MOLECULAR AND CELLULAR BIOLOGY, May 2009, p. 2876–2888
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 10
Normal Function of the Yeast TOR Pathway Requires the Type 2C
Protein Phosphatase Ptc1?†
Asier Gonza ´lez,1,2Amparo Ruiz,1‡ Antonio Casamayor,1,2and Joaquín Arin ˜o1,2*
Departament de Bioquímica i Biologia Molecular1and Institut de Biotecnologia i Biomedicina,2
Universitat Auto `noma de Barcelona, Bellaterra 08193, Barcelona, Spain
Received 13 November 2008/Returned for modification 15 December 2008/Accepted 24 February 2009
Yeast ptc1 mutants are rapamycin and caffeine sensitive, suggesting a functional connection between Ptc1
and the TOR pathway that is not shared by most members of the type 2C phosphatase family. Genome-wide
profiling revealed that the ptc1 mutation largely attenuates the transcriptional response to rapamycin. The lack
of Ptc1 significantly prevents the nuclear translocation of Gln3 and Msn2 transcription factors to the nucleus,
as well as the dephosphorylation of the Npr1 kinase, in response to rapamycin. This could explain the observed
decrease in both the basal and rapamycin-induced expression of several genes subjected to nitrogen catabolite
repression (GAT1, MEP1, and GLN1) and stress response element (STRE)-driven promoters. Interestingly,
this decrease is abolished in the absence of the Sit4 phosphatase. Epitasis analysis indicates that the mutation
of SIT4 or TIP41, encoding a Tap42-interacting protein, abolishes the sensitivity of the ptc1 strain to rapamycin
and caffeine. All of these results suggest that Ptc1 is required for normal TOR signaling, possibly by regulating
a step upstream of Sit4 function. According to this hypothesis, we observe that the mutation of PTC1 drastically
diminishes the rapamycin-induced interaction between Tap42 and Tip41, and this can be explained by
lower-than-normal levels of Tip41 in ptc1 cells. Ptc1 is not necessary for the normal expression of the TIP41
gene; instead, its absence dramatically affects the stability of Tip41. The lack of Ptc1 partially abolishes the
rapamycin-induced dephosphorylation of Tip41, which may further decrease Tap42 binding. Reduced Tip41
levels contribute to the ptc1 phenotypes, although additional Ptc1 targets must exist. All of these results provide
the first evidence showing that a type 2C protein phosphatase is required for the normal functioning of the
Type 2C Ser/Thr protein phosphatases (PP2C) are a group
of monomeric enzymes widely found in animals, plants, and
fungi (35, 62, 66, 69). The function(s) and regulatory mecha-
nisms for each specific PP2C isoform are largely unknown, and
its study constitutes a major challenge, particularly because of
the very important cellular roles played by this type of enzyme.
PP2C activities regulate a plethora of signaling networks that
control cell differentiation, proliferation, growth, survival, and
metabolism. As a general rule, they act as inhibitors of cellular
stress signaling (35). Because of the lack of regulatory or tar-
geting subunits (with a few exceptions, such as the Ptc1 scaffold
protein Nbp2 ), functional diversity most likely is achieved
by the expression of multiple, probably specialized isoforms.
For instance, the 16 human PP2C genes can generate at least
22 isoforms (35). In plants, PP2C genes comprise one of the
largest gene families, with more than 70 members in Arabidop-
sis thaliana (62). In the yeast Saccharomyces cerevisiae, there
are seven different PP2C isoforms (Ptc1 to Ptc7). Several of
these enzymes (Ptc1 to Ptc4) have been characterized in con-
nection with the HOG osmotic responsive pathway as Hog1
phosphatases (38, 43, 57, 71, 75), but it is now widely accepted
that Ptc phosphatases play many diverse roles in yeast cells,
such as tRNA splicing (52), mitochondrial and endoplasmic
reticulum inheritance (20, 53), and the dephosphorylation of
cyclin-dependent kinases (11). Yeast Ptc1 is the closest ho-
molog of human Wip1, a phosphatase that may have oncogenic
properties by reducing the cellular activities of p38, p53, and
ATM (35). Among Ptc1 cellular tasks, a role in tolerance to
lithium ions has been described. This function can be ex-
plained, at least in part, by the positive effect that Ptc1 exerts
on the expression of ENA1, which encodes a major determi-
nant for sodium/lithium efflux in yeast (56). Recent work (23)
has demonstrated that, under standard growth conditions, the
transcriptional profile of yeast cells lacking PTC1 is very dif-
ferent from those devoid of PTC2, PTC3, PTC4, or PTC5,
suggesting that, from a functional point of view, Ptc1 markedly
differs from other members of the family. However, in spite of
the growing body of knowledge, our understanding of the func-
tional role, isoform specificity, and regulatory properties of
these enzymes still is rather limited.
The target of rapamycin (TOR) pathway plays a key role in
the regulation of cell growth in eukaryotes in response to
nutrient availability. In yeast cells, the TOR pathway is active
when there are plenty of nutrients in the medium, whereas it is
inactivated by nutrient scarcity (i.e., less favorable nitrogen
sources, such as proline) or by the exposure of the cells to the
antifungal macrocyclic lactone rapamycin (see reference 27 for
a review). TOR controls the expression of a large number of
genes that are transcribed by all three RNA polymerases. The
* Corresponding author. Mailing address: Departament de Bio-
química i Biologia Molecular, Ed. V, Universitat Auto `noma de Bar-
celona, Bellaterra 08193, Barcelona, Spain. Phone: 34-93-5812182.
Fax: 34-93-5812006. E-mail: Joaquin.Arino@UAB.ES.
† Supplemental material for this article may be found at http://mcb
‡ Present address: Columbia University, Departments of Genetics &
Development and Microbiology, New York, NY 10032.
?Published ahead of print on 9 March 2009.
profound effect triggered by changes in the activity of the TOR
pathway was revealed by several transcriptional studies based
on DNA microarray analysis using cells treated with rapamycin
(8, 21, 25, 63). Rapamycin inhibits the expression of all ribo-
somal genes, including 35S, 5S, and tRNAs, as well as ribo-
somal proteins. An important set of genes whose expression is
potently activated by rapamycin treatment comprises those
related to nitrogen catabolite repression (NCR). These genes
encode proteins that are required for the adaptation to scarce
or less preferred nitrogen sources (for a review, see reference
12). The inhibition of TOR also induces genes required for the
degradation of biomolecules (at the ribosome or mitochon-
dria) in a process called autophagy (32). Similarly, treatment
with rapamycin alters the expression of genes regulated by the
mitochondrial signaling pathway, known as the retrograde re-
sponse (RTG) (6), and affects a large number of stress-related
genes by promoting entry into the nucleus of the stress-acti-
vated factors Msn2/Msn4 (3).
A well-characterized element mediating the effect of the
TOR pathway on downstream NCR genes is the GATA-type
transcription factor Gln3 (39). It is commonly accepted that an
excess of nitrogen activates both Tor1 and Tor2 kinases, which
in turn phosphorylate Tap42, thus promoting a functional as-
sociation with type 2A protein phosphatases (Pph21 and
Pph22) as well as with type 2A-like enzymes (Sit4, Pph3,
and Ppg1) (17, 30, 70). Rapamycin treatment inactivates Tor1
and Tor2, and this triggers the disassembling of the Tap42-
phosphatase complex from Tor kinases, thus allowing the de-
phosphorylation of Gln3. The dephosphorylated form of Gln3
dissociates from Ure2, which otherwise retains the transcrip-
tion factor in the cytoplasm, and is imported into the nucleus
(14, 18, 27, 74). Nuclear Gln3 mediates NCR-sensitive tran-
scription by binding to GATA-containing promoters, such as
those of GAP1 and MEP1 genes (encoding a general amino
acid permease and ammonium permease, respectively), GLN1
(encoding glutamine synthetase, which incorporates ammonia
into glutamate to form glutamine), or GDH1 (encoding a glu-
tamate dehydrogenase activity that synthesizes glutamate from
ammonia and ?-ketoglutarate).
