Download The TEA Transcription Factor Tec1 Links TOR and MAPK Pathways

INVESTIGATION
The TEA Transcription Factor Tec1 Links TOR
and MAPK Pathways to Coordinate Yeast Development
Stefan Brückner,* Sandra Kern,* Raphael Birke,* Irene Saugar,†,‡ Helle D. Ulrich,†
and Hans-Ulrich Mösch*,1
*Department of Genetics, Philipps Universität, D-35043 Marburg, Germany, †Cancer Research UK London Research Institute, Clare
Hall Laboratories, South Mimms, Herts EN6 3LD, United Kingdom, and ‡Centro de Biología Molecular Severo Ochoa, University of
Madrid, 28049 Madrid, Spain
ABSTRACT In Saccharomyces cerevisiae, the TEA transcription factor Tec1 controls several developmental programs in response to
nutrients and pheromones. Tec1 is targeted by the pheromone-responsive Fus3/Kss1 mitogen-activated protein kinase (MAPK) cascade, which destabilizes the transcription factor to ensure efficient mating of sexual partner cells. The regulation of Tec1 by signaling
pathways that control cell division and development in response to nutrients, however, is not known. Here, we show that Tec1 protein
stability is under control of the nutrient-sensitive target of rapamycin complex 1 (TORC1) signaling pathway via the Tip41-Tap42-Sit4
branch. We further show that degradation of Tec1 upon inhibition of TORC1 by rapamycin does not involve polyubiquitylation and
appears to be proteasome independent. However, rapamycin-induced Tec1 degradation depends on the HECT ubiquitin ligase Rsp5,
which physically interacts with Tec1 via conserved PxY motives. We further demonstrate that rapamycin and mating pheromone
control Tec1 protein stability through distinct mechanisms by targeting different domains of the transcription factor. Finally, we show
that Tec1 is a positive regulator of yeast chronological lifespan (CLS), a known TORC1-regulated process. Our findings indicate that in
yeast, Tec1 links TORC1 and MAPK signaling pathways to coordinate control of cellular development in response to different stimuli.
F
OR cells to appropriately respond to diverse external
signals, intracellular signal-specific pathways are often
interconnected to allow integration of multiple signals and
to ensure activation of the correct cellular programs. The
budding yeast Saccharomyces cerevisiae is well suited to
study the mechanisms of signal integration in eukaryotes,
because numerous evolutionarily conserved signaling
pathways are present in this organism, including a pheromone-responsive mitogen-activated protein kinase (MAPK)
pathway (Bardwell 2005) as well as nutrient-sensitive
target of rapamycin complex 1 (TORC1) (De Virgilio and
Loewith 2006) and Ras/protein kinase A (PKA) pathways
(Tamaki 2007). All of these pathways have been implicated
in the control of cell division and development (Zaman
et al. 2008), but their interconnection is not understood in
detail.
Copyright © 2011 by the Genetics Society of America
doi: 10.1534/genetics.111.133629
Manuscript received March 8, 2011; accepted for publication August 4, 2011
Supporting information is available online at http://www.genetics.org/content/
suppl/2011/08/12/genetics.111.133629.DC1.
1
Corresponding author: Department of Genetics, Philipps Universität Marburg, Karl von
Frisch Straße 8, D-35043 Marburg, Germany. E-mail: [email protected]
The pheromone-responsive MAPK pathway of S. cerevisiae has been well studied (Chen and Thorner 2007). It
controls not only sexual mating, but also vegetative adhesion required for the formation of biofilms and filaments
(Liu et al. 1993; Roberts and Fink 1994), and therefore is
also referred to as the mating and adhesive/filamentous
growth MAPK cascade. Execution of these distinct cellular
programs is under control of both shared and programspecific components. The shared components include the
two MAPKs Fus3 and Kss1, the MAPK kinase Ste7, and the
transcription factor Ste12, which positively control expression
of both mating and vegetative adhesin genes (Roberts and
Fink 1994). The program-specific components include the
transcription factor Tec1, which belongs to the TEA domain
(TEAD) family of transcriptional regulators, which control
cellular development in many eukaryotes (Andrianopoulos
and Timberlake 1991; Anbanandam et al. 2006). In S. cerevisiae, Tec1 is required for adhesion and the expression of
the vegetative adhesin gene FLO11 (Gavrias et al. 1996;
Mösch and Fink 1997; Lo and Dranginis 1998; Rupp et al.
1999). Tec1 is not required for expression of most matingspecific genes, e.g., the sexual agglutinin gene FUS1 (Zeitlinger
et al. 2003). The mating and vegetative adhesion programs
Genetics, Vol. 189, 479–494 October 2011
479
not only differentially control gene expression, but also cell
division. Whereas mating depends on sexual partner cells
arresting in G1, robust biofilms and filaments are best
formed by dividing cells. In haploid cells, this difference is
reflected by the fact that G1 cyclins are down-regulated
when yeast cells switch from vegetative adhesion to mating
(Wittenberg et al. 1990; Brückner et al. 2004). The cyclin
gene CLN1 is activated by the transcription factor Tec1 during
biofilm formation (Madhani et al. 1999). In contrast, Tec1dependent activation of CLN1 is lost during mating due to
Fus3-mediated phosphorylation of the transcription factor
at residue Thr273 within a conserved CPD motif (Cdc4phosphodegron), which confers ubiquitylation of Tec1 by
the ubiquitin-ligase SCFCdc4 and subsequent degradation
(Bao et al. 2004; Brückner et al. 2004; Chou et al. 2004). A
block of Tec1-degradation during mating not only interferes
with down-regulation of CLN1 expression, but also with an
efficient G1 arrest (Brückner et al. 2004). In addition, Tec1 is
stabilized by complex formation with Ste12 by a mechanism
that is independent of the CPD, but involves the C-terminal
part of Tec1 (Chou et al. 2006; Heise et al. 2010).
In S. cerevisiae, cell division and cellular development are
also controlled by the nutrient-sensitive TORC1 signaling
network, which is negatively regulated by the macrocyclic
lactone rapamycin (Crespo and Hall 2002; De Virgilio and
Loewith 2006). Inhibition of TORC1 by higher doses of rapamycin causes downregulation of G1 cyclins and leads to
a G1 arrest (Heitman et al. 1991; Barbet et al. 1996). Lower
doses of rapamycin lead to the inhibition of filament formation (Cutler et al. 2001) and to an extension of yeast chronological lifespan (CLS) (Powers et al. 2006). Numerous
TORC1-regulated processes have been elucidated and
a number of signaling pathways are known that either act
downstream of TORC1 or that function in parallel, but share
common targets (De Virgilio and Loewith 2006). Important
signaling components acting downstream of TORC1 include
the PP2A-like protein phosphatases Sit4 and Pph21/22,
their regulatory subunit Tap42, and the Tap42-interacting
protein Tip41 (Düvel and Broach 2004). These components
regulate a major branch of the TORC1 network and control
genes involved in stress regulation, nitrogen catabolite repression, and retrograde signaling, and they connect TORC1
with the general amino acid control system (Rohde et al.
