Sch9 Is a Major Target of TORC1
in Saccharomyces cerevisiae
Jo ¨rg Urban,1Alexandre Soulard,4Alexandre Huber,1Soyeon Lippman,5Debdyuti Mukhopadhyay,2
Olivier Deloche,3Valeria Wanke,3Dorothea Anrather,6Gustav Ammerer,6Howard Riezman,2James R. Broach,5
Claudio De Virgilio,3Michael N. Hall,4and Robbie Loewith1,*
1Department of Molecular Biology
2Department of Biochemistry
3Department of Microbiology and Molecular Medicine, Centre Me ´dical Universitaire
University of Geneva, Geneva, CH-1211, Switzerland
4Department of Biochemistry, Biozentrum, University of Basel, Basel, CH-4056, Switzerland
5Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
6Max F. Perutz Laboratories, Department of Biochemistry, University of Vienna, Dr. Bohrgasse 9, A1030 Vienna, Austria
The Target of Rapamycin (TOR) protein is a Ser/
Thr kinase that functions in two distinct multi-
protein complexes: TORC1 and TORC2. These
conserved complexes regulate many different
aspects of cell growth in response to intracellu-
lar and extracellular cues. Here we report that
the AGC kinase Sch9 is a substrate of yeast
TORC1. Six amino acids in the C terminus of
Sch9 are directly phosphorylated by TORC1.
Phosphorylation of these residues is lost upon
rapamycin treatment as well as carbon or nitro-
gen starvation and transiently reduced follow-
ing application of osmotic, oxidative, or thermal
stress. TORC1-dependent phosphorylation is
required for Sch9 activity, and replacement of
residues phosphorylated by TORC1 with Asp/
Glu renders Sch9 activity TORC1 independent.
Sch9 is required for TORC1 to properly regulate
ribosome biogenesis, translation initiation, and
entry into G0phase, but not expression of Gln3-
dependent genes. Our results suggest that
Sch9 functions analogously to the mammalian
In eukaryotes two distinct, conserved, multiprotein com-
plexes known as TOR complex 1 (TORC1) and TORC2
function as major regulators of cell growth (Wullschleger
et al., 2006). In both complexes, Target of Rapamycin
(TOR),alargeSer/Thrproteinkinase belonging tothe fam-
ily of phosphatidylinositol kinase-related kinases (Keith
and Schreiber, 1995), functions as the catalytic subunit.
TORC1, but not TORC2, is directly inhibited by the macro-
cyclic lactone rapamycin (Jacinto et al., 2004; Loewith
et al., 2002; Sarbassov et al., 2004).
S. cerevisiae TORC1 contains Lst8, Kog1, Tco89, and
either Tor1 or Tor2 (Loewith et al., 2002; Reinke et al.,
2004; Wedaman et al., 2003). Based primarily on the
observed phenotypic similarities between rapamycin-
treated and nutrient-starved cells it is generally believed
that TORC1 couples nutrient cues to the cell growth ma-
chinery (Rohde et al., 2001). TORC1 activity also appears
to be influenced by a number of other noxious stresses
and, in metazoans, by extracellular mitogens (Crespo
et al., 2001; Kim et al., 2002; Sarbassov and Sabatini,
2005). Under favorable conditions, yeast TORC1 pro-
motes growth by stimulating translation initiation and, via
transcription factors such as Ifh1, Crf1, and Sfp1, expres-
sion of genes required for synthesis and assembly of the
translation machinery. In addition to promoting anabolic
processes, TORC1 also antagonizes entry into G0phase,
the induction of stress response programs, and catabolic
processes including autophagy and expression of gene
products required for the metabolism of nonpreferred nu-
trients. Again, TORC1 regulates many of these processes
by influencing the localization/activity of transcription fac-
tors including Gln3 (nitrogen discrimination pathway),
Rtg1/Rtg3 (retrograde signaling), and Msn2/Msn4 (stress
response) (De Virgilio and Loewith, 2006).
How TORC1 activity is linked to its diverse downstream
targets is not well understood. Several processes are
thought to be regulated through modulation of protein
phosphatase 2A activity; however, the direct link between
TORC1 and the phosphatases has not been clearly de-
fined (Duvel et al., 2003). The lack of well-characterized
TORC1 substrates with defined phosphorylation sites has
also hampered the identification of physiological stimuli
and upstream regulatory components that control TORC1
Activity of many members of the AGC protein kinase
family (homologous to protein kinases A, G, and C)
Molecular Cell 26, 663–674, June 8, 2007 ª2007 Elsevier Inc. 663
requires both phosphorylation of the kinase domain acti-
phobic motif’’ (HM: F-X-X-F/Y-S/T-F/Y) found C terminal
of the catalytic domain to a pocket in the kinase domain.
The latter often depends on HM phosphorylation (Gold
et al., 2006).
Mounting evidence suggests that TOR complexes
phosphorylate the HM of several AGC kinases. In vivo
and in vitro data show that mammalian TORC1 (mTORC1)
and mTORC2 phosphorylate the HM in S6K1 and PKB/
Akt, respectively (Burnett et al., 1998; Isotani et al.,
1999; Sarbassov et al., 2005). S. cerevisiae TORC2 and
a Schizosaccharomyces pombe TOR complex have also
been reported to phosphorylate the HM in the AGC
kinases Ypk1/2 and Gad8, respectively (Kamada et al.,
2005; Matsuo et al., 2003). Both mammalian and yeast
TOR complexes phosphorylate additional residues adja-
cent to the HM, including a sequence termed the ‘‘turn
motif,’’ which are often followed by a Pro (Kamada et al.,
2005; Matsuo et al., 2003; Montagne and Thomas,
2004). Phosphorylation of the turn motif is thought to
further stabilize the interaction between the HM and its
binding pocket (Gold et al., 2006).