The ability of Tap42 to interact with Sit4 is greatly affected
by the presence of the dephosphorylated form of Tip41 (29). It
has been reported that in yeast cells lacking TIP41, Gln3 re-
mains in the cytoplasm after rapamycin treatment (29). Simi-
larly, the deletion of TIP41 prevents the nuclear migration of
Msn2 in a strain expressing the temperature-sensitive tap42-
106 allele (59). It has been shown that the dephosphorylation
of Tip41, which would promote its binding to Tap42, also is
Sit4 dependent. This would provide a feedback loop that would
enhance the association of Tip41 with Tap42 (29). It has been
proposed recently, on the basis of a combined experimental
and mathematical approach, that the Tip41-Tap42 complex
could act as a specificity factor that would trigger the rapa-
mycin-induced and PP2A/Sit4-mediated dephosphorylation of
downstream targets, such as phosphorylated Gln3 (33). Gln3
localization has been postulated to be negatively regulated by
the Npr1 kinase (15), although this may be an indirect effect. It
must be noted that, although useful, the models described
above do not explain all experimental observations. For in-
stance, there are discrepancies regarding the role of Sit4 in
regulating the phosphorylation state of Gln3 (67) or even on
the relationship between the Gln3 phosphorylation state and
its nuclear localization (68).
Existing evidence indicated that the lack of Ptc1 phos-
phatase activity results in sensitivity to rapamycin (47, 55, 73),
and we observed that ptc1 cells also were sensitive to caffeine,
a compound that has been proposed to inhibit the TOR path-
way (34). Interestingly, the functional mapping of the ENA1
promoter in response to salt stress in a ptc1 mutant (to be
reported elsewhere) allowed linking the absence of the phos-
phatase with a responsive region (around nucleotide ?1400 to
?800) that is rich in putative GATA sequences. Since it was
shown several years ago that the TOR pathway could play a
role in tolerance to sodium and lithium by regulating the ex-
pression of ENA1 (13), we considered that our results may
reflect a potential functional link between Ptc1 and the TOR
pathway. Here, we demonstrate that Ptc1 is required for nor-
mal TOR signaling, and this could be explained, at least in
part, by the requirement of functional Ptc1 to ensure normal
levels of the Tap42-interacting protein Tip41. To our knowl-
edge, this is the first report supporting a role of type 2C
phosphatases in TOR-mediated signaling.
MATERIALS AND METHODS
Escherichia coli and yeast growth conditions. Yeast cells (strains are listed in
Table 1) were grown at 28°C in YPD medium (10 g/liter yeast extract, 20 g/liter
peptone, and 20 g/liter dextrose) or, when carrying plasmids, in synthetic com-
plete medium (1) containing 2% glucose and lacking the appropriate selection
requirements. For the analysis of the transcriptional response to low ammonium
concentrations, cells were resuspended in synthetic medium containing 2% glu-
cose and supplemented only with 10 mM ammonium sulfate, 40 mg/liter methi-
onine, 20 mg/liter histidine, and 100 mg/liter leucine (designated low ammonium
medium). The sensitivity of yeast cells to rapamycin (Calbiochem) or caffeine
(Merck) was evaluated by growth on agar plates (drop tests) or in liquid cultures
as previously described in (7, 48).
E. coli DH5? cells were used as the plasmid DNA host and were grown at 37°C
in Luria-Bertani broth supplemented with 50 ?g/ml ampicillin, when required.
Bacterial and yeast cells were transformed using standard methods (16). Stan-
dard recombinant DNA techniques were performed as described elsewhere (58).
Gene disruptions and plasmid construction. Single kanMX deletion mutants
in the BY4741 background (MATa his3?1 leu2? met15? ura3?) were generated
in the context of the Saccharomyces Genome Deletion Project (72) and were
kindly provided by Jose ´ L. Revuelta (Universidad de Salamanca, Spain). The
ptc1::nat1 disruption cassette (56) was used to transform the appropriate strains.
Positive clones were selected in the presence of 100 ?g/ml nourseothricin
TABLE 1. Yeast strains used in this worka
Name Relevant genotype
MATa his3?1 leu2? met15? ura3?
BY4741 sit4::kanMX4 ptc1::nat1
BY4741 tor1::kanMX4 ptc1::nat1
BY4741 tip41::kanMX4 ptc1::nat1
BY4741 ure2::kanMX4 ptc1::nat1
BY4741 gln3::kanMX4 ptc1::nat1
BY4741 gat1::kanMX4 ptc1::nat1
MATa ura3-52 leu2-3112 his3-?1 trp1-?239
DBY746 PTC1 URA3-STRE(7x)-lacZ
DBY746 ptc1 URA3-STRE(7x)-lacZ
aSingle kanMX deletion mutants in the BY4741 background were generated
in the context of the Saccharomyces Genome Deletion Project (72) and are not
VOL. 29, 2009 Ptc1 REGULATES THE TOR PATHWAY2877
(Werner BioAgents). Strain AGS66, which contains an integrated STRE(7x)-
lacZ reporter system (41) at the URA3 locus, was made as follows. Wild-type
strain DBY746 was transformed with plasmid pGM18/17 (a kind gift of F.
Estruch, U. Valencia), which was previously linearized at the URA3 gene marker
by digestion with NotI. Positive clones were selected in synthetic medium in the
absence of uracil. An identical strategy was used to generate strain AGS67,
except that in this case the transformed strain was the ptc1::nat1 derivative
The construction of plasmid YEp195-PTC1 was described earlier (45). To
obtain YCp33-PTC1, an EcoRI/SalI 1.56-kbp fragment that was released from
YEp195-PTC1, containing the PTC1 gene, was subcloned into the same restric-
tion sites of plasmid YCplac33 (a URA3 marker). To generate the D58N allele
of PTC1, the YEp195-PTC1 construct was used as a template for PCR to change
the TTG codon, encoding an Asp residue at position 58, to a TTA codon,
encoding an Asn residue. The 1.56-kbp amplification fragment was digested with
EcoRI/SalI and cloned into these same sites of plasmid YEplac195 to yield
YEp195-PTC1[D58N]. The entire amplification fragment was sequenced to ensure
the absence of unexpected mutations. For the low-level expression of the mu-
tated version of Ptc1, the EcoRI/SalI fragment was released from YEp195-
PTC1[D58N]and subcloned into the same restriction sites of plasmid YCplac33 to
To analyze the expression of GAP1, GLN1, GDH1, and MEP1 promoters, lacZ
translational fusions were constructed as follows. The regions comprising nucle-
otides ?968 to ?21 (pGAP1-LacZ) and ?853 to ?21 (pGLN1-LacZ) were
amplified by PCR with added KpnI and XbaI sites and cloned into the same sites
of plasmid YEp357 (46). Regions comprising ?904 to ?21 (pGDH1-LacZ) and
?700 to ?21 (pMEP1-LacZ) were amplified by PCR with artificial EcoRI and
XbaI sites and cloned into these sites of plasmid YEp357. The oligonucleotides
employed are listed in Table 2. Plasmids pEJ27 (YEp195-GST-TAP42) and
pEJ120 (YEp181-TIP41-HA) were a generous gift from M. Hall (University of
Basel, Switzerland). Plasmid pEJ23 (YEplac181-HA-NPR1) was a generous gift
from E. Jacinto (University of Medicine and Dentistry, Piscataway, NJ). All three
plasmids have been described previously (29). Plasmid YCp111-TIP41-HA was
generated by cloning the entire 2.1-kbp EcoRI-HindIII insert present in pEJ120
(containing the TIP41 promoter region and open reading frame, the carboxyl-
terminal 3? HA tag, and the CYC1 transcription terminator) into the same sites
of YCplac111 (a LEU2 marker).