2008). A further important branch of TORC1 signaling is
regulated by the protein kinase Sch9, which is a direct target
of TORC1 and controls ribosome synthesis and cell size
(Urban et al. 2007). In addition, Sch9 regulates replicative
lifespan (RLS) (Kaeberlein et al. 2005) and CLS (Fabrizio
et al. 2001; Powers et al. 2006; Wanke et al. 2008). Finally,
amino acid uptake is also regulated by TORC1 and involves
the nitrogen-regulated protein kinase Npr1 and the HECT
ubiquitin ligase Rsp5 (Schmidt et al. 1998; De Craene et al.
2001; Crespo et al. 2004).
The TORC1 signaling network is interconnected to
a number of other signaling cascades that control cell
division and development (Rohde et al. 2008). A prominent
480
S. Brückner et al.
example is the Ras/PKA pathway, which includes the small
GTP-binding protein Ras2 and the cAMP-dependent protein
kinase A, composed of the regulatory subunit Bcy1 and the
catalytic subunits Tpk1, Tpk2, and Tpk3 (Zaman et al.
2008). With respect to controlling cell division, the Ras/
PKA pathway has been suggested to control G1 progression
and entry into stationary phase by targeting the transcription factors Msn2/Msn4 (Smith et al. 1998) and the protein
kinase Rim15 (Pedruzzi et al. 2003), regulatory components
that are also targets of TORC1. These findings lead to the
current view that the TORC1 and Ras/PKA pathways act in
parallel in the control of cell division (Rohde et al. 2008).
With respect to controlling cellular development, the Ras/
PKA pathway is known to regulate adhesive and filamentous
growth via the catalytic subunit Tpk2, which targets the transcription factors Flo8 and Sfl1 to regulate expression of
FLO11 (Robertson and Fink 1998; Pan and Heitman 2002).
Like TORC1, the Ras/PKA pathway also regulates chronological yeast lifespan (Reinders et al. 1998; Longo 2003).
In this study, we explored the possibility that the
pheromone-responsive Fus3/Kss1 MAPK cascade and the
nutrient-sensitive TORC1 pathways might be interconnected, as both pathways control G1 arrest, vegetative adhesion, and filamentous growth. We focused on Tec1,
because this transcription factor is an activator of G1 cyclin
and vegetative adhesin genes, which are down-regulated
during mating through degradation of Tec1. In contrast,
nutrient starvation causes down-regulation of G1 cyclin
genes, but induces expression of the adhesin gene FLO11,
raising the question of how Tec1 is regulated by known
nutrient-sensitive signaling pathways. Here we show that
the TORC1 signaling pathway regulates Tec1 protein stability and Tec1-target gene expression by a mechanism that is
distinct from the stability control exerted by the Fus3/Kss1
MAPK cascade and that does not involve the Ras/PKA pathway. We also find that Tec1 controls chronological lifespan.
We suggest that in S. cerevisiae, the Fus3/Kss1 MAPK and
the TORC1 pathways are interconnected by Tec1 to ensure
proper control of cell division and cellular development in
response to pheromone and nutrients.
Materials and Methods
Yeast strains and growth conditions
All yeast strains used in this study are described in Table 1.
Strains carrying tec1D::HIS3, fus3D::TRP1, tpk1D::loxPkanMX-loxP, tpk2D::loxP-kanMX-loxP, tpk3D::loxP-kanMXloxP, npr1D::URA3, bul2D::URA3, bul1D::kanMX4, ste7D::
loxP-kanMX-loxP, sit4D::loxP-kanMX-loxP, rsp5D::kanMX4,
and pdr5Δ::kanMX deletion alleles were obtained by transformation using respective deletion cassettes and verified by
Southern blot analysis. The URA3(P)-OLE1 allele was introduced by targeting the integrative plasmid BHUM1053 to
the leu2::hisG locus of RH2754.
Standard yeast culture medium was prepared essentially
as described (Guthrie and Fink 1991). For rapamycin-based
experiments, cultures grown to exponential growth phase
were treated with 200 ng/ml of rapamycin (LC Laboratories) for 70 min or with solvent alone. For pheromone induction, 1 mm synthetic a-factor (Novabiochem) was
applied for 60 min. Adhesive growth tests were performed
as described by Roberts and Fink (1994). For nitrogen and
amino acid starvation, cultures were grown in SC medium to
exponential growth phase, washed with water, incubated in
fresh SC medium (no starvation) or in SC medium lacking
ammonium sulfate and/or amino acids (starvation medium), and grown for 70 min before further investigations.
Aging assay
Yeast chronological lifespan was analyzed as previously
described (Wanke et al. 2008). Briefly, cultures of yeast
strain YHUM928 (tec1Δ) carrying one of the plasmids
YCplac33, BHUM169, or BHUM1306 were grown overnight,
diluted to an OD600 of 0.2, and grown into exponential
growth phase before 2 ng/ml of rapamycin or drug vehicle
was added. Colony-forming units (CFU ml21) were determined by collecting culture aliquots regularly every day and
plating serial dilutions on solid medium.
Plasmids
Plasmids used in this study are listed in Table 2 and primers
in Supporting Information, Table S1. Plasmid BHUM570
was constructed by subcloning of a 3.1-kb PstI/BamHI fragment containing TEC1(P)-BglII-TEC1 from pME2068 into
YEplac195, followed by insertion of a GFPuv fragment from
pME1771 into the BglII site. BHUM576 was the result of
a PCR-based mutagenesis (Köhler et al. 2002) of TEC1 in
pME2068, where a frameshift led to a stop codon.
BHUM750 was isolated from a yeast two-hybrid library
(Fashena et al. 2000) and identified as RSP5G372-E809, representing the C-terminal half of RSP5. BHUM752 was
obtained by cloning a PCR-based EcoRI/BamHI fragment
using primers SB/TEC1-2 and SB/TEC1-3 in EcoRI/BamHIdigested pEG202. For BHUM765, the ORF of RSP5 was amplified using primers SB/RSP5-1 and SB/RSP5-2 and cloned
as a SalI fragment into pYGEX-2T. Blasting the URA3 marker
of BHUM765 with a LEU2 fragment from pUC4-ura3::LEU2
(kind gift from Y. Kassir, Haifa, Israel) resulted in
BHUM1305. BHUM1521 was obtained from BHUM1305
by removing the SalI fragment containing RSP5. Plasmid
BHUM1158 was obtained by PCR amplification of the
URA3 promoter using primers URA3-1 and URA3-2 and insertion of the resulting fragment into pRS305 after SalI/
BamHI digestion. For plasmid BHUM1053, OLE1 was amplified with primer OLE1-1 and OLE1-2 and inserted into
BamHI/XbaI-digested
BHUM1158.