In this study we queried whether the yeast AGC kinase
Sch9 could be a direct substrate for TORC1. Previous
studies have revealed phenotypic similarities between
sch9 cells and rapamycin-treated cells including nuclear
localization and activation of the Rim15 kinase and de-
creased expression of genes encoding proteins required
for ribosome biogenesis (Jorgensen et al., 2004; Pedruzzi
et al., 2003). Furthermore, tor1 and sch9 cells also share
report suggested that Sch9 becomes partially dephos-
phorylated upon rapamycin treatment (Jorgensen et al.,
Here we show that Sch9 is directly phosphorylated by
TORC1 at multiple C-terminal sites and by the yeast
PDK1 orthologs in the activation loop. Both phosphoryla-
tion events are independently required for Sch9 activity.
Phosphorylation of TORC1 sites is abolished under either
nitrogen or carbon starvation and transiently reduced
when cells are subjected to various stress conditions.
These observations support the notion that TORC1 activ-
ity is regulated by nutrient abundance and inhibited by
noxious stress. Using TORC1-independent versions of
Sch9, we found that Sch9 is a major effector of TORC1
that appears to function similarly to the mTORC1 sub-
Chemical Fragmentation Reveals Multiple
Rapamycin-Sensitive Phosphorylation Sites
in the Sch9 C Terminus
Analysis of Sch9 phosphorylation using SDS-PAGE mi-
gration shifts has been complicated by both the large
size of Sch9 (?100 kDa) and by the presence of multiple
phosphorylation sites. To circumvent these challenges
we tested various chemical reagents used for frag-
mentation of proteins (Burgess et al., 2000), of which
NTCB (2-nitro-5-thiocyanatobenzoic acid) proved to be
particularly useful. NTCB selectively cyanylates Cys resi-
dues, and under alkaline conditions this is followed by
chain cleavage at the modified residues. This allowed us
to analyze smaller fragments that contained fewer phos-
phorylated residues and were better resolved by immuno-
blotting. Treatment of C-terminally HA-tagged Sch9 in
a crude yeast extract yielded a highly reproducible
cleavage at some of the nine cysteines found in Sch9
Phosphorylation of Sch9 was decreased in cells treated
with rapamycin (Rap) or wortmannin (WM) but was in-
creased in the presence of a sublethal dose of cyclo-
heximide (CHX). Cleavage with NTCB revealed that this
included a dephosphorylation or hyperphosphorylation
of the Sch9 C terminus, respectively (Figure 1A). Treat-
ment with l phosphatase showed that the ladder of
bands migrating around 50 kDa in SDS-PAGE represents
multiple phosphoisoforms of the same fragmentation
product (Figure 1B). This was subsequently found to
include the activation loop phosphorylation site T570
(Liu et al., 2005) and thus probably encompasses amino
acids 554–824 of Sch9 and the 5HA tag (see Figures 2A
We next tested whether physiological conditions pre-
dicted to regulate TORC1 activity also affect the phos-
phorylation of the Sch9 C terminus. Shifting cells from
a medium containing glucose and Gln to media lacking
either a carbon ornitrogen source caused arapid dephos-
phorylation that was quickly reversed upon readdition of
the missing nutrient. The centrifugation step required for
the medium change also led to a transient partial dephos-
phorylation of the Sch9 C terminus (Figure 1C). Changing
the nitrogen source from NH4+to urea resulted in a rapid
dephosphorylation followed by a complete rephosphory-
lation in less than 2 hr (data not shown). Transferring cells
grown in a low-phosphate medium into a phosphate-free
medium also caused a dephosphorylation, although with
much slower kinetics (Figure 1D). Again, this was quickly
reversed upon phosphate readdition. Further analysis
showed that the C terminus of Sch9 was transiently de-
phosphorylated when cells were subjected to various
stress conditions, including high salt, redox stress, or
a shift to a higher temperature (Figure 1E).
We next wished to determine whether the extent of
Sch9 C-terminal phosphorylation correlated with nutrient
quality (Figure 1F). Sch9 was found to be slightly less
phosphorylated when cells were supplied with raffinose
or ethanol plus glycerol compared to glucose or galactose
as carbon sources. NH4+- and Pro-based media also
supported slightly less phosphorylation compared to
Gln- or urea-based media. However, the extent of Sch9
phosphorylation did not always correlate with growth
rate; cells grew faster in NH4+- versus Pro-based medium,
but Sch9 C-terminal phosphorylation was similar under
664 Molecular Cell 26, 663–674, June 8, 2007 ª2007 Elsevier Inc.
Sch9 Is a Major Target of TORC1 Signaling
TORC1 Phosphorylates Six Residues in the
C Terminus of Sch9
To identify the sites phosphorylated in Sch9, we purified
Sch9 from actively growing yeast and mapped potential
phosphorylation sites by mass spectrometry (see Table
S3 in the Supplemental Data available with this article
online). Building on these results, we started an extensive
mutational analysis that identified seven Ser/Thr residues
in the C terminus that when changed to Ala caused obvi-
ous alterations in SDS-PAGE migration of fragmented
Sch9 (Figure 2A). Cumulative substitution of these Ser/
Thr residues to Ala led to a progressive loss of phosphor-
ylated species, and a version of Sch9 lacking all seven
sites yielded a C-terminal fragment upon NTCB cleavage
that migrated as a single band (Figure 2B; uncropped
image, Figure S1).
Experiments with a variety of constructs containing
multiple Ser/Thr to Ala substitutions generally suggested
that the various sites can be phosphorylated indepen-
dently; only the phosphorylation of T723 seemed to de-
pend to some extent on prior phosphorylation of S726
(data not shown). With the exception of T570 in the activa-
tion loop, phosphorylation of the remaining six C-terminal
sites was sensitive to rapamycin treatment (Figure 2C,see
also Figure 4A) demonstrating that it occurred in a
Figure 2D shows the domain structure of Sch9 and the
position of the phosphorylated residues in the C-terminal
fragment. In addition to T570, the sites that were identified
included T737 in the classical HM of AGC kinases and two
Ser/Thr-Pro sites, T723 and S726. Two more sites were
found in the C-terminal extension (CE) beyond the HM of
Sch9 (S758 and S765). These show similarity to the HM,
especially the presence of bulky hydrophobic residues
at positions ?4, +1, and +2. Finally, S711 was found to
be phosphorylated as well. This residue is also followed
by two hydrophobic amino acids but is not preceded by
a hydrophobic residue at position ?4.
first queried whether TORC1 components can physically
interact with Sch9. Although we were unable to coimmu-
action between Tor1 and Sch9 using a two-hybrid ap-
proach (data not shown). Next we asked whether Sch9 is
a substrate for TORC1 in vitro. Indeed, TORC1 purified
from yeast phosphorylated recombinant Sch9 (Figure 2E).
