). Thus, UBI4 leader-dependent expression
of CLN3 causes rapamycin-treated cells to traverse
Expression of UBI4-CLN3 Confers
The finding that TOR may normally modulate synthe-
sis of CLN3 (among other proteins) as part of a star-
vation response suggested that cells containing the
UBI4-CLN3 fusion might be sensitive to starvation. To
test this suggestion, UBI4-CLN3 strain NB36 and con-
trol strain NB37 were grown to stationary phase
(starved) and samples were removed daily for assess-
ment of cell viability and the percentage of budded
cells. As NB36 (UBI4-CLN3) cells entered stationary
phase (cell number no longer increased) (Figure 8A),
their ability to form colonies on rich medium rapidly
decreased (Figure 8B). In contrast, starved NB37 con-
trol cells retained high viability for the duration of the
experiment. The starvation sensitivity of NB36 (UBI4-
CLN3) was most likely due to this strain's inability to
arrest in Gl (GO), as suggested by the observations
that it stopped dividing at a higher cell density (-1.5-
fold) than the control strain (Figure 8A) and with a
high percentage of budded cells (Figure 8C). Strain
NB38 containing the wild-type CLN3 gene in high
dosage behaved in this experiment like control strain
NB37. These findings support the involvement ofTOR
in nutrient sensing, and also confirm that modulating
the level of translation is part of the regulated entry
into stationary phase (GO).
We have shown that loss of TOR function (rapamycin
treatment or TOR depletion) causes yeast cells to ar-
rest in early Gl and to exhibit, by all criteria examined,
characteristics of starved cells entering stationary
phase, or GO. We have also demonstrated that loss of
TOR function causes a general inhibition of translation
initiation. Providing the transcript for the Gi cyclin
CLN3 under the translational control of the UBI4 5'
region suppresses the rapamycin-induced Gl arrest
and confers starvation sensitivity. These results sug-
gest the following model for the role of TOR in cell
cycle control (Figure 9). In response to nutrient avail-
ability, TOR stimulates general translation initiation,
including translation of Gi-regulatory transcripts such
as those for CLN3 and other Gi cyclins. This then
drives cells through Gi and into S phase. In the con-
verse situation, the absence of nutrients causes inacti-
vation of TOR, which leads to loss of translation and a
subsequent early Gi arrest and entry into GO. It is
important to emphasize that TOR is required for gen-
eral translation and that the role of TOR in cell cycle
control, as proposed here, is just part of a greater role
in general growth control. We would also like to stress
that, although it accounts for all our data, the model is
largely speculative and is intended only as a frame-
work to bring together our and other findings.
Several lines of evidence suggest that TOR is part of
a signaling pathway. First, the TORs are homologous
to PI kinases, enzymes implicated in signaling. Sec-
ond, because loss of TOR rapidly causes a starvation
response, TOR is likely involved in sensing and relay-
ing the availability of nutrients. Indeed, constitutively
activating the proposed pathway by providing CLN3
independently of upstream components (CLN3 under
the translational control of the UBI4 untranslated
leader) causes starvation sensitivity. Third, the mam-
malian counterpart of TOR (FRAP/RAFT1/RAPT1/
mTOR) appears to mediate an intermediate step in
a defined, rapamycin-sensitive signal transduction
pathway required for cell proliferation (Brown et al.,
1994; Chiu et al., 1994; Downward, 1994; Sabatini et al.,
1994; Sabers et al., 1995). The putative TOR pathway is
novel because it acts in early Gl, and TOR is not part
of the RAS/cAMP pathway.
The observed inhibition of translation initiation is
likely a direct consequence of loss of TOR function
and the cause (rather than an effect) of the cell cycle
arrest, for the following reasons. First, the reduction in
translation is the earliest effect observed upon loss of
TOR function. Second, a specific block in translation
initiation, either by mutation of an initiation factor
or by treatment with a low concentration of cyclo-
Vol. 7, January 1996
N.C. Barbet et al.
heximide, causes yeast cells to arrest in early Gi
(Hartwell and Unger, 1977; Johnston et al., 1977;
Pringle and Hartwell, 1981; Hanic-Joyce et al., 1987;
Brenner et al., 1988; Hubler et al., 1993; Barnes et al.,
1995). Third, and most important, allowing transla-
tion initiation of an appropriate, cell cycle-control-
ling transcript is sufficient to suppress the rapamy-
cin-induced Gl arrest. Fourth, TOR in mammalian
cells probably activates translation initiation and Gl
progression in response to mitogens (Downward,
1994; see INTRODUCTION). Thus, the TOR path-
way in yeast appears to control translation initiation
and, thereby, early Gl progression.