BHUM1122
was
obtained by site-directed mutagenesis using primers SKTEC1-3 and SK-TEC1-4, pME2068 as template, and the
QuickChange Site-Directed Mutagenesis kit (Stratagene,
Amsterdam, The Netherlands). BHUM1306 was the result
of three subsequently performed site-directed mutagenesis
steps using primers SB-TEC1-m1f, SB-TEC1-m1r, SB-TEC1-
m2f, SB-TEC1-m2r, SB-TEC1-m3f, SB-TEC1-m3r, and
pME2068 as the first template.
Two-hybrid screen and analysis
For yeast two-hybrid analysis, plasmids pEG202 and pJG4-5,
a yeast genomic library cloned in vector pJG4-5 (Fashena
et al. 2000), and the yeast strain EGY48-p1840 were used
(kindly provided by Erica Golemis and Roger Brent). For the
isolation of Tec1-interaction partners, yeast strain EGY48p1840 carrying TEC1281-486 as a bait (BHUM752) was transformed with the yeast genomic library on pJG4-5 to obtain
5 · 106 transformants. Transformants were collected as
a pool, and Leu-prototrophic strains were selected by
growth on SC medium lacking Leu, Trp, and His, and containing 2% galactose and 1% raffinose, and assayed for
b-galactosidase activity as described (Fashena et al. 2000).
Library plasmids were isolated as described (Hoffman and
Winston 1987) and analyzed by DNA sequencing using the
ABI Prism Big Dye terminator sequencing kit and an ABI
310 Genetic Analyzer (Applied Biosystems, Weiterstadt,
Germany). Interactions were verified by reintroducing library plasmids into the parental strain EGY48-p1840 carrying either BHUM752 or pEG202, followed by a growth
test for Leu prototrophy.
Two-hybrid interactions between Tec1 and Rsp5 were
quantified by measuring specific b-galactosidase activities
of strain EGY48-p1840 that was cotransformed with the
plasmids pEG202 or BHUM752 together with either pJG4-5
or BHUM750. Transformants were grown on SC 2His 2Trp
containing 2% galactose and 1% raffinose to an optical density at 600 nm of between 1 and 2 before b-galactosidase
assays were performed as described below. All assays were
performed in triplicate on at least three independent transformants for each combination of plasmids.
b-Galactosidase assays
Yeast strains carrying plasmids for two hybrid analysis or the
TEC1-lacZ reporter were grown to the exponential growth
phase in appropriate media, and extracts were prepared and
assayed for b-galactosidase activity as described previously
(Brückner et al. 2004). b-Galactosidase activity was normalized to the total protein in each extract with the following
formula: (optical density at 420 nm · 1.7)/(0.0045 · protein
concentration · extract volume · time). Assays were performed in triplicate on at least three transformants, and the
mean values were calculated. Standard deviations did not
exceed 20%.
Protein analysis
Purification of glutathione S-transferase fusion proteins
from yeast: Extracts of strains expressing glutathione Stransferase (GST) or GST-RSP5 together with Tec1 variants
were prepared from cultures grown for 4 hr to exponential
growth phase in appropriate SC medium. When required,
200 ng/ml rapamycin was added 20 min prior to protein
extraction. Cells were harvested by centrifugation (5 min,
Tec1 Links TOR and MAPK Pathways
481
Table 1 Yeast strains used in this study
Strain
BY4741
CDV316
CY3938
CY4029
EGY48-p1840
IP31
JC19-1a
KT1960
RH2500
RH2754
WCG4a
WCG4/11/22
Y01133
Y01276
Y01579
Y01856
Y02394
Y02412
Y02708
Y03578
Y03665
Y04010
Y04065
Y04077
Y04173
Y04245
Y04291
Y04596
Y04814
Y04859
Y04883
Y05277
Y05336
Y05459
Y05701
Y06834
Y06902
Y07006
Y07045
Y07122
Y07299
Y2864
YHI29/14
YHUM119
YHUM120
YHUM389
YHUM397
YHUM407
YHUM476
YHUM482
YHUM622
YHUM793
YHUM928
YHUM1089
YHUM1093
YHUM1104
YHUM1341
YHUM1376
YHUM1803
YHUM2039
YM105
YM106
YM107
YP0607WH
482
Description
Source
MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 gal1Δ::HIS3 sch9Δ::natMX4
CY4029 with sit4Δ::HIS3
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 SSD1-v1
MATa ura3 his3 trp1 leu2 lexAop-lacZ-URA3
KT1960 with rim15Δ::kanMX2
MATa npr1::HIS3MX in TB50a
MATa ura3-52 leu2::hisG his3::hisG trp1::hisG
RH2754 with tec1Δ::HIS3
MATa ura3-52 leu2::hisG his3::hisG trp1::hisG
MATa his3-11,15 leu2-3,112 ura3
WCG4a with pre1-1 pre2-2
BY4741 with skp2Δ::kanMX4
BY4741 with yjl149wΔ::kanMX4
BY4741 with ubr2Δ::kanMX4
BY4741 with dia2Δ::kanMX4
BY4741 with rup1Δ::kanMX4
BY4741 with siz2Δ::kanMX4
BY4741 with hrt3Δ::kanMX4
BY4741 with mfb1Δ::kanMX4
BY4741 with ydr306cΔ::kanMX4
BY4741 with pph3Δ::kanMX4
BY4741 with ydr131cΔ::kanMX4
BY4741 with san1Δ::kanMX4
BY4741 with ylr224wΔ::kanMX4
BY4741 with siz1Δ::kanMX4
BY4741 with tom1Δ::kanMX4
BY4741 with sap4Δ::kanMX4
BY4741 with ubr1Δ::kanMX4
BY4741 with ufd4Δ::kanMX4
BY4741 with tul1Δ::kanMX4
BY4741 with mdm30Δ::kanMX4
BY4741 with asi3Δ::kanMX4
BY4741 with tip41Δ::kanMX4
BY4741 with rog3Δ::kanMX4
BY4741 with hul4Δ::kanMX4
BY4741 with grr1Δ::kanMX4
BY4741 with yak1Δ::kanMX4
BY4741 with sap190Δ::kanMX4
BY4741 with asi1Δ::kanMX4