This phosphorylation was strongly diminished if TORC1
expressing only a catalytically inactive version of Tor1
(Tor1D2275A). Recombinant Sch9 lacking all six C-terminal
sites (Sch96A) was much less phosphorylated compared
Figure 1. The Sch9 C Terminus Is Phosphorylated in
a TORC1-, Nutrient-, and Stress-Dependent Manner
(A) Chemical fragmentation analysis using NTCB shows that the C
terminus of Sch9 becomes dephosphorylated in response to rapa-
mycin (Rap, 200 ng/ml) and wortmannin (WM, 5 mM) and hyperphos-
phorylated in response to cycloheximide (CHX, 25 mg/ml) treatment
(all 30 min).
(B) Incubation with l phosphatase (PP) in the absence or presence of
phosphatase inhibitors (PPi) shows that the ladder of bands derived
from the Sch9 C terminus represents differently phosphorylated iso-
forms of the same fragmentation product. Samples were prepared
from CHX-treated cells.
(C) The Sch9 C terminus becomes reversibly dephosphorylated in
response to carbon (?Glc) and nitrogen (?Gln) starvation. 2% Glc or
0.2% Gln was readded after 40 min of starvation. Aliquots were taken
at the indicated times following medium change.
(D) Reversible dephosphorylation of Sch9 following transfer from low
phosphate (0.37 mM) to phosphate-free medium. Phosphate (0.37 mM)
was readded after 90 min of starvation.
(E) The Sch9 C terminus becomes temporarily dephosphorylated in
response to high salt, redox, and temperature stress.
(F) Phosphorylation of the Sch9 C terminus is reduced in cells growing
on less preferred nitrogen or carbon sources. (A)–(F) show anti-HA
immunoblots of untreated (A and B) and NTCB-treated (A–F) protein
extracts obtained from WT cells containing a plasmid-based copy of
SCH9-5HA. Only the C-terminal fragment is shown in (C)–(F).
Molecular Cell 26, 663–674, June 8, 2007 ª2007 Elsevier Inc. 665
Sch9 Is a Major Target of TORC1 Signaling
to Sch9WT, suggesting that we have identified the majority
of the residues in Sch9 that are modified by TORC1.
Phosphorylation of Sch9 was specific to TORC1: puri-
fied TORC2, although able to phosphorylate a physiologi-
cal substrate, Ypk2 (Kamada et al., 2005), was unable to
phosphorylate Sch9 in vitro (Figure S2).
To determine whether each of the six sites in the Sch9 C
terminus could be phosphorylated by TORC1, we ‘‘added
back’’ Ser/Thr residues to the Sch96Amutant and asked
whether this improved their phosphorylation. A compari-
son between Sch96Aand a version containing S711
(Sch95A) showed that S711 was poorly phosphorylated
in the in vitro assay (Figure 2E). Sch9 versions containing
any of the other sites in addition to S711 were more
strongly phosphorylated than Sch95A, indicating that at
least five sites in the Sch9 C terminus can be directly
phosphorylated by TORC1 (Figure 2F). Among these, the
HM-like sites S758 and S765 in the CE appeared to be
particularly good substrates in vitro while the Ser/Thr-
Pro sites T723 and S726 were less used.
TORC1 Phosphorylation Sites Are Critical
to Sch9 Function
To analyze the importance of TORC1-dependent phos-
phorylation of Sch9 in vivo, we took advantage of the ob-
servation that sch9 cells grew slowly on YPD and not at all
Figure 2. TORC1 Phosphorylates at Least Five C-Terminal Residues of Sch9
(A) Changing T570 or six other Ser/Thr residues to Ala alters the appearance of the C-terminal fragment.
(B) Cumulative mutation of these sites reduces the number of phosphorylated species until the C-terminal fragment migrates as a single band. The
uncropped picture is shown in Figure S1.
(C) Phosphorylation of all six C-terminal sites is sensitive to rapamycin treatment. Samples for (A)–(C) were prepared from CHX-treated WT cells
expressing Sch9-5HA. NTCB-treated protein extracts were analyzed by immunoblotting using anti-HA antibody. In (B) and (C), different amounts
of protein were loaded per lane to get comparable signal intensity.
(D) Domain structure of Sch9 and localization of the mapped phosphorylation sites and the putative NTCB cleavage site. A comparison of the
surrounding amino acids reveals that the phosphorylated residues either lie in a sequence resembling the HM or are followed by a Pro. RD, regulatory
domain; CE, C-terminal extension.
(E) TORC1 phosphorylates bacterially expressed Sch9 in vitro, while TORC1 purified from cells expressing only inactive Tor1D2275Aor from rapa-
mycin-treated cells has reduced activity toward Sch9. Phosphorylation is strongly diminished when the six C-terminal sites are replaced with Ala.
Sch95Acontaining only S711 is not a good substrate for TORC1.
(F) TORC1 phosphorylates recombinant Sch9 in vitro at T723, S726, T737, S758, and S765. Values in (E) and (F) show the relative amount of
radioactivity incorporated into Sch9.
666 Molecular Cell 26, 663–674, June 8, 2007 ª2007 Elsevier Inc.
Sch9 Is a Major Target of TORC1 Signaling
SCH9 or the acidic residue-substituted alleles SCH93E
(T737E, S758E, S765E) and SCH92D3E(T723D, S726D,
T737E, S758E, S765E) into sch9 cells restored normal
growth on both YPD and YPGal, while the Ala-substituted
allele SCH95A(T723A, S726A, T737A, S758A, S765A)
failed to complement the growth defect. The SCH93E
and SCH92D3Ealleles also conferred a slight resistance
to rapamycin (Figure 3A).