?-Galactosidase assays. To evaluate the promoter activity of diverse NCR-
sensitive genes in response to rapamycin, yeast cells were grown to saturation in
the appropriate dropout medium and then inoculated into 5 ml of YPD and
incubated for 4 h to reach an A660of 0.8 to 1. Aliquots of 1 ml were centrifuged
and resuspended in the same volume of YPD (noninduced cells) or YPD con-
taining 200 ng/ml rapamycin. Growth was resumed for 60 (in the case of GAP1,
GLN1, and GDH1 promoter activity assays) or 90 min (when MEP1 promoter
activity was assessed). Cells were collected and processed for ?-galactosidase
assay as described previously (51). The same protocol was used to evaluate the
stress response element (STRE) response in strains AGS66 and AGS67 after 60
min of incubation with rapamycin. The activity of the NCR-sensitive promoters
in cells growing under ammonium limitation was assessed by inoculating cells
(A660of 0.005 to 0.01) in 5 ml of low ammonium medium. Cultures were grown
overnight until an A660of 0.8 to 1, cells were collected, and ?-galactosidase
activity was measured as described above.
RNA purification, cDNA synthesis, and DNA microarray experiments. For
RNA purification, 30 ml of yeast culture was grown at 28°C in YPD medium until
an A660of 0.6 to 0.8 and, when required, was treated with 200 ng/ml rapamycin
or drug vehicle alone (90% ethanol and 10% Tween P20) for 1 h. Cells were
harvested by centrifugation and washed with cold water. Dried pellets were kept
at ?80°C until RNA purification. Total RNA was extracted using the RiboPure-
Yeast kit (Ambion) by following the manufacturer’s instructions. RNA quality
was assessed by electrophoresis in a denaturing 0.8% agarose gel and quantified
by measuring the A260in a BioPhotometer (Eppendorf).
Transcriptional analyses using DNA microarrays developed in our laboratory
(2) were performed exactly as described previously (23). For each experimental
condition (the presence versus the absence of rapamycin in both wild-type and
ptc1 mutant cells), a dye swapping was performed. The scanner ScanArray 4000
(Packard Instrument Co.) was used to obtain the Cy3 and Cy5 images with a
resolution of 10 ?m. The fluorescent intensity of the spots was measured and
processed using GenePix Pro 6.0 software (Molecular Devices). Spots with either
a diameter smaller than 120 ?m or fluorescence intensity for Cy3 and Cy5 of less
than 150 U were not considered for further analysis. A given gene was considered
to be induced when the plus/minus rapamycin signal ratio was equal or higher
than 2.0-fold, whereas it was considered to be repressed when this average was
equal or less than 0.50-fold. The GEPAS server (version v3.1) was used to
preprocess the data (26).
TABLE 2. Oligonucleotides used in this study
5? RT-PCR for GAP1 (?1352 from ATG)
3? RT-PCR for GAP1 (?1561 from ATG)
5? RT-PCR for MEP1 (?1116 from ATG)
3? RT-PCR for MEP1 (?1317 from ATG)
5? RT-PCR for GLN1 (?782 from ATG)
3? RT-PCR for GLN1 (?1018 from ATG)
5? RT-PCR for CPS1 (?1268 from ATG)
3? RT-PCR for CPS1 (?1516 from ATG)
5? RT-PCR for TIP41 (?638 from ATG)
3? RT-PCR for TIP41 (?830 from ATG)
5? RT-PCR for ACT1 (?400 from ATG)
3? RT-PCR for ACT1 (?495 from ATG)
5? oligonucleotide (?968) for cloning GAP1 promoter in YEp357
(URA3-lacZ), contains artificial KpnI sequence
3? oligonucleotide (?21) for cloning GAP1 in YEp357
(URA3-lacZ), contains artificial XbaI sequence
5? oligonucleotide (?853) for cloning GLN1 promoter in YEp357
(URA3-lacZ), contains artificial KpnI sequence
3? oligonucleotide (?21) for cloning GLN1 promoter in YEp357
(URA3-lacZ), contains artificial XbaI sequence
5? oligonucleotide (?700) for cloning MEP1 promoter in YEp357
(URA3-lacZ), contains artificial EcoRI sequence
3? oligonucleotide (?21) for cloning MEP1 promoter in YEp357
(URA3-lacZ), contains artificial XbaI sequence
5? oligonucleotide (?904) for cloning GDH1 promoter in
YEp357 (URA3-lacZ), contains artificial EcoRI sequence
3? oligonucleotide (?21) for cloning GDH1 promoter in YEp357
(URA3-lacZ), contains artificial XbaI sequence
2878GONZA ´LEZ ET AL.MOL. CELL. BIOL.
RT-PCR assays. For reverse transcription-PCR (RT-PCR), yeast cells were
grown in YPD to an A660of 0.5 to 0.8. Cultures were collected at 4°C, and total
RNA was prepared as described above. RT-PCRs were performed using the
Ready-To-Go RT-PCR bead kit (GE Healthcare) and 100 ng of total RNA
(except for TIP41 and MEP1 amplifications, for which 200 ng was used). Specific
pairs of oligonucleotides were used (Table 2) to determine the levels of TIP41
(26 amplification cycles); MEP1 (30 amplification cycles); and GAP1, GLN1,
CPS1, and ACT1 (25 amplification cycles).
Microscopy techniques. The indirect immunofluorescence detection of tagged
Gln3 was accomplished as follows. TB123 and AGS39 cells were grown in YPD
medium until an A660of 0.8 to 1 was reached. Cultures then were treated for
different times (10, 20, 30, and 45 min) with 200 ng/ml rapamycin or drug vehicle
and fixed with 3.7% formaldehyde for 60 min. Cells were prepared for immu-
nofluorescence as described previously (49), incubated overnight at 4°C with
anti-c-myc antibody (9E10 monoclonal antibody; BabCO; a generous gift of H.
Martín, University Complutense of Madrid, Spain) at a final dilution of 1:500
and subsequently with 1:100 diluted Alexa Fluor 488 goat anti-mouse immuno-
globulin G antibody (Invitrogen). For Msn2 subcellular localization experiments,
wild-type BY4741 or MAR143 (ptc1) was transformed with plasmid pMSN2-
GFP (a generous gift of F. Estruch, University of Valencia, Spain), a YCplac111-
based vector that contains a C-terminal Msn2-green fluorescent protein fusion
(24). Cells were grown in YPD containing 4% glucose as the carbon source until
an A660of 0.8 to 1.0 was reached. After this, 500 ?l of the culture was treated with
200 ng/ml of rapamycin or drug vehicle for 15 min and fixed for 5 min by adding
50 ?l of 37% formaldehyde. Cells were harvested, washed three times with
phosphate-buffered saline (PBS), and concentrated 10-fold before visualization.
In all cases the cells were visualized with a fluorescein filter using a Nikon Eclipse
E800 fluorescence microscope (magnification, ?1,000). Digital images were cap-
tured with an ORCA-ER 4742-80 camera (Hamamatsu) using the Wasabi
Preparation of cell extracts and immunoblotting. Yeast strains were grown on
YPD to an A660of 0.8 to 1 at 28°C. For Tip41 detection, whole-cell lysates (10
ml of culture) were prepared by resuspending the cells in 200 ?l of extraction
buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 1 mM
dithiothreitol, 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, and complete
inhibitor mixture; Roche Applied Science). For Npr1 detection, lysates were
made by resuspending cells in PBS buffer (pH 7.4) containing 10 mM NaF, 10
mM sodium pyrophosphate, 10 mM ?-glycerophosphate, 1% Nonidet NP-40,
and the protease inhibitor mixture mentioned above (28). One volume of acid-
washed glass beads (Sigma) was added, and cells were broken at 4°C by vigorous
shaking (five times for 25 s each, with intervals of 1 min on ice) in a Fast Prep cell
breaker (setting 5.5; Bio 101 Inc., Vista, CA). After sedimentation at 500 ? g for
10 min at 4°C, the cleared lysate was recovered and the protein concentration
quantified by the Bradford assay. Total proteins (30 to 90 ?g) were fractionated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in
10% polyacrylamide gels and transferred to nitrocellulose membranes (Hybond
C-Extra; Amersham Biosciences).