BY4741 with ssm4Δ::kanMX4
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 gal1Δ::HIS3
WCG4a with pre1-1 pre4-1
MATa ura3-52 leu2::hisG his3::hisG trp1::hisG FRE(Ty1)::lacZ::LEU2
YHUM119 with ras2Δ::ura3::HIS3
YHUM119 with tpk1Δ::loxP tpk2Δ::loxP-kanMX-loxP
YHUM119 with tpk1Δ::loxP tpk3Δ::loxP-kanMX-loxP
YHUM119 with tpk2Δ::loxP tpk3Δ::loxP-kanMX-loxP
MATa ura3Δ leu2Δ his3Δ trp1Δ
MATa leu2Δ his3Δ trp1Δ
YHUM119 with ste7Δ::loxP-kanMX-loxP
RH2754 with fus3Δ::TRP1 tec1Δ::HIS3
MATa ura3-52 tec1Δ::HIS3 his3::hisG
YHUM476 with npr1Δ::URA3
YHUM476 with bul2Δ::URA3 bul1Δ::kanMX4
RH2754 with URA3(P)-OLE1::LEU2
YHUM1104 with rsp5Δ::kanMX4
RH2754 with sit4Δ::kanMX4
MATa tec1Δ::HIS3 pdr5Δ::kanMX ura3-52 leu2::hisG his3::hisG trp1::hisG
RH2500 with sit4Δ::kanMX4
RH2754 with kss1Δ::hisG
RH2754 with fus3Δ::TRP1
RH2754 with kss1Δ::hisG fus3Δ::TRP1
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 pph21Δ::TRP1 pph22Δ::HIS3
EUROSCARF
C. DeVirgilio, lab collection
Luke et al. (1996)
Luke et al. (1996)
Gyuris et al. (1993)
Pedruzzi et al. (2003)
Crespo et al. (2004)
Pedruzzi et al. (2003)
Köhler et al. (2002)
Köhler et al. (2002)
Heinemeyer et al. (1993)
Heinemeyer et al. (1993)
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
Wang et al. (2004)
Gerlinger et al. (1997)
Mösch et al. (1999)
Mösch et al. (1999)
This study
This study
This study
MICROBIA
MICROBIA
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Madhani et al. (1997)
Madhani et al. (1997)
Madhani et al. (1997)
Sakumoto et al. (2002)
S. Brückner et al.
Table 2 Plasmids used in this study
Plasmid
BHUM29
BHUM169
BHUM277
BHUM570
BHUM576
BHUM750
BHUM752
BHUM765
BHUM1053
BHUM1122
BHUM1158
BHUM1177
BHUM1305
BHUM1306
BHUM1521
pEG202
pHU821
pJG4-5
pME1771
pME2065
pME2068
pME2083
pME2096
pME2102
pME2280
pME2676
pRS305
pYGEX-2T
YCplac33
YEplac181
YEplac195
Description
Source
GAL1(P)::TEC1 in URA3-based CEN vector
TEC1 in YCplac33
TEC1 in YEplac181
TEC1(P)-GFPuv-TEC1 in YEplac195
TEC1(P)-TEC1Δ355-486 in YCplac33
B42-RSP5G372-E809 in pJG4-5
lexA-TEC1K281-Y486 in pEG202
GAL1(P)-GST-RSP5 in pYGEX-2T
URA3(P)::OLE1 in pRS305
TEC1(P)-TEC1K54A in YCplac33
pRS305 with 1.0-kb URA3-promoter
TEC1 in YEplac195
GAL1(P)-GST-RSP5 in LEU2-based 2mm vector
TEC1(P)::TEC1P411A P429A P448A (TEC1AxY) in YCplac33
GAL1(P)-GST in LEU2-based 2-mm vector
Vector for construction of lexA fusion proteins
His-UBI with CUP1 promoter in YEplac181
Vector for construction of B42 fusion proteins
BUD8(P)-GFPuv-BUD8
TEC1-lacZ in URA3-based 2-mm vector
TEC1(P)-TEC1 in YCplac33
TEC1(P)-TEC1Δ258-486 in YCplac33
TEC1(P)-TEC1P274S in YCplac33
TEC1(P)-TEC1T273M in YCplac33
3myc-TEC1 in YEplac195
GST-TEC1M1-E280 in pETM30
LEU2-based integrative vector
GAL1(P)-GST URA3-based 2-mm vector
URA3-based CEN vector
LEU2-based 2-mm vector
URA3-based 2-mm vector
Mösch et al. (1999)
Köhler et al. (2002)
Köhler et al. (2002)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Gyuris et al. (1993)
Davies et al. (2010)
Gyuris et al. (1993)
Taheri et al. (2000)
Köhler et al. (2002)
Köhler et al. (2002)
Köhler et al. (2002)
Köhler et al. (2002)
Köhler et al. (2002)
Köhler et al. (2002)
Brückner et al. (2004)
Sikorski and Hieter (1989)
Schlenstedt et al. (1995)
Gietz and Sugino (1988)
Gietz and Sugino (1988)
Gietz and Sugino (1988)
3000 rpm), washed in 2% galactose solution, and transferred to SC medium containing 2% galactose. After incubation for 5 hr at 30, cultures were chilled on ice, harvested
by centrifugation at 4, washed once in B buffer [50 mm
HEPES (pH 7.5), 50 mm KCl, 5 mm EDTA (pH 7.5)], resuspended in 300 ml ice-cold B buffer containing protease
inhibitors (50 mm DTT, 1 mm PMSF, 0.5 mm TPCK,
0.5 mm TLCK, 0.5 mm Pepstatin A) and transferred to 2 mlreaction tubes. Cells were broken by vortexing with glass
beads at 4 for 10 min, followed by addition of 300 ml B
buffer plus protease inhibitors and Triton X-100 to a final
concentration of 0.1%. Samples were mixed again by vortexing at 4 for 1 min, followed by centrifugation for 5 min at
13,000 rpm to remove glass beads and large cell debris. A
total of 10 ml of extracts was removed to determine total
protein concentration. For input control, 50 ml of the supernatant were transferred to a 1.5 ml-reaction tube and denatured by addition of SDS sample buffer and heating for 5 min
at 95. A total of 200 ml of the remaining extract was mixed
with 800 ml B buffer plus protease inhibitors plus 0.1% Triton
X-100 and 100 ml 50% glutathione sepharose and incubated
for 2 hr at 4. Beads were repeatedly washed in B buffer plus
0.1% Triton X-100 and collected to purify GST fusion proteins
and any associated proteins. Samples were denatured by
heating at 95 for 5 min in SDS sample buffer.
Preparation of total cell extracts: Preparation of total cell
extracts was performed as described (Kushnirov 2000).
Immunoblot analysis: Equal amounts of proteins were
subjected to SDS–PAGE using 12% gels (Laemmli 1970).