Because deletion of GLN3 and GAT1 renders cells re-
sistant to low doses of rapamycin (Beck and Hall, 1999),
we also performed our complementation studies in a (pro-
totroph) sch9 gat1 gln3 background. An added advantage
of using this strain was that it appeared to be phenotypi-
cally more stable than sch9 cells (data not shown). Com-
pared to sch9 cells, sch9 gat1 gln3 cells grew markedly
better on YPD and slowly on YPD + rapamycin but they
still failed to grow on YPGal. Importantly, introduction of
SCH93Eand SCH92D3Ealleles but not of SCH9WTin this
background allowed cells to grow on YPGal + rapamycin
(Figure 3A). This shows that Sch9 function depends on
TORC1-mediated phosphorylation of its C terminus and
that substitution of the C-terminal TORC1 phosphoryla-
tion sites of Sch9 with acidic amino acids yields Sch9 pro-
teins that appear to function independently of TORC1.
Further detailed analysis indicated that, with the possi-
ble exception of S711, all of the TORC1 phosphorylation
sites in Sch9 play a positive role in Sch9 function with
T737in the HMbeing the mostimportant site (Figure S3A).
A version containing only a substitution of the HM site
with glutamate (T737E) conferred rapamycin-resistant
growth, but simultaneous replacement of several TORC1
sites with acidic residues (3E and 2D3E) resulted in
a higher level of rapamycin resistance (Figure S3B).
In order to analyze how these mutations effect the Sch9
kinase activity, WT and mutated versions of Sch9-3HA
were isolated from yeast cells treated with drug vehicle
or rapamycin and tested for their ability to phosphorylate
a peptide (GRPRTSSFAEG; Cross et al., 1995), which is
known to be phosphorylated by various AGC kinases.
Sch9WTobtained from mock-treated cells was able to
phosphorylate the peptide while no activitywas measured
when Sch9WTwas isolated from rapamycin-treated cells
(Figure 3D). Sch9k.d.(K441A; Morano and Thiele, 1999)
and Sch95Ashowed no activity toward the substrate while
Sch92D3Eactivity was, for unknown reasons, increased by
prior rapamycin treatment. Together, these results dem-
onstrate that phosphorylation by TORC1 is necessary for
both Sch9 function in vivo and catalytic activity in vitro.
Pkh Kinases Phosphorylate T570 in the Activation
Loop of Sch9
Activity of many AGC kinases requires phosphorylation of
a Ser/Thr residue in the activation loop by PDK kinases
(Mora et al., 2004). To analyze the phosphorylation in the
Sch9 activation loop, phosphospecific antibodies against
phospho-T570 were generated. Immunoblotting showed
that these antibodies detected Sch9WTexpressed in
sch9gat1 gln3 cells, butnot the Ala-substituted Sch9T570A
(Figure 4A). In vivo, T570 was phosphorylated similarly in
Sch9WT, the inactive Sch95A, or the TORC1-independent
versions Sch93Eand Sch92D3Eas well as in Sch9WTafter
rapamycin treatment (Figure 4A). This suggests that
phosphorylation of the HM is not required to facilitate
subsequent phosphorylation of the activation loop. The
finding that both Sch9T570Aand Sch95Aare inactive (see
Figure 3B), although they still are phosphorylated by
TORC1 and Pkh kinases, respectively (see Figures 2A
and 4A), demonstrates that both activation loop phos-
phorylation and phosphorylation of the C terminus by
TORC1 are independently required for Sch9 activity.
In yeast, PDKs are encoded by the PKH1 and PKH2
genes (Casamayor et al., 1999). To determine whether the
activation loop in Sch9 is phosphorylated by Pkh kinases,
we performed an in vitro kinase assay and found that
recombinant GST-Sch9 was efficiently phosphorylated
by GST-Pkh2 purified from yeast cytosol. A preparation
Figure 3. Mutation of Several TORC1 Phosphorylation Sites
to Ala or Glu/Asp Renders Sch9 Inactive or Independent of
(A) SCH9 alleles with multiple Ser/Thr to Ala mutations fail to suppress
Alleles with multiple Ser/Thr to Asp/Glu mutations yield rapamycin-
resistant growth on YPGal in sch9 gat1 gln3 cells. Serial 10-fold
dilutions were spotted onto the indicated medium and incubated for
2 to 3 days at 30?C.
(B) Sch9WTisolated from untreated, but not from rapamycin-treated,
yeast shows activity toward the peptide GRPRTSSFAEG. Sch92D3E
activity is rapamycin insensitive while Sch9k.d.or Sch95Adisplay little
or no activity. Cultures were treated with CHX (10 min, 25 mg/ml) prior
to harvest, and the kinase activity was determined in duplicate. Error
bars represent the standard error.
Molecular Cell 26, 663–674, June 8, 2007 ª2007 Elsevier Inc. 667
Sch9 Is a Major Target of TORC1 Signaling
of the catalytically inactive protein, Pkh2K208R(Inagaki
et al., 1999), did not show any activity toward Sch9, and
the activation loop mutant Sch9T570Awas not a substrate
for Pkh2, indicating that Pkh2 directly phosphorylates
Sch9 at T570 (Figure 4B).
Immunoblot analysis showed that Pkh activity is also
required for T570 phosphorylation in vivo. Relative to WT
cells, at permissive temperature phosphorylation of T570
was reduced in pkh1 cells and strongly reduced in pkh1ts
pkh2 cells carrying the temperature-sensitive allele
PKH1D398G(Inagaki et al., 1999). After incubation at non-
permissive temperature, phosphorylation of the Sch9
activation loop was undetectable in pkh1tspkh2 cells
(Figure 4C). This shows that Sch9 is phosphorylated by
both Pkh1 and Pkh2 in vivo.