Membranes were incubated for 2 h with anti-HA antibody (Roche Applied
Science) at a 1:500 dilution, anti-glutathione S-transferase (anti-GST) (Z-5;
Santa Cruz Biotechnology Inc.) at a 1:2,000 dilution, or anti-actin (I-19; Santa
Cruz Biotechnology Inc.) at a 1:2,000 dilution, followed by the secondary horse-
radish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G
antibody (Amersham Biosciences) at a 1:20,000 dilution. The immunocomplexes
were visualized using an ECL Western blotting detection kit. Chemilumines-
cence was detected using LAS-3000 equipment (Fuji) and quantified using Multi
Gauge software, version 3.0.
GST pull-down assays. Five milligrams of the crude protein extract, prepared
as described above, was mixed with 50 ?l of a slurry of glutathione-Sepharose
(Amersham Biosciences) affinity matrix for 2 h at 4°C with gentle shaking.
Samples were centrifuged and the supernatant was removed. Beads were resus-
pended in 200 ?l of extraction buffer, transferred to MultiScreen filter plates
(Millipore), and extensively washed with the same buffer (without protease
inhibitors). Proteins retained by the affinity system were fractionated by SDS-
PAGE, followed by immunoblotting using anti-HA antibody. Membranes were
stripped and then incubated with anti-GST antibody.
Cycloheximide chase assays. Yeast strains were grown on synthetic medium
lacking leucine until saturation and then were inoculated (A660of 0.2 to 0.3) into
YPD and further grown for 4 h. Cultures then received 200 ng/ml of rapamycin
or vehicle, and incubation was resumed for 10 min. Cycloheximide or vehicle
(ethanol) (25 ?g/ml) was added, and aliquots were taken at the indicated times.
Cells were processed for Tip41 and actin immunodetection as described above.
Two-dimensional gel electrophoresis. The phosphorylation state of Tip41 was
monitored by two-dimensional electrophoresis according to previously published
methods (28, 29), with some modifications. Cell cultures (100 ml) were grown in
YPD until an A660of 0.6 to 0.8 and treated for 30 min with 100 ng/ml of
rapamycin or drug vehicle. Cells were harvested by centrifugation and resus-
pended in 1 ml of TNE lysis buffer (0.1 M Tris-HCl [pH 8.0], 10 mM NaF, 10 mM
sodium pyrophosphate, 10 mM ?-glycerophosphate, 1% Nonidet NP-40, 1 mM
EDTA, 2 mM phenylmethylsulfonyl fluoride, complete protease inhibitor mix-
ture [Roche Applied Science], and 0.1 ?g/ml RNase A) and mechanically dis-
rupted as described above. Homogenates were centrifuged for 10 min at 500 ?
g, and supernatants were recovered. HA-Tip41 was immunoprecipitated from 8
(wild-type cells) or 12 mg (ptc1 cells) of crude protein extracts after incubation
with 0.5 ?g of anti-HA antibody and 40 ?l of protein G Sepharose beads (GE
Healthcare) for 3 h at 4°C. Immunoprecipitates were washed three times with
800 ?l TSNE (TNE plus 0.15 M NaCl) and three times with 1 ml TNE. Samples
were resuspended in 150 ?l of elution buffer (8 M urea, 4 M thiourea, 0.1 M
Tris-HCl [pH 8.0], 1% Nonidet NP-40, 1 mM EDTA, and 4% ?-mercaptoeth-
anol) and then incubated at 37°C for 30 min with mild agitation. Samples then
were centrifuged at 400 ? g for 1 min, the supernatant was recovered, and 75 ?l
of a solution containing 6% 3-[(3-cholamidopropyl)-dimethylammonio]-1-pro-
panesulfonate (CHAPS), 3% dithiothreitol, 3% immobilized pH gradient (IPG)
buffer (pH 4 to 7) (GE Healthcare), and 0.06% bromophenol blue was added.
Immobiline DryStrip IPG gel strips (GE Healthcare) (24 cm; pH 4 to 7) were
rehydrated in a solution containing 8 M urea, 2% CHAPS, 0.6% DeStreak
rehydration solution (GE Healthcare), and 0.5% IPG buffer and placed in the
manifold ceramic tray, and 150 ?l of the sample was loaded in the sample cups.
First-dimension isoelectric focusing was performed on an Ettan IPGphor II unit
(GE Healthcare) using the following protocol: 500 V for 1 h, 1,000 V for 1 h, and
8,000 V for 48 h (50 mA per strip). Usually, a total of 225,000 to 250,000 V/h was
reached. Strips were equilibrated for 15 min in 9 ml of 50 mM Tris-HCl (pH 6.8),
6 M urea, 30 glycerol, 2% SDS, 1% dithiothreitol, and 0.002% bromophenol
blue) and then for an additional 15 min with the same solution lacking dithio-
threitol and supplemented with 2.5% iodoacetamide. The second dimension was
performed on an SDS–10% polyacrylamide resolving gel (25 mM Tris-HCl [pH
6.8], 192 mM glycine, and 0.1% SDS as a running buffer). Proteins were detected
by immunoblotting using Immobilon-P membranes (Millipore) and anti-HA
antibody as described above. The position and intensity of the spots was analyzed
using Melanie 7.0 software (GeneBio SA). A comparison of the relative intensity
of the different spots among several experiments was done using the %vol
parameter, which is a normalized value that remains independent of variations
due to protein loading and/or staining, particularly for gels with a similar spot
Microarray data accession number. Microarray data described in the manu-
script have been deposited in the Gene Expression Omnibus database under
accession number GSE11530.
Evidence for involvement of Ptc1 in the TOR pathway. The
phenotype of the sensitivity of ptc mutants to rapamycin is
rather specific for the Ptc1 isoform, as deduced from growth
analysis on agar plates, since it is not observed in cells lacking
PTC2 to PTC5 or in the ptc7 strain (Fig. 1A). We confirm the
recent observation that the lack of PTC6 confers some degree
of sensitivity to rapamycin (55), although it is far less pro-
nounced than that found in ptc1 cells. Interestingly, the same
sensitivity pattern is observed (Fig. 1A) when cells are treated
with different concentrations of caffeine, a likely inhibitor of
the TOR pathway (34). The overexpression of PTC1, but not
that of PTC2 or PTC3, conferred tolerance to toxic concentra-
tions (20 ng/ml) of rapamycin (not shown). In addition, the
growth of ptc1 mutants is somewhat impaired on media con-
taining low concentrations of ammonium or less-preferred N
sources, such as proline (data not shown). These results rein-
forced the notion that Ptc1 function is related to the TOR
pathway. The phenotypes of ptc1 mutants in the presence of
rapamycin or caffeine were not altered by the lack of the Hog1
kinase gene or the adaptor protein Nbp2 (data not shown),
indicating that these effects were not mediated by the HOG
VOL. 29, 2009Ptc1 REGULATES THE TOR PATHWAY 2879
The regulation of the TOR pathway involves diverse protein
phosphatase activities, although so far all of them belong to the
type 2A family. Type 2C proteins are rather different from the
PP2A or Sit4 phosphatases in primary sequence and regulatory
properties. Therefore, we wanted to test whether or not the
loss of Ptc1 catalytic activity could justify the phenotypes ob-
served in the ptc1 mutant. To this end, we prepared a version
of Ptc1 in which Asp58, a residue that previously has been
shown to be catalytically relevant (71), was changed to Asn. As
observed in Fig. 1B, the expression of the mutated Ptc1 version
was unable, even at a high copy number, to rescue the rapa-
mycin- and caffeine-sensitive phenotype of the ptc1 strain.
Therefore, Ptc1 catalytic activity appears to be necessary for
normal TOR function.
It has been shown that the inhibition of the TOR pathway
(i.e., by the short-term treatment of yeast cells with rapamycin)
has profound effects on the global transcriptional profile (8, 21,
25, 63). We hypothesized that if Ptc1 was required for normal
TOR function, Ptc1-deficient strains may exhibit an altered
transcriptional profile when challenged with rapamycin. There-
fore, RNA was prepared from wild-type and ptc1 cells that had
been incubated for 1 h with 200 ng/ml of rapamycin or vehicle,
and DNA microarray analysis was performed. Under our
working conditions, rapamycin increased by at least twofold
the expression of 667 genes in wild-type cells (13.8% of genes
with a measurable expression level), whereas the treatment
decreased the mRNA level of 721 genes (14.9%). Gene ontol-
ogy analysis shows that, as previously documented, many in-
duced genes fall into the NCR, Msn2/Msn4-regulated stress
response, or retrograde response category, whereas genes en-
coding cytoplasmic (but not mitochondrial) ribosomal proteins
and the so-called Ribi regulon, which includes nonribosomal
proteins that are involved in ribosome synthesis and matura-
tion (31), were largely repressed. A comparison of the re-
sponse to rapamycin in wild-type and ptc1 cells is shown in Fig.