Proteins were separated and then transferred to nitrocellulose membranes (Schleicher und Schuell, Dassel, Germany)
by electrophoresis for 1 hr at 100 V using a Mini-PROTEAN
3 electrophoresis system (Bio-Rad, Munich, Germany). Tec1
variants as well as GST fusion proteins or Cdc28 were
detected using enhanced chemiluminescence (ECL) technology (Amersham, Buckinghamshire, UK) after incubation of
membranes with polyclonal rabbit anti-Tec11-280 antibodies
(Heise et al. 2010), goat polyclonal anti-Cdc28 antibodies,
or polyclonal rabbit anti-GST antibodies (Santa Cruz Biotechnology). Phosphorylation of Fus3 and Kss1 was detected
using a Phospho-p44/42 MAPK (Thr202/Tyr204) antibody
from Cell Signaling Technology. As secondary antibodies
peroxidase-coupled goat antirabbit, goat antimouse, or donkey antigoat immunoglobulin G (Santa Cruz Biotechnology)
were used. For quantification of signals, a scanner and the
Quantity One software (Bio-Rad, Munich, Germany) or a
Chemo Star Imager (INTAS Science Imaging Instruments,
Göttingen, Germany) and the Lab Image 1D software (Kapelan
Bio-Imaging, Leipzig, Germany) were used.
Tec1 Links TOR and MAPK Pathways
483
Ubiquitylation analysis: Cultures of yeast strain
YHUM1803 carrying plasmids pME2280 (3myc-TEC1) or
BHUM1177 (TEC1) as well as YEplac181 (empty vector)
or pHU821 (His-UBI), respectively, were grown to exponential phase in the presence of 0.1 mm CuSO4 and treated with
10 mm MG132. An equivalent volume of DMSO was added
to untreated control cultures. After 2 hr, an additional 5 mm
MG132 (or DMSO) was added along with 200 ng/ml rapamycin where indicated, and incubation was continued for
70 min. Extracts were prepared under denaturing conditions, and total ubiquitin conjugates were isolated by NiNTA affinity chromatography as described (Davies et al.
2008, 2010). Ubiquitylated forms of Tec1 were detected in
the isolated material by anti-myc Western blotting. In parallel, Tec1, total ubiquitin conjugates, and Pgk1 (as a loading
control) were detected in the total extracts with monoclonal
antibodies 9E10 (anti-myc), P4D1 (antiubiquitin), and 22C5
(anti-Pgk1).
RNA analysis
Yeast cultures were grown to exponential growth phase
prior to addition of rapamycin (200 ng/ml) or drug vehicle
and incubation for 70 min. Total RNA was then prepared
following the instruction manual of Trizol reagent (Invitrogen, Karlsruhe, Germany) and 8 mg was separated on
a 1.2% agarose gel. After transfer to a positively charged
nylon membrane TEC1, FLO11, CLN1, and ACT1 transcripts
were detected by using gene-specific, DIG-labeled DNA
probes, following the instruction manual for DIG filter hybridization (Roche Diagnostics, Mannheim, Germany).
GFP fluorescence microscopy
Yeast strains expressing GFP-TEC1 were grown to mid-log
phase in YNB medium before addition of rapamycin to 200
ng/ml. Cells were harvested at appropriate time points and
immediately viewed in vivo on a Zeiss Axiovert microscope
using (i) differential interference microscopy (DIC) and (ii)
fluorescence microscopy using a GFP filter set (AHF Analysentechnik, Tübingen, Germany). Cells were photographed
using a Hamamatsu Orca ER digital camera and the Improvision Openlab software (Improvision, Coventry, UK).
Results
Reduction of Tec1 protein stability in response to
rapamycin treatment
We directly tested whether Tec1 is under control of the
TORC1 pathway by treatment of yeast cells with rapamycin.
For this purpose, we used polyclonal anti-Tec1 antibodies
that specifically detect endogenous Tec1 protein levels in
yeast extracts (Heise et al. 2010). We found that inhibition
of TORC1 by addition of rapamycin to yeast cultures led to
a roughly fivefold decrease in endogenous Tec1 protein levels within 70 min after addition of the drug (Figure 1A).
Addition of rapamycin did not significantly alter either
484
S. Brückner et al.
TEC1 transcript levels (Figure 1B) or expression of a TEC1lacZ reporter gene (Figure 1C), indicating a post-translational mechanism. To measure the effect of rapamycin on
Tec1 protein stability, we performed a promoter shutoff experiment using the TEC1 gene under the control of the glucose-repressible GAL1 promoter. We found that rapamycin
reduces the half-life of Tec1 approximately threefold from
67 to 24 min (Figure 1, D and E). These data suggest that
Tec1 protein stability is under positive control of TORC1.
Rapamycin-induced degradation of Tec1 depends on
the TORC1 network
To further explore how TORC1 regulates Tec1 stability, we
screened an array of yeast mutant strains carrying deletions
in genes encoding known TORC1-controlled signaling components for defects in rapamycin-induced degradation of
Tec1. We first tested components of the TORC1-regulated
type 2A and 2A-related protein phosphatase modules including the catalytic PP2A subunits Pph21/Pph22 and
Pph3 as well as the PP2A-related catalytic subunit Sit4.
We found that rapamycin-induced degradation of Tec1
was blocked in sit4Δ, but not in pph21Δ pph22Δ or pph3Δ
mutant strains (Figure 2A), indicating that TORC1 controls
Tec1 via Sit4. This conclusion is further supported by our
finding that Tec1 protein degradation is blocked in strains
lacking the Sit4 regulatory subunit Sap190 and the nonessential protein Tip41, which controls Sit4 activity in response to rapamycin (Jacinto et al. 2001). We then
investigated the involvement of a number of protein kinases
that have been reported to act either downstream or in
parallel to TORC1 and Sit4 including Npr1, Rim15, Yak1,
and Sch9. We found that rapamycin-induced Tec1 degradation was affected in npr1Δ strains, but not in mutants strains
lacking any of the other protein kinases tested (Figure 2B).
This finding indicates that Npr1 might be involved in the
TORC1-mediated control of Tec1. Finally we tested whether
the Ras/PKA pathway might be involved in conferring control of Tec1 stability by rapamycin. However, rapamycin-induced Tec1 degradation was not affected by deletions in any
of the genes encoding the catalytic PKA subunits Tpk1,
Tpk2, or Tpk3 or in the absence of Ras2 (Figure 2C). Tec1
degradation was also not altered in strains expressing the
hyperactive RAS2Val19 allele (data not shown). Together
these data suggest that control of Tec1 stability by rapamycin is mediated by the TORC1-controlled protein phosphatase Sit4 and the protein kinase Npr1, but does not involve
action of the Ras/PKA pathway.