TORC1 Is Active at the Vacuole
Sch9 was found previously to be concentrated at the
vacuolar membrane, and this localization was shown to
be rapamycin insensitive (Jorgensen et al., 2004). Consis-
tently, the localization of GFP-Sch9 was not significantly
altered by the introduction of mutations at TORC1 phos-
phorylation sites (5A, 3E, and 2D3E) (Figure 5A). We found
that functional, GFP-tagged TORC1 components Tco89
and Kog1 (Figure 5A and data not shown) also localized
to the vacuolar membrane. Although the localization of
TORC1 is currently debated, the existence of a pool of
TORC1 at the vacuolar membrane is consistent with sev-
eral reports (discussed in De Virgilio and Loewith, 2006).
Thus, we wished to corroborate our localization data and
determine whether TORC1 is active at the vacuole.
Figure 4. Pkh Kinases Phosphorylate the Activation Loop of
(A) Phosphospecific antibodies directed toward the activation loop
specifically recognize Sch9 phosphorylated at T570. Mutating the C-
terminal TORC1 sites to Ala or Asp/Glu or treating cells with rapamycin
(30 min, 200ng/ml) does not affect the phosphorylation of T570 in vivo.
The indicated versions of plasmid-encoded 6HA-Sch9 were ex-
pressed in sch9 gat1 gln3 cells, and protein extracts were analyzed
(B) Pkh2, but not inactive Pkh2K208R, phosphorylates recombinant
GST-Sch9 at T570 in vitro.
(C) Phosphorylation of T570 in 6HA-Sch9, expressed from the endo-
temperature (26?C) and undetectable after a shift to nonpermissive
temperature (20 min, 37?C).
Figure 5. The C-Terminal Portion of Sch9 Artificially Tethered
to the Surface of the Vacuole Is an Efficient Substrate for
(A) Tco89-GFP and versions of GFP-Sch9 are concentrated at the
vacuolar surface. A fusion protein consisting of Vac8 and the Sch9
C-terminal 116 amino acids is found exclusively on the vacuole. The
vacuolar lumen was stained with blue CMAC.
(B) Immunoblotting using anti-HA antibody of untreated and NTCB-
treated protein extract shows that the Vac8-cSch9-3HA fusion protein
is phosphorylated at its TORC1 sites in a rapamycin-sensitive manner.
(C) Immunoblot of NTCB-treated protein extracts shows that phos-
phorylation of the TORC1 sites in Vac8-cSch9-3HA is stimulated by
CHX (25 mg/ml, 10 min) and reduced in response to high salt treatment
(0.5 M NaCl, 10 min). It is also reduced under carbon or nitrogen
starvation (30 min).
668 Molecular Cell 26, 663–674, June 8, 2007 ª2007 Elsevier Inc.
Sch9 Is a Major Target of TORC1 Signaling
To test directly whether TORC1 can phosphorylate
Sch9 at the vacuole, we fused the C-terminal portion of
Sch9 (cSch9 = aa 709–824) and a tag onto the C terminus
of Vac8, a palmitoylated protein that resides on the vacu-
olar membrane (Wang et al., 1998). Vac8-cSch9-GFP ex-
pressed in WT cells localized to the surface of the vacuole
as expected (Figure 5A). Immunoblotting of untreated and
NTCB-treated protein extracts showed that the C termi-
nus of Vac8-cSch9-3HA became highly phosphorylated
in a rapamycin-sensitive manner. No rapamycin-sensitive
modification occurred in a construct containing alanines
at all six TORC1 phosphorylation sites (Vac8-cSch96A-
3HA; Figure 5B). Further analyses showed that the
TORC1 sites in this construct were hyperphosphorylated
upontreatment withCHXanddephosphorylated following
(Figure 5C). Following readdition of Gln to nitrogen-
starved cells, Vac8-cSch9-3HA and Sch9-5HA were re-
phosphorylated with similar kinetics (data not shown).
Similar results were obtained when the cSch9-GFP or
cSch9-3HA sequences were fused to the first 134 amino
acids of Sna4, a small proteolipid of the vacuolar mem-
brane (data not shown), confirming that our findings are
not specific for Vac8. These experiments indicate that
the pool of TORC1 at the vacuolar surface is active. In
the future, variants of these reporter constructs may be
useful to probe for TORC1 activity at other loci.
TORC1 Regulates the Ribi and RP Regulons in Part
To begin toinvestigate whichof themanydifferent TORC1
readouts are mediated by Sch9, we used global transcrip-
tional analysis to compare the rapamycin response of WT
with those expressing plasmid-encoded Sch92D3E(WT/
Sch92D3Eshould act in a dominant manner to attenuate
the response of genes that are regulated by TORC1 via
Sch9. An analysis of all genes whose expression changed
response to rapamycin, with one set of genes responding
maximally after 20 or 30 min and a second, essentially
nonoverlapping set of genes responding maximally at
later time points (Figure S4).
Thus we separately analyzed early- and late-responsive
genes whose expression changed at least 3-fold in WT/
WT cells (Figure 6A). At early time points following
Figure 6. Sch9 Is Required for TORC1 to Properly Regulate
Transcription and Entry into G0Phase
(A) Microarray analysis comparing WT cells (W303) containing a
plasmid-based copy of SCH9WT(WT/WT) or SCH92D3E(WT/2D3E)
uncovers the Sch9 dependence of rapamycin-induced changes in
transcription. Genes showing at least 3-fold change in expression
were classified as either early- or late-responsive genes by comparing
the rapamycin response in WT/WT cells at early (20/30 min) and late
(90/120 min) time points. Genes were sorted according to the maximal
For genes above the black line the rapamycin-induced change in
expression is diminished R2-fold in WT/2D3E compared to WT/WT
cells (i.e., dif R 1 or % ?1). Ribosome biosynthesis genes and genes
encoding RPs as well as genes, whose expression is regulated by
Gln3/Gat1 or Msn2/4, are indicated. Color codes show the log2of
the expression change relative to untreated cells (green-red) and the
corresponding dif value (blue-gold).
(B) The rapamycin-induced (200 ng/ml, 30 min) translocation of GFP-
Rim15C1176Yinto the nucleus is blocked in sch9 rim15 cells expressing
plasmid-encoded Sch92D3E. In cells expressing Sch95A, Rim15 local-
izes constitutively to the nucleus.