2A. It can be observed that the deletion of PTC1 decreases the
number of genes induced or repressed by rapamycin treat-
ment. Remarkably, a lack of Ptc1 seems to lead to a general
attenuation of changes triggered by rapamycin (see Tables S1
and S2 in the supplemental material). For instance, 91 genes
were induced at least fourfold in both wild-type and ptc1 cells,
but the increase in their induction averages only 6.5-fold for
the ptc1 mutant and 9.2-fold for the wild-type strain. Similarly,
the decrease in expression for the 251 genes repressed at least
fourfold in both strains is much more pronounced in wild-type
cells (9.9-fold) than in the mutant strain (7.6-fold). The atten-
uation of the transcriptional response to rapamycin caused by
the lack of Ptc1 can clearly be observed by plotting the 150
genes showing the highest degree of induction (Fig. 2B, upper)
or repression (Fig. 2B, lower) in wild-type cells and comparing
their expression level to that found in ptc1 cells. It is evident
that many of the highly induced genes, which include compo-
nents of the NCR and the mitochondrial retrograde pathways
as well as Msn2/Msn4-responsive downstream targets, de-
crease their expression in the absence of the phosphatase.
Likewise, genes whose expression is dramatically decreased by
rapamycin, including numerous ribosomal proteins as well as
the Ribi regulon, clearly are less repressed in ptc1 cells. These
results indicate that the lack of Ptc1 has a remarkable and
general effect on the transcriptional program elicited by the
inhibition of the TOR pathway and suggest that this specific
type 2C phosphatase is necessary for normal signaling through
Ptc1 is required for normal Gln3- and Msn2-mediated tran-
scriptional responses and nuclear localization. To gain further
insight into the role of Ptc1 in the transcriptional response
under TOR regulation, we selected for further characteriza-
tion two well-known transcription factors: Gln3, which is
largely responsible for the expression of NCR genes that are
required for adaptation to nonpreferred nitrogen sources, and
Msn2, which mediates TOR-regulated stress responses. As
shown in Fig. 3A, lacZ translational fusions of the promoters of
four NCR genes (GAP1, MEP1, GLN1, and GDH1) were in-
troduced into wild-type and ptc1 strains. When cells were chal-
lenged with rapamycin, it became evident that the response to
the drug was attenuated in Ptc1-deficient cells. The expression
of three of these genes (plus CPS1, another NCR-regulated
gene) also was investigated by RT-PCR (Fig. 3B), with similar
results. Interestingly, when a Myc13-tagged version of Gln3
was expressed in wild-type and ptc1 cells and cultures were
incubated with rapamycin, it was observed that the ability of
Gln3 to enter into the nucleus after drug treatment was sub-
stantially impaired (Fig. 4A). Thus, after 10 min of incubation
FIG. 1. Sensitivity to rapamycin and caffeine of the diverse yeast
type 2C protein phosphatase mutants. (A) Wild-type strain BY4741
and the different ptc mutants were spotted onto YPD plates containing
the indicated concentrations of rapamycin or caffeine. Growth was
monitored after 60 h of incubation at 28°C. (B) The BY4741 strain and
its ptc1 derivative were transformed with an empty centromeric
YCplac33 vector (YCp-Ø) or with the wild type or a catalytically
inactive form of Ptc1 (PTC1[D58N]) cloned in either centromeric (YCp)
or high-copy-number (YEp) vectors. Cells were spotted as described
above and incubated for 48 h.
2880GONZA ´LEZ ET AL.MOL. CELL. BIOL.
with the drug, most Gln3 was nuclear in the wild-type strain,
whereas its location was largely cytosolic in the ptc1 strain. The
time course monitoring of Gln3 distribution in ptc1 cells
showed that Gln3 entered the nucleus only 20 to 30 min after
the addition of rapamycin, although cytosolic Gln3 still was
perceived all during the experiment (data not shown). This was
consistent with the observed decrease in NCR-sensitive gene
expression and suggests that the lack of Ptc1 impairs TOR-
mediated signaling on Gln3. The effect of the ptc1 deletion on
Msn2-mediated signaling was monitored by integrating an
STRE-containing reporter into wild-type strain DBY746 and
its isogenic ptc1 derivative. As shown in Fig. 4B, the STRE-
driven response to rapamycin also is markedly decreased in
ptc1 cells. As observed for Gln3, the rapamycin-triggered entry
of Msn2 into the nucleus is markedly blocked in cells lacking
the phosphatase. All of these observations confirm that signal-
ing through the TOR pathway is impaired in Ptc1-deficient
Lack of Ptc1 results in hyperphosphorylated Npr1. The
Npr1 kinase is required, under specific conditions, to maintain
Gln3 in a cytosolic location, and the function of this kinase can
be regulated by phosphorylation (15, 29, 61). Therefore, we
considered it necessary to investigate the phosphorylation state
of Npr1 in ptc1 cells. As shown in Fig. 5, in cells exposed to
rapamycin for 30 min, Npr1 shows higher mobility, which cor-
responds to dephosphorylated forms of the protein. As re-
ported previously (29), this shift is not observed in sit4 cells.
Figure 5 also shows that the dephosphorylation of Npr1 is
blocked in ptc1 cells. Remarkably, the lack of Ptc1 resulted in
lower-than-normal amounts of Npr1 (note that, in ptc1 lanes,
threefold more protein was loaded). In contrast, we did not
observe the increase in Npr1 levels previously reported for sit4
cells (29). Therefore, our results indicate that Ptc1 is necessary
for the rapamycin-induced dephosphorylation of Npr1. This
observation fits with the impaired entry in the nucleus of Gln3
in Ptc1-deficient cells.
Genetic interactions between the ptc1 mutation and relevant
mutations in the TOR pathway. A genetic approach was taken
to get insight into the role of Ptc1 in the TOR pathway. To this
end, the PTC1 gene was deleted in strains carrying mutations
in different components of the pathway (Fig. 6A), and the
tolerance to rapamycin and caffeine was tested. As observed in
Fig. 6B, the deletion of PTC1 in a tor1, ure2, gln3, gat1, or npr1
(not shown) mutant background still resulted in increased sen-
sitivity to both drugs when tested on YPD agar plates. How-
ever, the hypertolerant phenotype of the tip41 mutant was not
affected at all by a lack of Ptc1. Similarly, the sit4 ptc1 double
mutant exhibited a sit4 phenotype, which was slightly more
FIG. 2. Global transcriptional response analysis to rapamycin in wild-type (WT) and ptc1 cells. (A) Venn diagram showing the number of genes
whose expression was considered to be induced (top) or repressed (bottom) by rapamycin in wild-type ptc1 cells for a set of 4,677 genes, with valid
data for both strains. (B) Plots of the log2values for the changes in the level of expression induced by rapamycin in both wild-type (open circles)
and ptc1 strains for the 150 most upregulated (top) and 150 most downregulated (bottom) genes in the wild-type strain. The expression values for
the ptc1 strain are shown according to the TOR-dependent regulon to which each gene belongs. For the induced genes, the following categories
were used: the NCR family (closed squares), as defined previously (21); the RTG group (open squares) comprises the genes described as
documented targets for the Rtg1 or Rtg3 transcription factors in YEASTRACT (44), as well as those identified elsewhere (19); and the
documented targets of Msn2 or Msn4 described in YEASTRACT, plus those identified elsewhere (9), are represented as open diamonds. Genes
not included in these categories are designated others (closed triangles). The localization of representative genes for each family in the plot is
shown. The genes downregulated by rapamycin in the wild-type strain are classified into one of three possible families: Ribi regulon (closed
squares), which include the genes described previously (31), ribosomal proteins (open squares), and others (closed triangles).