Given the role of TORC1 in nutrient sensing, we also
tested how nitrogen and amino acid deprivation affects
Tec1. In comparison to rapamycin treatment, starvation for
nitrogen and amino acids caused a significant increase in
Tec1 protein levels when measured at a comparable time
point (Figure S1). Moreover, starvation-induced up-regulation of Tec1 was not efficiently blocked in strains lacking
SIT4. This suggests that Tec1 protein levels are nitrogen
regulated by TORC1-independent mechanisms.
Figure 1 Regulation of Tec1 by rapamycin. (A) Tec1
protein levels in yeast strains RH2500 (tec1Δ) and
RH2754 (TEC1) 70 min after addition of rapamycin
(+) or drug vehicle (2) to exponentially growing cultures. Tec1 and Cdc28 proteins were detected by
immunoblot analysis using specific anti-Tec1 and antiCdc28 antibodies. Relative Tec1 levels (Tec1 norm)
are indicated and were obtained by normalizing Tec1
to Cdc28 signals. (B) TEC1 transcript levels in yeast
strain RH2754 70 min after addition of rapamycin (+)
or drug vehicle (2). TEC1 and ACT1 transcripts were
determined by Northern blot analysis and relative TEC1
transcript levels were obtained by using ACT1 as an
internal standard. (C) TEC1-promoter activity. TEC1lacZ expression was measured in yeast strain RH2754
carrying the pME2065 plasmid 0, 30, and 60 min after
addition of rapamycin. Bars depict specific b-galactosidase activities and are means of three independent
measurements. (D) Stability of Tec1 in the absence
and presence of rapamycin. TEC1 expression was induced in yeast strain RH2500 carrying GAL1(P)-TEC1
(BHUM29) by growth in galactose containing medium
for 6 hr. Glucose was added to repress TEC1 transcription. At time point zero rapamycin (+) or drug vehicle
(2) were added and Tec1 protein levels were measured
by immunoblot analysis at the indicated time points.
Protein levels of Cdc28 served as an internal standard in all extracts. (E) Quantification of Tec1 protein assayed in D. Relative Tec1 levels in the absence
( ) or presence (n) of rapamycin are shown in arbitrary units and were obtained by normalizing Tec1 to Cdc28 signals. The Tec1/Cdc28 ratio at time
point 0 was set to 100%. Half-life times (t1/2) are indicated.
•
Control of Tec1 stability in the absence of the Fus3/Kss1
MAPK cascade
We next tested whether rapamycin-induced degradation of
Tec1 involves elements of the Fus3/Kss1 MAPK cascade,
because Tec1 is known to be degraded in response to mating
pheromone. However, strains carrying deletions in the
MAPK genes KSS1 and FUS3 or in the MAPK kinase gene
STE7 are not suppressed with respect to rapamycin-dependent degradation of Tec1 (Figure 3A). This finding indicates
that rapamycin-induced degradation of Tec1 does not involve action of the Fus/Kss1 MAPK cascade. We also measured how rapamycin affects the intracellular amounts of
the phosphorylated forms of Fus3 and Kss1 by using phospho-specific antibodies against the two MAPKs. As expected,
vegetatively growing cells contain low levels of phosphorylated Fus3 and Kss1, whereas high levels of the activated
MAPK forms are present in pheromone-stimulated cells (Figure 3B). Interestingly, addition of rapamycin to vegetative
cells led to a significant decrease in phosphorylated Kss1,
but not Fus3. Moreover, this effect was dependent on the
presence of Fus3, because the amount of phosphorylated
Kss1 did not decrease in response to rapamycin in cells lacking FUS3. In pheromone-stimulated cells, rapamycin led to
a reduction of the phosphorylated forms of both Kss1 and
Fus3 (Figure 3B). These data indicate that the TORC1 pathway might feed into Fus3/Kss1 MAPK cascade at or above
the level of the two MAPKs. However, this cross-pathway
control does not appear to be crucial for pheromone-induced
degradation of Tec1. Finally, we measured how concomitant
addition of both rapamycin and mating pheromone to yeast
affects Tec1 protein levels. This experiment revealed that
the two effectors seem to act in a synergistic manner (Figure
3C), and it indicates that rapamycin and mating pheromone
control Tec1 stability by distinct mechanisms.
Rapamycin-induced Tec1 degradation does not involve
polyubiquitylation and appears to be
proteasome independent
Previous studies have shown that pheromone treatment
triggers polyubiquitylation of Tec1 and proteasomedependent degradation (Bao et al. 2004; Chou et al. 2004).
We therefore tested whether rapamycin-induced Tec1 degradation also involves ubiquitylation of the transcription factor.
For this purpose, we expressed an epitope-tagged version of
TEC1 (3myc-TEC1) together with an epitope-tagged variant of
the ubiquitin gene (His-UBI) and performed pull-down
experiments under denaturing conditions to detect ubiquitylated forms of Tec1. These experiments revealed that
rapamycin treatment did not induce detectable polyubiquitylation of the transcription factor, not even under conditions of proteasome inhibition, where total ubiquitin
conjugates accumulate to a high level (Figure 4A). However, we detected a monoubiquitylated form of Tec1, whose
levels decreased in response to rapamycin. Analysis of the
total protein extracts further revealed that treatment of cells
with the proteasome inhibitor MG132 did not prevent degradation of Tec1 (Figure 4A). Because this finding indicates that
rapamycin might induce a proteasome-independent degradation pathway, we also measured rapamycin-induced Tec1
degradation in temperature-sensitive proteasome mutants.
Tec1 Links TOR and MAPK Pathways
485
Figure 2 Rapamycin-induced Tec1 degradation depends on Sit4, Tip41,
and Npr1. Tec1 protein levels were determined in the yeast strains of the
indicated genotype as described in Figure 1. (A) CY4029 (control),
CY3938 (sit4Δ), Y07045 (sap190Δ), Y04596 (sap4Δ), Y05459 (tip41Δ),
YP0607WH (pph21Δ pph22Δ), and Y04010 (pph3Δ). (B) YHUM482 (control), Y07006 (yak1Δ), YHUM1089 (npr1Δ), KT1960 (control), IP31
(rim15Δ), Y2864 (control), and CDV316 (sch9Δ). (C) YHUM119 (control),
YHUM120 (ras2Δ), YHUM407 (tpk2Δ tpk3Δ), YHUM397 (tpk1Δ tpk3Δ),
and YHUM389 (tpk1Δ tpk2Δ). Numbers indicate relative Tec1 levels (Tec1
norm) and were obtained by normalizing Tec1 to Cdc28 signals.
Indeed, genetic inactivation of different proteasomal subunits
was not sufficient to efficiently block Tec1 degradation (Figure 4B). Taken together, these data indicate that rapamycin
triggers Tec1 degradation independent of polyubiquitylation
and the proteasome.