(C) FACS analysis of sch9 cells containing the indicated plasmid-
based alleles of SCH9 following treatment with rapamycin (4 hr, 200
ng/ml) or drug vehicle. Expression of Sch92D3Eprevents the rapamy-
cin-induced cell-cycle arrest, observed as accumulation of cells with
a 1n DNA content. An enhanced portion of cells expressing Sch95A
have a 1n DNA content even in the absence of rapamycin.
glycogen accumulation upon rapamycin treatment (6 hr, 200 ng/ml)
compared to cells expressing Sch9WT.
Molecular Cell 26, 663–674, June 8, 2007 ª2007 Elsevier Inc. 669
Sch9 Is a Major Target of TORC1 Signaling
rapamycin treatment the expression of 272 genes was
reduced R3-fold. Repression of a majority (181) of these
genes was attenuated at least 2-fold in WT/2D3E cells
compared to WT/WT cells, suggesting that TORC1-
dependent phosphorylation of Sch9 contributes to the
regulation of these genes. This group included predomi-
nantly genes that encode factors involved in the synthesis
of ribosomes, tRNAs, and nucleotides (SGD), most of
which have previously been assigned to the ribosome bio-
genesis (Ribi) regulon (Jorgensen et al., 2004). Quantita-
tive PCR analyses confirmed that the effect of rapamycin
on the expression of several Ribi genes (PWP1, UTP13,
DIP2, and CIC1) was reduced in WT/2D3E compared to
WT/WT cells (Figure S5A). Further experiments in the
TB50 genetic background showed that the effect of rapa-
mycin on the expression of these Ribi genes is reduced in
sch9 gat1 gln3 cells lacking Sch9 activity or expressing
only TORC1-independent versions of Sch9 compared to
cells expressing Sch9WT. It is not known why rapamycin
treatment caused a stronger reduction in Ribi gene
expression in the W303 compared to the TB50 genetic
background. The data for TB50 cells lacking Sch9 also
revealed a strong Sch9-independent component in the
regulation of Ribi gene expression upon rapamycin treat-
ment (Figure S5B).
At later time points (R90 min), the expression of
308 genes was downregulated R3-fold in WT/WT cells.
Repression of 113 of these genes was significantly (R2-
fold) dependent on Sch9. Among these, genes encoding
ribosomal proteins (RPs) figured prominently. These
data are consistent with previous work (Jorgensen et al.,
2004), which demonstrated that both TORC1 and Sch9
regulate the expression of Ribi and RP genes.
The expression of relatively few genes was increased
more than 3-fold at early time points (93), and most of
these appeared to be regulated independently of Sch9.
The expression of 237 genes was increased at late time
points. Among these, the upregulation of 55 genes was
diminished more than 2-fold in WT/2D3E cells compared
to WT/WT cells. Genes regulated by Msn2/4 (http://
www.yeastract.com) were concentrated in this group,
suggesting that TORC1 regulates Msn2/4 activity (Beck
and Hall, 1999) in part via Sch9 (Figure 6A).
Importantly, not all TORC1-regulated transcription pro-
grams appear to depend on Sch9. For example, most
Gln3-regulated genes (Scherens et al., 2006), whose ex-
pression increased more than 3-fold following rapamycin
treatment, did not show a significant dependence on
Sch9 (Figure 6A). qPCR analyses confirmed that the
genes GLN1 and GAP1 was similar in SCH9WT, SCH93E,
and SCH92D3Ecells (Figure S5C). The expression of the
in sch9 gat1 gln3 cells containing different alleles of SCH9
(Figure S5D). Consistent with these results, both Gln3-
13myc and Rtg1-GFP translocated normally into the nu-
cleus after rapamycin treatment of sch9 cells expressing
Sch93E(data not shown). Thus Sch9 is not required for
TORC1 to negatively regulate the activity of Gln3/Gat1
(nitrogen discrimination pathway) or Rtg1/3 (retrograde
TORC1 Inhibits G0Entry via Sch9
Both TORC1 and Sch9 prevent entry into G0by inhibiting
nuclear translocation and activation of the Rim15 kinase
(Pedruzzi et al., 2003). When treated with rapamycin,
SCH9WTcells contained nuclear Rim15 (Figure 6B), ar-
rested with a 1n DNA content (Figure 6C), and exhibited
G0-specific phenotypes such as accumulation of the car-
bon reserve glycogen (Figure 6D). These readouts were
partially blocked in SCH92D3Ecells while cells lacking
Sch9 or expressing Sch95Aconstitutively localized Rim15
to the nucleus and accumulated glycogen even in the ab-
genes like GRE1 following rapamycin treatment was
only moderately reduced in cells expressing Sch92D3Ein
our microarray experiments. These results suggest that
To analyze whether Sch9 phosphorylation by TORC1 is
also required to complement the small cell phenotype of
sch9 cells (Jorgensen et al., 2004), we measured the
peak volume of sch9 gat1 gln3 cells expressing plasmid-
encoded versions of Sch9. Cells expressing Sch95A
were similar in size to cells containing a control plasmid
(30.0 versus 31.0 mm3) and significantly smaller than
cells expressing Sch9WT(43.1 mm3). Cells expressing
Sch93Eand Sch92D3Eyielded a peak volume of 40.2 and
36.6 mm3, respectively (data not shown).
TORC1 Regulates Translation Initiation in Part via
In yeast, rapamycin treatment leads to a rapid decrease of
translation initiation (Barbet et al., 1996) and TORC1 has
been proposed to regulate translation initiation via several
potential targets including eIF2a phosphorylation (Cher-
kasova and Hinnebusch, 2003) and eIF4G stability (Berset
et al., 1998).
To investigate whether Sch9 is required for TORC1 to
regulate translation initiation, we analyzed polysome
profiles generated from mock- or rapamycin-treated
SCH9WT, sch9, sch95A, and SCH92D3Ecells (Figure 7A).