VOL. 29, 2009Ptc1 REGULATES THE TOR PATHWAY2881
sensitive than the wild-type strain but clearly more tolerant
than the single ptc1 mutant. These phenotypes were fully con-
firmed in liquid medium (not shown). Conversely, the trans-
formation of the above-mentioned single mutants with plasmid
YEp195-PTC1 resulted in increased rapamycin tolerance in all
cases, with the only exceptions being tip41 and sit4 (data not
shown). This genetic interaction also was observed by examin-
ing the response to the nitrogen starvation of the four NCR
promoters described above in the ptc1, sit4, and ptc1 sit4
strains. As shown in Fig. 7, the deletion of PTC1 substantially
impairs the response from GAP1, GLN1, GDH1, and MEP1
promoters (similarly to what was observed for rapamycin) (Fig.
3A). The deletion of SIT4 also decreased the promoter re-
sponses, although the effect was less prominent than in the case
of the PTC1 mutation. Remarkably, the activity measured for
all four promoters in the ptc1 sit4 double mutant was virtually
identical to that observed in the sit4 strain. Therefore, our
experiments indicate that the sit4 mutation (and also probably
the tip41 mutation) is epistatic to the ptc1 mutation. This sug-
gests that Ptc1 acts on the TOR pathway by regulating Tip41 or
Sit4 function (or both).
ptc1 cells display altered Tap42-Tip41 interaction, probably
due to lower-than-normal Tip41 protein levels. The interaction
between Tip41 and Tap42 is an important event that regulates
signaling through the TOR pathway (29). Therefore, we con-
sidered it necessary to explore whether the lack of Ptc1 affects
such an interaction. To this end, wild-type and ptc1 cells were
treated with rapamycin, and the drug-induced interaction
between Tip41 and Tap42 was investigated by means of GST-
Tap42 pull-down experiments followed by the detection of
Tip41 by immunoblotting (Fig. 8A, upper). As can be ob-
served, the ptc1 mutation greatly impaired the formation of the
Tip41-Tap42 complex. This observation was in keeping with
the phenotypes observed upon the deletion of the phosphatase
gene. Notably, the examination of Tip41 levels in whole-cell
extracts revealed that they are clearly lower in phosphatase-
deficient cells than in wild-type cells, irrespective of the pres-
ence of the drug in the medium. Therefore, the failure to form
a complex with Tap42, as was observed in ptc1 cells, might be
caused, at least in part, by smaller-than-normal amounts of
Tip41. To identify the molecular basis for this effect of the ptc1
mutation, we examined by RT-PCR the mRNA levels of TIP41
in wild-type and ptc1 cells. However, no significant differences
were observed (data not shown). We considered the possibility
that Ptc1 plays a role in regulating Tip41 stability. To this end,
wild-type and ptc1 cells were treated with cycloheximide to
arrest translation, and the amount of Tip41 was monitored at
different times by immunoblotting. Figure 8B shows that in
wild-type cells, Tip41 levels remained rather stable upon the
blockage of translation. In sharp contrast, the amount of Tip41
decreased quite rapidly in Ptc1-deficient cells, suggesting that
the lack of the phosphatase significantly affects the stability of
the Tip41 protein. A similar experiment was carried out by
treating the cells with 200 ng/ml of rapamycin for 10 min prior
to cycloheximide addition. As shown in Fig. 8B, treatment with
rapamycin does not affect Tip41 stability in wild-type cells or
enhance Tip41 degradation in ptc1 cells, even after relatively
long periods of incubation (180 min).
FIG. 3. Decreased response of diverse NCR-sensitive genes to rapamycin treatment. (A) The indicated constructs were introduced into
wild-type BY4741 (WT) and its ptc1 derivative, and cells were treated with 200 ng/ml rapamycin (Rap) (open bars) for 60 (for GAP1, GLN1, and
GDH1 promoters) or 90 min (for MEP1). Control cells (closed bars) received only the solvent. ?-Galactosidase activity was measured as indicated
in the text. Data are means ? standard errors of the means from six independent clones. (B) RT-PCR experiments were performed using
oligonucleotides specific for the indicated genes (see Materials and Methods). Amplification fragments were run on 2% agarose gels. ACT1 is
included for comparison.
2882GONZA ´LEZ ET AL.MOL. CELL. BIOL.
The results presented above clearly show that Ptc1 is re-
quired for the normal expression of Tip41. We then wondered
whether the altered level of Tip41 explains some of the phe-
notypes caused by the ptc1 mutation. As shown in Fig. 3, a lack
of Ptc1 results in the impaired expression of several NCR
genes. We hypothesized that if the amount of Tip41 could be
increased, the ptc1 phenotype should be alleviated. As ob-
served in Fig. 8C, the high-copy-number expression of Tip41 in
a ptc1 strain increases the rapamycin-induced expression
driven from the GAP1 promoter, thus counteracting the effect
of the ptc1 mutation, although the expression levels do not
reach those of the wild-type strain. It is remarkable that the
overexpression of Tip41 in wild-type cells does not increase but
instead results in a moderate decrease of the GAP1 promoter
activity. Similar results were obtained when expression from
the MEP1 promoter was tested (data not shown). The expres-
sion levels of Tip41-HA in PTC1 and ptc1 cells when the
protein is expressed from low-copy-number (centromeric) or
high-copy-number (episomal) plasmids are shown in the inset
of Fig. 8C. Interestingly, whereas the expression of TIP41 in
high-copy-number plasmids is decreased by the mutation of
PTC1 (Fig. 8A, lower, and C, inset, compare lanes 3 and 5), the
FIG. 4. ptc1 mutation impairs rapamycin-induced Gln3 and Msn2 entry into the nucleus. (A) TB123 (PTC1 GLN3-myc13-kanMX) and AGS39
(ptc1 GLN3-myc13-kanMX) cells were exposed to 200 ng/ml rapamycin (Rap) or to solvent for 10 min and processed for indirect immunofluo-
rescence using anti-Myc antibodies. Samples also were stained with DAPI to reveal the position of the nuclei (magnification, ?1,000). (B) The
upper panel shows wild-type (WT) strain AGS66 and its ptc1 isogenic derivative AGS67, which contain integrated STRE-LacZ reporters. They
were treated for 1 h with 200 ng/ml rapamycin (open bars) or solvent (closed bars), and ?-galactosidase activity was measured. Data are means ?
standard errors of the means from six independent clones. The lower panel shows strains BY4741 (WT) and MAR143 (ptc1), which were
transformed with plasmid pMsn2-GFP (24) and incubated with 200 ng/ml rapamycin for 15 min. The localization of Msn2 was followed by
fluorescence microscopy (magnification, ?1,000).
FIG. 5. LackofPtc1affectsNpr1levelsandphosphorylationstate.Wild-
type BY4741 (WT) and its ptc1 (MAR143) or sit4 derivative were trans-
formed with plasmid pEJ23, which carries an N-terminally HA-tagged ver-
sion of Npr1. Cells were incubated for 30 min with 100 ng/ml rapamycin or
vehicle, and extracts were prepared. Thirty micrograms of protein [90 ?g in
incubated with anti-HA monoclonal antibodies. Faster-migrating bands cor-
respond to the dephosphorylated forms of Npr1.
VOL. 29, 2009 Ptc1 REGULATES THE TOR PATHWAY2883
amount of Tip41 in this case still is clearly higher than that of
the protein expressed from a centromeric plasmid in PTC1
cells (which should closely mimic Tip41 wild-type levels). Since
the expression of TIP41 from centromeric plasmids restores
the wild-type phenotype of a tip41 mutant when Ptc1 is present
(not shown), our result indicates that the restoration of Tip41
levels is not enough to fully eliminate ptc1 defects related to
the signaling of the TOR pathway.
Lack of Ptc1 slightly alters Tip41 phosphorylation pattern.