Identification of the HECT ubiquitin ligase Rsp5 as
a Tec1-interacting protein
We next aimed at identifying components that might
participate in degradation of Tec1 in response to rapamycin.
For this purpose, we analyzed an array of 22 yeast mutant
strains carrying single deletions in nonessential genes
encoding components of different protein modifying systems
(Figure S2). However, none of the mutants tested was significantly suppressed by rapamycin-induced degradation of
Tec1. This indicates that none of the tested E3 ubiquitin
ligases, F-box proteins, or SUMO/Smt3 ligases, which are
lacking in these mutants, are involved in the process. To
more directly identify proteins involved in Tec1 protein degradation, we performed a yeast two-hybrid screen. For this
purpose, a lexA-TEC1 construct lacking the Tec1 DNA-binding domain (TEAD) was used that alone led to only a weak
induction of a lexA-driven lacZ reporter gene (Figure 5, A
and B). Roughly 100,000 B42-fused yeast cDNAs (Fashena
et al. 2000) were screened for a two-hybrid interaction with
lexA-TEC1 and led to the identification of the HECT ubiquitin
ligase Rsp5 as a potential interaction partner for Tec1 (Fig-
486
S. Brückner et al.
Figure 3 Rapamycin-induced down-regulation of Tec1 is independent of
the Fus3/Kss1 MAPK cascade. (A) Tec1 protein levels were determined in
yeast strains RH2754 (control), YM105 (kss1Δ), YM106 (fus3Δ), YM107
(kss1Δ fus3Δ), and YHUM622 (ste7Δ) as described in Figure 1. Numbers
indicate relative Tec1 levels (Tec1 norm) and were obtained by normalizing Tec1 to Cdc28 signals. (B) Influence of rapamycin on Fus3 and Kss1.
Yeast strains RH2754 (FUS3) and YM106 (fus3Δ) were grown in the
absence (2) or presence (+) of either rapamycin or pheromone or both
and protein extracts were prepared. Phosphorylated forms of Fus3 and
Kss1 were detected by immunoblot analysis using phospho-specific antibodies. Cdc28 served as a loading control. (C) Concomitant effect of
rapamycin and pheromone on Tec1. Tec1 protein levels were determined
in yeast strain RH2754 grown for 70 min in the absence (2) or presence
(+) of either rapamycin or pheromone or both. Relative Tec1 protein levels
(Tec1 norm) are indicated.
ure 5B). Sequence analysis of the isolated RSP5 cDNA
revealed that it encoded residues 372–809 of Rsp5, encompassing the WW domain that confers interaction of this ubiquitin ligase with proteins that contain PxY motifs (Hoppe
et al. 2000; Shcherbik et al. 2004). Analysis of the Tec1
protein sequence revealed that it harbors three PxY sequence motifs in the C-terminal part (Figure 5A). To further
investigate the Tec1–Rsp5 physical interaction, we performed GST coaffinity purification experiments using Tec1
and Rsp5 proteins. We found that Tec1 could be copurified
with GST–Rsp5 and that the interaction remained stable
upon rapamycin treatment (Figure 5, C and D and Figure
S3). Moreover, mutation of the three PxY motifs of Tec1 to
AxY sequences or deletion of the C-terminal part of Tec1
efficiently blocked interaction of the transcription factor
with Rsp5 (Figure 5C). Together, these data demonstrate
that the ubiquitin ligase Rsp5 is a specific Tec1-interacting
protein.
Dependence of rapamycin-induced degradation of Tec1
on Rsp5 and C-terminal PxY motifs
To address the physiological significance of the Tec1–Rsp5
interaction, we measured rapamycin-induced degradation of
Tec1 in strains lacking RSP5. Normally, yeast strains carrying
Figure 4 Rapamycin-triggered Tec1 degradation does not
appear to involve polyubiquitylation or proteasomal degradation. (A) Ubiquitylation of Tec1 in response to rapamycin. Cultures of strain YHUM1803 carrying plasmids
pME2280 (3myc-TEC1) or BHUM1177 (TEC1) as well as
YEplac181 (empty vector) or pHU821 (His-UBI), respectively, were grown in the presence of 0.1 mm CuSO4
and treated with MG132 and/or rapamycin as indicated.
Extracts were prepared under denaturing conditions, and
total ubiquitin conjugates were isolated by Ni-NTA affinity
chromatography. Ubiquitylated forms of Tec1 were
detected in the isolated material by anti-myc Western blotting together with a whole cell extract (WCE) as a control
(top). Total levels of Tec1 (anti-myc) and total ubiquitin
conjugates (anti-Ubi) in the cell extracts were detected in
parallel (bottom). Detection of Pgk1 (anti-Pgk1) served as
a loading control. (B) Requirement of proteasome function. Tec1 protein levels were determined in temperature-sensitive yeast strains WCG4a (control), WCG4/11/
22 (pre1-1 pre2-2), and YHI29/14 (pre1-1 pre4-1) carrying
plasmid BHUM277 were grown for 70 min at 25 or 37
and in the absence (2) or presence (+) of rapamycin. Tec1
protein was then analyzed by immunoblot analysis.
a full deletion of RSP5 are not viable, due to insufficient
expression of the OLE1 gene that is required for production
of essential amounts of oleic acid (Hoppe et al. 2000). To
circumvent this problem, we constructed a strain that
expresses the OLE1 gene from the Rsp5-independent URA3
promoter. In this genetic background, a full deletion of the
RSP5 gene could be introduced, resulting in a viable rsp5Δ
mutant strain. Using this strain, we found that rapamycininduced degradation of Tec1 was efficiently blocked in the
absence of RSP5 (Figure 6A). In contrast, deletion of RSP5
did not block the increase of Tec1 protein levels observed in
response to nitrogen and amino acid starvation (Figure S1).
Thus, Rsp5 might be involved in destabilization of the transcription factor in response to rapamycin. This conclusion is
supported by our finding that rapamycin-induced degradation of Tec1 is blocked by deletion of the C-terminal domain
of Tec1, which is required for association of Tec1 with Rsp5
(Figure 6B). Moreover, mutation of the three PxY motifs in
Tec1 to AxY sequences also generated a rapamycin-resistant
variant of Tec1 (Figure 6B). Importantly, the Tec1AxY variant, which does not interact with Rsp5, is fully functional
in vivo when assayed in an adhesion test (Figures 6C and
Figure S4). We also measured whether destabilization of
Tec1 depends on Bul1, Bul2, Rog3, or Rup1, which represent Rsp5-interacting proteins that have been suggested to
confer substrate specificity (Yashiroda et al. 1998; Andoh
et al. 2002; Kee et al. 2005). However, rapamycin-induced
degradation of Tec1 was not affected in strains lacking these
components (Figure 6A).