As expected, SCH9WTcells showed a rapid arrest of
translation initiation following rapamycin treatment as in-
dicated by a 66% decrease in the polysome to 80S mono-
some (P/M) ratio compared to untreated cells. In cells ex-
pressing SCH92D3Eonly a slight reduction in the P/M ratio
occurreduponrapamycin treatment (21%decrease). Pro-
teinsynthesis appeared to bealready compromised in un-
treated sch9 and sch95Acells as judged by the reduced
polysome content, and the P/M ratio of these cells was
less reduced by rapamycin treatment (19% and 46% de-
crease, respectively) compared to SCH9WTcells.
eIF2aphosphorylation increased 4-fold insch9cellsex-
pressing Sch9WTupon treatment with rapamycin, but only
2.7-fold when rapamycin was added to cells expressing
670 Molecular Cell 26, 663–674, June 8, 2007 ª2007 Elsevier Inc.
Sch9 Is a Major Target of TORC1 Signaling
phosphorylation of eIF2a was strongly elevated in sch9
and sch95Acells (5.6- and 5.4-fold, respectively, as com-
pared to SCH9WTcells) and this phosphorylation was only
slightly further enhanced by rapamycin (Figure 7B).
In contrast, Sch9 does not seem to be involved in the
rapamycin-induced turnover of the translation factor eIF4G,
because eIF4G was equally abundant in SCH9WT, sch9,
sch95A, and SCH92D3Eyeast and was degraded upon ra-
pamycin treatment with similar kinetics in all of these cells
(data not shown). Together, these results demonstrate
that some but not all aspects of the regulation of transla-
tion initiation by TORC1 are mediated by Sch9.
Lastly, we wished to address whether Sch9 may func-
tion similarly to mammalian S6 kinase and phosphorylate
the yeast S6 ortholog (see Discussion). Sch9, but not
catalytically inactive Sch9k.d., efficiently phosphorylated
Rps6 in vitro (Figure 7C). Rps62A, which contains two
amino acid substitutions (S232A and S233A) previously
shown to abolish phosphorylation of Rps6 in vivo (Kruse
et al., 1985), was not phosphorylated by Sch9. This sug-
gests that Sch9 is indeed a genuine S6 kinase.
In this study, we have confirmed and extended previous
work (Jorgensen et al., 2004) by showing that Sch9 is a di-
rect substrate for TORC1 and a major component of the
TORC1 signaling pathway in S. cerevisiae. TORC1 regu-
lates ribosome biosynthesis and thus cell-size control in
large part via Sch9. Sch9 is also required for TORC1 to
properly regulate entry into stationary phase and transla-
tion initiation, while other processes like the expression
of Gln3/Gat1 and Rtg1/3 target genes appear to be regu-
lated by TORC1 independently of Sch9. This is consistent
with previous studies that demonstrated that TORC1 uses
distinct effector pathways to regulate the expression of
(Duvel et al., 2003). In the future it will be very important to
identify and characterize Sch9 substrates.
In addition to the seven sites described here, Sch9 is
phosphorylated at many more residues in its N terminus,
apparently due in part to autophosphorylation (J.U. and
R.L., unpublished data). It is likely that inputs from other
signaling pathways are integrated with those of TORC1
and Pkh kinases to regulate Sch9 activity. Indeed, cross-
talk between TORC1 and other nutrient-responsive sig-
naling pathways appears to be a recurring theme in cell
growth control (Schneper et al., 2004). Last, but not least,
localization and stability of Sch9 warrant further study, in
particular the ligand-binding properties of its C2 domain.
How Is TORC1 Regulated?
Phosphorylation of the C terminus of Sch9 is sensitive to
struct thatcontainsonlytheC-terminal116amino acidsof
Sch9 tethered to the vacuolar membrane. However, it is
possible that changes in phosphatase activity contribute
to the regulation of Sch9 phosphorylation as well. The
next challenge will be to determine, at the molecular level,
how growth cues regulate TORC1. An intriguing observa-
tion is the finding that at least a portion of TORC1 is active
at the surface of the vacuole. The vacuole is a major
nutrient reservoir in yeast, and therefore the vacuolar
membrane would be an ideal location for a sensor of intra-
cellular nutrients and for the compartmentalization of
nutrient-responsive signaling pathways.
It is also noteworthy that Sch9 C-terminal phosphoryla-
tion does not always correlate with growth rate. Thus,
although TORC1 activity is required for growth, factors
in additionto TORC1contribute todetermine steady-state
Figure 7. Sch9 Mediates Aspects of Translation Initiation
Regulation by TORC1
(A) Changes in polysome profiles caused by rapamycin treatment
a TORC1-independent version of Sch9. The positions corresponding
to the 40S and 60S subunits, the 80S monosomes, and polysomal
ribosomes are indicated in the profile of untreated SCH9WTcells. The
ratio between polysome and 80S monosome peaks (P/M) is indicated.
(B) Cells containing SCH92D3Eshow less rapamycin-induced (30 min,
200 ng/ml) phosphorylation of eIF2a (Sui2) compared to SCH9WTcells,
while eIF2a is constitutively phosphorylated in sch9 and sch95Acells.
Numbers indicate the increase of the eIF2a-P/eIF2a ratio relative to
untreated WT cells.
(C) Sch9 phosphorylates the yeast S6 ortholog Rps6, but not the Ala-
substituted Rps62Ain vitro.
Molecular Cell 26, 663–674, June 8, 2007 ª2007 Elsevier Inc. 671
Sch9 Is a Major Target of TORC1 Signaling
growth rate. The reduction in TORC1 activity following
nutrient starvation or the application of stress conditions
elicits, in addition to a reduction in protein synthesis, a de-
repression of genes encoding proteins required for the
utilization of alternative nutrient sources and stress re-
sponse factors. In this way TORC1 plays an important
conditions. Once cells have successfully adapted their
metabolism to the availability of nutrients or acquired
tolerance to environmental stress, TORC1 is reactivated
and growth resumes. Developmental decisions such as
entry into G0phase (Pedruzzi et al., 2003) or sporulation
(Colomina et al., 2003) may require a prolonged inactiva-
tion of TORC1.
Is Sch9 an S6K1 Ortholog?