Tip41 is phosphorylated in vivo at multiple sites, and it has
been reported that hyperphosphorylated Tip41 cannot prop-
erly bind Tap42. Since Ptc1 is a protein phosphatase, it was
reasonable to evaluate the Tip41 phosphorylation state in ptc1
cells, both under basal conditions and in cells treated with
rapamycin. HA-tagged Tip41 was immunoprecipitated, and
samples were subjected to two-dimensional electrophoresis,
essentially as reported previously (29). Under these conditions,
Tip41 was detected in wild-type cells as nine different spots
focused around pH 5.5 (Fig. 9, spots A to I). The higher
number of spots observed here compared to those from a
previous study (29) probably is due to the higher resolution of
our methodology (i.e., larger strips and the use of a narrower
pH range [4 to 7] for the first dimension). In fact, the number
of species detected here approaches the number of in silico
predicted phosphorylatable sites (score, ?0.9) according to the
NetPhos 2.0 server. As shown in Fig. 9 (lower), the major
change observed after the treatment of wild-type cells with
rapamycin for 30 min was an increase in spot A (focusing at
higher pH and, therefore, corresponding to the most dephos-
phorylated form detected) and a decrease in spots G and C.
These changes are compatible with the dephosphorylation of
Tip41 induced by rapamycin treatment. Interestingly, when
ptc1 cells are treated with the drug, the signals for the relatively
highly phosphorylated spot D increased, whereas the relative
intensity for spot A was unchanged. This suggests that the
rapamycin-induced dephosphorylation of Tip41 is impaired in
The results presented in this work clearly demonstrate a
functional link between the type 2C phosphatase Ptc1 and the
TOR pathway. We show here that cells lacking Ptc1 present a
general attenuation of signaling through this pathway. This
seems to be a specific feature of the Ptc1 isoform, since the
mutation of other type 2C phosphatases does not cause caf-
feine or rapamycin sensitivity (perhaps with the exception of
Ptc6), and the overexpression of other isoforms (such as PTC2
or PTC3) does not confer tolerance to rapamycin. We have
observed that a ptc1 ptc6 double mutant is more sensitive to
rapamycin and caffeine than the single mutants (data not
shown). This suggests independent roles of both phosphatases
in the TOR pathway. The role of Ptc6 in the TOR pathway
currently is being investigated in our laboratory. These findings
reinforce the previous notion that, from a functional point of
FIG. 6. Epistatic analysis of ptc1 and mutations affecting the TOR
pathway. (A) A simplified model of signaling through the TOR path-
way (focused on the regulation of NCR genes) based on previous
models (15, 29, 33). (B) Rapamycin and caffeine sensitivity of diverse
mutants in genes relevant in the TOR pathway in the presence (?) or
the absence of PTC1 (?). Cultures were spotted on YPD plates con-
taining the indicated concentrations of the drugs, and growth was
monitored after 2 days. WT, wild type.
FIG. 7. Effect of the ptc1 and sit4 mutations in the transcriptional
response to the ammonium starvation of several NCR-sensitive genes.
Strains BY4741 (PTC1 SIT4) and its ptc1, sit4, and ptc1 sit4 derivatives
were transformed with the indicated NCR-sensitive reporters. Cells
were grown overnight on either standard synthetic (closed bars) or low
ammonium medium (open bars), and ?-galactosidase activity was mea-
sured. Data are means ? standard errors of the means from three
2884 GONZA ´LEZ ET AL.MOL. CELL. BIOL.
view, Ptc1 is far different from the other members of the type
2C phosphatase family (23). In any case, we show that ptc1
defects are caused by the lack of its phosphatase activity.
Therefore, it can be established that the functional alterations
attributable to this mutant are caused by an imbalance in the
phosphorylation state of one or more Ptc1 targets.
The requirement of Ptc1 for correct TOR signaling is evi-
denced by the wide effect of the ptc1 mutation on the tran-
scriptional response to rapamycin. We show that the lack of
Ptc1 causes a quite general attenuation of the transcriptional
response that affects virtually all known TOR targets (8, 21, 25,
63). In some cases, such as the NCR genes, we provide a likely
FIG. 8. Lack of Ptc1 decreases Tip41 stability and Tip41-Tap42
rapamycin-induced interaction. (A) BY4741 and its ptc1 derivative
were transformed with plasmids pEJ27 (GST-Tap42) and pEJ120
(Tip41-HA). Cultures were grown overnight on synthetic medium
lacking leucine and uracil and then inoculated into YPD (A660, 0.2 to
0.3) and grown for 4 h. Cells were treated with rapamycin (100 ng/ml)
or drug vehicle for 30 min, and cell extracts were prepared and GST-
Tap42 affinity purified as described in Materials and Methods. Purified
material (upper) or cell extracts (lower; 40 ?g of proteins) were sub-
jected to SDS-PAGE and immunoblotted with anti-HA antibodies to
reveal the presence of Tip41. Membranes were stripped and incubated
with anti-GST antibody to detect Tap42. (B) A BY4741 strain lacking
the TIP41 gene (PTC1) was deleted for the PTC1 gene (strain
MAR164; ptc1), and both strains were transformed with plasmid
YCp111-TIP41-HA. Cultures received 200 ng/ml of rapamycin or ve-
hicle, and incubation was resumed for 10 min. Cycloheximide then was
added to the cultures (25 ?g/ml) to halt translation, and samples were
taken at the indicated times and processed for immunoblotting using
anti-HA antibodies. Membranes were stripped and incubated with
anti-actin antibodies as a loading and transfer control. The experiment
was repeated three times, with similar results. (C) Wild-type BY4741
(WT) and MAR143 (ptc1) were transformed with plasmid pEJ120
(expressing Tip41-HA in high copy number; filled bars) or the equiv-
alent empty plasmid (YEplac181; open bars). These strains then were
transformed with plasmid pGAP1-lacZ. Cells were grown and treated
with rapamycin as illustrated in the legend to Fig. 3. ?-Galactosidase
activity generated from the GAP1 promoter was measured as de-
scribed in the text. Data are means ? standard errors of the means
from nine independent clones. The inset shows the levels of Tip41-HA
in tip41 cells in the presence (lanes 2 and 3) or the absence (lanes 4 and
5) of the PTC1 gene when the gene is expressed from low-copy-number
(lanes 2 and 4) or high-copy-number plasmids (lanes 3 and 5). Lane 1
corresponds to tip41 cells carrying an empty plasmid. Actin levels are
included as loading controls.
FIG. 9. Phosphorylation pattern of Tip41 in Ptc1-deficient cells.
The upper panel shows wild-type BY4741 (WT) and ptc1 cells trans-
formed with plasmid pEJ120, which were incubated for 30 min with
100 ng/ml rapamycin (Rap) or vehicle. Extracts were prepared for
isoelectrofocusing as described in Materials and Methods. The first
dimension was run using Immobiline DryStrip (pH 4 to 7) strips, and
the second dimension was performed in SDS–10% polyacrylamide
gels. Gels were transferred to membranes, and Tip41 was detected
using anti-HA antibodies. Only the relevant region of the immunoblot
is shown, and the different Tip41 forms are labeled (A to I). pHs are
indicated on the top, and the molecular mass standard is on the right.
The lower panel shows the vol% parameter (i.e., relative volume of a
spot) for each spot, calculated using Melanie 7.0 software. The
means ? standard errors from the means from three independent
experiments are represented. The intensity of spots G, F, and E in the
ptc1 mutant was too low to be integrated.