The observation that phosphorylation of the yeast
equivalent of S6 (S10) is not important for growth
(Zinker and Warner, 1976; Kruse et al., 1985; Johnson
and Warner, 1987) suggests that TOR is not regulat-
ing translation initiation in yeast through S6 (see
INTRODUCTION). One alternative possibility is
that the TOR pathway controls translation initiation
through the initiation factor eIF-4E (or an associated
subunit). eIF-4E is the cap-binding subunit of the
eIF-4F complex, which also contains eIF-4A, an
RNA helicase, and eIF-4y, a protein of unknown
function (Rhoads, 1988; Lanker et al., 1992; Linder,
1992; Goyer et al., 1993; Redpath and Proud, 1994).
eIF-4F binds to the 5' cap structure of mRNA and
promotes unwinding of 5' secondary structure, fa-
cilitating binding of the 43S ribosomal preinitiation
complex to the mRNA. Several observations suggest
that TOR could control eIF-4E. First, analyses of
CDC33 (encodes eIF-4E) and TOR mutants indicate
that eIF-4E and TOR have remarkably similar roles.
Both have essential functions required for general
translation initiation (Altmann et al., 1989; Kunz et
al., 1993; see RESULTS). Furthermore, both have an
early Gl-specific function and an essential function
that is not Gl specific (Johnston et al., 1977; Pringle
and Hartwell, 1981; Brenner et al., 1988; Kunz et al.,
1993); protein synthesis is required at several points
in the cell cycle but is most limiting in GI (Burke
and Church, 1991). Second, in mammalian cells,
eIF-4E is the rate-limiting protein in translation
(Duncan et al., 1987) and a target for regulation.
Growth factors activate protein synthesis by trigger-
ing the phosphorylation and release of the eIF-4E-
an inability to arrest in GO. (A) Growth curve of NB36 cells (closed
circles) expressing UBI4-CLN3 and NB37 cells (open squares) ex-
pressing the UBI4 5' region alone. (B) Viability curve of NB36
(closed circles) and NB37 (open squares) strains. Cells reached
stationary phase after 3 days of growth. Strains were grown in SD
medium minus leucine for the indicated times. Viability was as-
sessedby plating103 cells on YPD medium andcounting colony-
forming units. (C) Percentage of budded cells in cultures of NB36
(closed circles) and NB37 (open squares).
The UBI4-CLN3 fusion confers starvation sensitivity and
Molecular Biology of the Cell
TOR Signaling Pathway
We thank Jim Broach, Elizabeth Craig, Bruce Futcher, Stephen Gar-
rett, Patrick Linder, Susan Lindquist, Kim Nasmyth, Kelly Tatchell,
and Johan Thevelein for plasmids, and also Jeannette Kunz, Marc
Bickle, members of the department, George Thomas, and Kim
Nasmyth for useful discussions during the course of this work. We
thank Brian Stevenson for critical reading of the manuscript. We
acknowledge Peter Erb for use of the FACScan and technical advice.
N.C.B. was supported by a Long Term EMBO Fellowship. This
work was supported by grants from the Swiss National Science
Foundation and the Canton of Basel to M.N.H.
(GI cyclins, etc.)
mycin (R) forms a complex with FKBP to inhibit TOR (Heitman et
al., 1993; Kunz et al., 1993). TOR is TOR1 and TOR2. PI is phosphati-
dylinositol. See DISCUSSION for further details. Because TOR is
required for general translation (see RESULTS), the role of TOR in
cell cycle control is just part of a greater role in general growth
control; the model proposed here focuses exclusively on that part of
TOR's role in general growth control that affects progression
through the Gl phase of the cell cycle.
Model of the TOR pathway in cell cycle control. Rapa-
inhibiting factor 4E-BP1 /PHAS-I (Haystead et al.,
1994; Hu et al., 1994; Lin et al., 1994; Pause et al.,
1994). Importantly, rapamycin blocks the phosphor-
ylation of 4E-BP1 and inhibits cap-dependent initi-
ation of translation (Beretta et al., 1996). Third, in
proliferating yeast and mammalian cells, eIF-4E and
an associated subunit are phosphorylated and there-
fore potentially subject to regulation by this type of
modification (Duncan et al., 1987; Joshi-Barve et al.,
1990; Morley et al., 1991; Rhoads et al., 1993; Redpath
and Proud, 1994; Zanchin et al., 1994). Fourth, trans-
lation of UBI4 appears to have, at least, reduced
dependence on eIF-4E (Brenner et al., 1988). Thus,
the block in translation initiation caused by loss of
TOR function may be due to a down regulation of
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