Many groups suggest that Sch9 is the yeast ortholog of
for the following reasons we suggest that Sch9 function
may be more closely related to that of mammalian S6K1.
(1) Like S6K1, Sch9 activity is regulated by TORC1. In
contrast, Akt is regulated by mTORC2 (Sarbassov et al.,
2005). S6K1 and Sch9 also share the unusual feature of
having an extended sequence beyond their HMs. S6K1
mutants lacking this domain have been found to be inap-
propriately activated by mTORC2 (Ali and Sabatini, 2005).
(2) S6K1 and Sch9 apparently perform similar functions,
most notably the regulation of translational initiation. In-
deed, we have found that Sch9 is able to phosphorylate
Rps6, the yeast ortholog of the mammalianRP S6, in vitro,
and thus by definition is an S6 kinase.
tion will not only enhance our understanding of TORC1
signaling in yeast but will also reveal additional functions
of mammalian S6 kinase. For example, both TORC1 and
Sch9 have been implicated in coupling nutrient excess
to decreased lifespan (Fabrizio et al., 2005; Kaeberlein
possibility that mTORC1 and S6K1 similarly influence
longevity in mammals.
Strains and Plasmids
Yeast strains and plasmids used in this study are listed in Tables S1
and S2. All strains except those used in Figures 4C, 6A, and 6B and
Figure S4 were made prototroph for amino acids and nucleotides by
introducing pJU 450 and a URA3-containing plasmid.
Western Blot and Chemical Fragmentation Analysis
Na2P2O4, and 10 mM b-glycerophosphate; PI: 13 Roche protease
inhibitor cocktail and 1 mM PMSF.
Cultures were mixed with TCA (final concentration 6%) and put on
ice for at least 5 min before cells were pelleted, washed twice with
cold acetone, and dried in a speed-vac. Cell lysis was done in 100 ml
of urea buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 6 M urea, 1% SDS,
sequent heating for 10 min to 65?C. For NTCB cleavage 30 ml of 0.5 M
CHES (pH 10.5) and 20 ml of NTCB (7.5 mM in H2O) were added and
samples incubated over night at RT before 1 vol of 23 sample buffer
(+20 mM TCEP and 0.53 PPi) was added. Further analysis was done
by SDS-PAGE and immunoblotting using anti-HA antibody 12CA5 or
TORC1 Kinase Assay
Preparation of Recombinant Sch9
GST-Sch9 fusion proteins were expressed in E. coli from a pGEX-6P
vector. After a 3 hr induction with 0.4 mM IPTG, a clarified bacterial
lysate was prepared and the fusion protein was bound to glutathione
Sepharose following standard procedures. Sch9 was cleaved from
the GST moiety overnight at 4?C with 24 units PreScission protease
(GE Healthcare) in 300 ml PreScission cleavage buffer containing
0.01% Tween 20 following manufacturer’s instructions. The superna-
tant was dialyzed against (13 PBS, 20% glycerol, and 0.5% Tween
20), aliquotted, and frozen at ?80?C.
TORC1 was purified from RL175-2d or RL176-1b cells treated with
drug vehicle or 200 nM rapamycin for 30 min. Cells grown at 30?C in
YPD (250 ml per assay point) to an OD600of ?1.0 were chilled on
ice, collected by centrifugation, washed with H2O, resuspended in
lysis buffer (13 PBS, 10% [w/v] glycerol, 0.5% [v/v] Tween 20, PI,
and PPi), transferred to 2 ml screw-cap tubes half-filled with glass
beads (0.5 mm), and disrupted in a Fast Prep machine at 4?C
(Bio101; 53 30 s at max. speed). Crude lysates were cleared of debris
with two 1000 3 g spins and protein concentrations normalized as
necessary. Extracts were precleared over CL-4B Sepharose before
7 ml of IgG Sepharose (GE Healthcare) per assay point was added
washed with cold lysis buffer, and aliquotted to 1.5 ml tubes. Kinase
reactions were performed in kinase buffer (13 PBS, 20% glycerol,
0.5% Tween 20, 4 mM MgCl2, 10 mM DTT, 2 mg/ml heparin, and PI
[?EDTA]) in a final volume of 30 ml containing ?350 ng of recombinant
Sch9. Assays were started with the addition of 100 mM ATP and 50 mCi
[g-32P]ATP, shaken for 20 min at 30?C, and terminated with the addi-
tion of 8 ml of 53 SDS-PAGE sample buffer. Samples were heated to
95?C for 5 min before being separated by SDS-PAGE, stained with
Coomassie, and analyzed using a BioRad Molecular Imager.
Supplemental Data include five figures, three tables, Supplemental
Experimental Procedures, and Supplemental References and can be
found with this article online at http://www.molecule.org/cgi/content/
We acknowledge financial support from the following: Novartis
Stiftung (A.H.), NIH CA41086 (J.R.B.), and the Cantons of Basel and
Geneva as well as the Swiss National Science Foundation (O.D.,
H.R., C.D.V., M.N.H., and R.L.). Part of this work was supported by
an FP5RTN grant (ACE) from the EC (G.A.). We also thank Danie `le Rifat
and Wolfgang Oppliger for technical assistance, Elisabetta Cameroni
for analyzing the polysome profiles, Philippe Demougin for help setting
up the qPCR protocol, Manuele Piccolis for help analyzing microarray
data, and Tom Sturgill, Didier Picard, and David Shore for their critical
comments on the manuscript.
Author contributions: J.U., Figures 1, 2A–2D, 3A, 4A, 4C, and 5, and
Figure S3; R.L., Figures 2E and 2F and Figure S2; A.H., Figures 3B and
7C; D.M., Figure 4B; S.L., Figure 6A and Figures S4 and S5A; A.S., Fig-
ures 6B and 6D and Figures S5B–S5D; V.W., Figure 6C; O.D., Figures
7A and 7B; D.A., Table S3; and manuscript preparation, J.U. and R.L.
Received: December 21, 2006
Revised: March 14, 2007
Accepted: April 23, 2007
Published: June 7, 2007
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Sch9 Is a Major Target of TORC1 Signaling