VOL. 29, 2009Ptc1 REGULATES THE TOR PATHWAY 2885
explanation, since the mutation of PTC1 clearly affects the
nuclear entry of the Gln3 transcription factor (Fig. 4A). The
inability to dephosphorylate Npr1 in response to rapamycin, as
observed in ptc1 cells (Fig. 5), may contribute to this effect. In
other cases, such as the effect on ribosomal proteins and the
Ribi regulon, the cause might be less direct. It has been pro-
posed that the induction by rapamycin of this set of genes
involves the entry of Tor1 into the nucleus (36). However, we
did not observe epistasis between the tor1 and the ptc1 muta-
tions (Fig. 6B). It must be noted that the expression of ribo-
somal protein genes and the Ribi regulon also has been placed
under the control of the protein kinase A (PKA) pathway (10,
42, 60, 76). Although there is no previous link between Ptc1
and PKA activity regulation, the observed effects could be
explained if a lack of Ptc1 resulted in the activation of PKA. In
this regard, we also observed the expression of diverse STRE-
regulated genes being attenuated by the ptc1 mutation. The
expression of these genes is activated upon the entry of the
Msn2/Msn4 transcription factors into the nucleus. Our data
indicate that a lack of Ptc1 substantially impairs the rapamycin-
induced nuclear entry of Msn2 as well as STRE-driven expres-
sion (Fig. 4B). Interestingly, there is some controversy about
whether or not Tap42 and Sit4 are required to direct Msn2 to
the nucleus upon rapamycin stimulation (3, 21, 59, 60). Since
there is long-standing evidence for regulation by PKA of
stress-promoted Msn2 nuclear localization (3, 24, 64), a hypo-
thetical regulation of the PKA pathway by Ptc1 would explain
our results. This notion is reinforced by our observation that
the STRE-driven response to heat shock also is decreased in
ptc1 cells (data not shown).
The very wide range of effects of the ptc1 mutation on the
transcriptional changes induced by rapamycin suggest that Ptc1
plays a role upstream in the TOR pathway. Indeed, the com-
bination of the ptc1 deletion with the mutation of several genes
involved in the pathway (Fig. 6B) indicated that Ptc1 could be
placed at the Tip41/Sit4 level. Tip41 is known to be a regula-
tory component of the pathway by binding to Tap42, and it has
been shown that rapamycin treatment enhances the interaction
between both proteins, which appears necessary for the re-
sponse to the drug (29). We show that in cells lacking Ptc1,
treatment with rapamycin fails to promote the interaction be-
tween Tip41 and Tap42. Surprisingly, we also observed that
whereas the level of Tap42 was normal, the amount of cellular
Tip41 clearly was lower in ptc1 mutants than in wild-type cells.
This effect was independent of the inhibition of the TOR
pathway, as it is not observed in wild-type cells treated with
rapamycin. Therefore, our data support the notion that a lack
of Ptc1 affects the stability of the Tip41 protein, and this could
explain the failure to observe an enhanced Tip41-Tap42 inter-
action in rapamycin-treated ptc1 cells. Interestingly, very re-
cent work has shown that Wip1, the closest homolog of Ptc1 in
humans (22), catalyzes the dephosphorylation of Mdm2, an E3
ubiquitin ligase involved in p53 proteosomal degradation. De-
phosphorylated Mdm2 has increased stability and enhanced
p53 binding, thereby facilitating p53 ubiquitination and degra-
dation (37). The possibility that Tip41 is ubiquitinated and that
Ptc1 affects this condition has been investigated in our labora-
tory, but no evidence supporting such a hypothesis has been
found. In any case, it is apparent that the effect of Ptc1 on
Tip41 stability cannot be attributed to a regulatory effect of
Ptc1 on Sit4, since cells lacking Sit4 do not show significant
changes in the amount of Tip41 (29 and our own data).
A key question is whether or not the low levels of Tip41
observed in Ptc1-deficient cells are sufficient to explain the
interference of the ptc1 mutation with the TOR pathway. Our
results indicate that, in ptc1 cells, an artificial increase of Tip41,
even well above wild-type levels, can increase the expression
from NCR-related gene promoters. However, the expression
levels reached do not match those of wild-type cells (Fig. 8C).
In addition, the sensitivity to rapamycin or caffeine of ptc1 and
ptc1 sit4 mutants essentially is unaltered by the expression of
high levels of Tip41 (data not shown). Therefore, the effects of
a lack of Ptc1 cannot be explained exclusively by a decreased
level of Tip41, indicating that the phosphatase must play ad-
ditional roles in the TOR pathway. In this regard, several
possibilities are worth considering. For instance, in addition to
maintain proper levels of Tip41 protein, Ptc1 might be neces-
sary to catalyze further posttranslational modifications that are
required to render Tip41 a functional protein. This hypothesis
is supported by the observations that Tip41 is phosphorylated
in vivo at multiple sites and that it is dephosphorylated upon
rapamycin treatment. It has been shown that a hyperphosphor-
ylated form of Tip41 cannot properly bind Tap42, and Sit4 has
been proposed as a likely Tip41 phosphatase acting upon the
rapamycin signal. However, the lack of Sit4 does not fully
abolish the dephosphorylation of Tip41 (29), suggesting that
further phosphatase activities target Tip41. The evidence that
a mutation of the Ptc1 catalytic site yields a functionally inac-
tive protein (Fig. 1B), and the observation that a lack of Ptc1
partially abolishes the rapamycin-induced dephosphorylation
of Tip41, support the notion that Ptc1 acts as a Tip41 phos-
phatase and raise the possibility that the Tip41 phosphoryla-
tion state (and, hence, its Tap42 binding ability) is controlled
by a pair of phosphatases, Sit4 and Ptc1 (Fig. 6A). We wish to
stress that although a lack of Ptc1 causes only a slight modifi-
cation in the phosphorylation pattern of Tip41, this may still
result in significant functional changes. For instance, rat liver
glycogen synthase can be phosphorylated at multiple sites, and
a recent report has demonstrated that the inability to phos-
phorylate a single Ser residue (site 2) is sufficient to constitu-
tively activate the enzyme (54).
Our results do not rule out the possibility that Ptc1 has other
targets within the TOR pathway that would be relevant for
signaling, such as Tap42, which also is regulated by phosphor-
ylation (30), or even that the phosphatase acts on other path-
ways functionally interacting with TOR (i.e., the PKA path-
way). Interestingly, very recent work has shown that mutants in
class C and D VPS genes display impaired TOR pathway sig-
naling (50, 77), suggesting that the integrity of the Golgi body-
to-endosome transport system is necessary for correct TOR
function. It must be noted that ptc1 mutants display an altered
vacuolar morphology that is reminiscent of class B vps mutants
(4, 23, 65), and that this defect has been proposed to be
responsible, at least in part, for a number of ptc1-related phe-
notypes (23). It is suggestive that about 50% of the class B
mutants (according to the classification in reference 5) were
found to be rapamycin sensitive in two recent genome-wide
screens (47, 73). In addition, a recent survey for high-copy-
number suppressors of diverse ptc1 phenotypes (A. Gonza ´lez
and J. Arin ˜o, unpublished results) has shown that the overex-
2886 GONZA ´LEZ ET AL.MOL. CELL. BIOL.
pression of VPS70 and VPS73, encoding proteins of uncharac-
terized function that are required for correct vacuolar protein
sorting, partially rescues the rapamycin- and caffeine-sensitive
phenotype of ptc1 cells. Therefore, our findings that Ptc1 is
required for correct TOR signaling may provide a new link
between the TOR pathway and intracellular trafficking.
In essence, we show that Ptc1 is a specific type 2C phos-
phatase isoform that is necessary for normal TOR signaling.
Whereas type 2A-related phosphatases have been involved in
the TOR pathway for more than 10 years, as far as we know
this is the first evidence for a role of type 2C enzymes in this
important and conserved pathway. It is worth noting that, in
this case, Ptc1 is necessary for the transmission of stress signals
triggered by the inhibition of TOR, which is in contrast to the
generally accepted concept that type 2C phosphatases act as
inhibitors of cellular stress signaling (35).
We thank laboratory members Raquel Serrano, Carlos Casado,
Anna Marco, and Maribel Marquina for support and advice. Thanks
are given to Mike Hall, Estela Jacinto, Humberto Martin, and Fran-
cisco Estruch for strains and reagents. The excellent technical assis-
tance of Anna Vilalta, María Jesu ´s A ´lvarez, and Montse Robledo is
This work was supported by grants BFU2005-06388-C4-04-BMC
and BFU2008-04188-C03-01 to J.A. and BFU2007-60342 to A.C.
(Ministerio de Educacio ´n y Ciencia, Spain, and Fondo Europeo de
Desarrollo Regional). J.A. is the recipient of an Ajut de Suport a les
Activitats dels Grups de Recerca (2005SGR-00542; Generalitat de
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