Finally, we tested how rapamycin and Rsp5 affect the
cellular localization of Tec1 in living cells by using a functional GFP-TEC1 fusion gene. We found that in the control
strain, a functional GFP-Tec1 protein is rapidly degraded
upon addition of rapamycin when analyzed by immunoblot
analysis (Figure 6D) or by fluorescence microscopy (Figure
6E). In contrast, rapamycin-induced degradation of GFPTec1 was blocked in the rsp5Δ deletion strain (Figure 6E),
corroborating the data obtained with strains expressing endogenous Tec1. However, addition of rapamycin did not
significantly alter the nucleo-cytoplasmic distribution of
GFP-Tec1, neither in the presence nor in the absence of
Rsp5 (Figure 6E), indicating that subcellular localization
of Tec1 is not under control of rapamycin.
In summary, these data demonstrate that Rsp5 is a Tec1interacting protein that is also required for rapamycin-induced destruction of Tec1.
Tec1 Links TOR and MAPK Pathways
487
Figure 5 Interaction between Tec1 and Rsp5. (A) The
linear diagram of Tec1 indicates the TEA DNA-binding
domain (TEAD), Cdc4-phosphodegron motif (CPD) and
the C-terminal portion (shaded) conferring Ste12 binding, transactivation, and Tec1 destabilization (Köhler
et al. 2002; Chou et al. 2004; Anbanandam et al. 2006;
Chou et al. 2006; Heise et al. 2010). The location of
the conserved PxY motifs for Rsp5 interaction is indicated, and the three motifs are shown in boldface type
in the protein sequence below. Underlined proline residues were exchanged for alanine in the AxY variant of
Tec1. Numbers indicate amino acid residues of Tec1. (B)
Two-hybrid interaction between Tec1 and Rsp5. Numbers indicate specific b-galactosidase activities obtained
in strain EGY48-p1840 expressing lexA (pEG202) or lexATEC1281-486 (BHUM752) in combination with B42 (pJG45) or B42-RSP5372-809 (BHUM750). Values are the means
of three independent measurements. (C) Copurification
of different Tec1 variants with GST–Rsp5. Protein extracts
were prepared from yeast strain YHUM793 expressing
GST (BHUM1521) or GST–Rsp5 (BHUM1305) together
with TEC1 (pME2068), TEC1AxY (BHUM1306), or TEC1ΔC
(pME2083). GST or GST–Rsp5 and associated proteins
were purified using glutathione-sepharose. Presence of
Tec1 protein in cell extracts (input) and in GST-purified
complexes (beads) was analyzed by immunoblotting using anti-Tec1 antibodies. GST and GST–Rsp5 were detected by antibodies against GST. (D) Influence of rapamycin on Tec1–Rsp5 interaction. Copurification of Tec1 with GST–Rsp5 was determined in yeast strain RH2754 carrying plasmids BHUM1305 (GST-RSP5) and BHUM29 (TEC1) as described in C
before (2) and 20 min after (+) addition of rapamycin.
Rapamycin and mating pheromone control Tec1
stability by distinct mechanisms
We then focused on the differences in the mechanisms that
confer destabilization of Tec1 in response to either rapamycin or mating pheromone. For this purpose, we measured
how rapamycin affects the stability of known pheromoneresistant variants of Tec1 that carry mutations within the
Cdc4-phosphodegron motif (Chou et al. 2004). We found
that the pheromone-resistant variants Tec1T273M and
Tec1P274S were rapidly degraded in response to rapamycin
(Figure 6A). Similarly Tec1K54A, a variant that blocks
sumoylation of Tec1 at residue Lys54 and confers higher
stability of the protein (Wang and Dohlman 2006), was
not resistant to rapamycin treatment (Figure 7A). We next
asked, how mating pheromone affects stability of the
Tec1AxY variant that we here uncovered to be resistant to
rapamycin. As shown in Figure 7B, Tec1AxY is rapidly degraded in response to pheromone treatment. In addition,
pheromone-induced Tec1 degradation was not efficiently
blocked in a rsp5Δ mutant strain (Figure 7C). These experiments demonstrate that pheromone- and rapamycin-induced
destabilization of Tec1 depend on distinct residues of the
transcription factors and indicate that rapamycin and pheromone are two cues that control Tec1 stability by distinct
mechanisms.
transcription of the two Tec1-target genes CLN1 and FLO11
(Brückner et al. 2004). Therefore, we measured how rapamycin affects expression of CLN1 and FLO11 in the absence
and presence of TEC1. In the case of CLN1, we found that
rapamycin caused a significant decrease in CLN1 transcript
levels, but only in the TEC1 strain and not in the tec1Δ
mutant, in which expression of this cyclin gene was very
low (Figure 8A). This finding indicates that rapamycininduced repression of CLN1 depends on TEC1 and suggests
that loss of CLN1 transcription might be caused by rapamycininduced degradation of Tec1. This conclusion is supported by
the fact that rapamycin does not cause a decrease in CLN1
transcript levels in a strain expressing the TEC1AxY variant
(Figure 8B). A different expression pattern was observed in
the case of the Tec1-target gene FLO11, which was slightly
up-regulated by rapamycin in the presence of Tec1 (Figure
8A). In the absence of Tec1, FLO11 transcript levels were very
low, even when rapamycin was added, indicating that Tec1 is
essential for efficient FLO11 expression, independent of the
TORC1 activity. Finally, rapamycin also caused a slight increase in FLO11 transcript levels in strains expressing TEC1AxY
or TEC1T273M (Figure 8B). In summary, these data show that
the Tec1-target genes CLN1 and FLO11 are differentially regulated by rapamycin and that regulation of these genes by
TORC1 involves Tec1.
Differential regulation of Tec1 targets CLN1 and FLO11
by rapamycin
Tec1 is a regulator of TORC1 controlled
chronological lifespan
We have previously shown that degradation of Tec1 in response to mating pheromone causes a significant loss in the
Our finding that repression of CLN1 gene expression by
rapamycin involves Tec1 prompted us to test whether
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S. Brückner et al.
Figure 6 Control of Tec1 by RSP5. (A) Tec1
protein levels were determined in yeast
strains YHUM1104 (control), YHUM1341
(rsp5Δ), YHUM1093 (bul1Δ bul2Δ), Y05701
(rog3Δ), and Y02394 (rup1Δ) grown in
the presence of rapamycin (+) or drug
vehicle (2) as described in Figure 1. (B)
Rapamycin-induced destruction of different
Tec1 variants. Tec1 protein levels were
determined in RH2500 carrying TEC1
(pME2068),
TEC1Δ355-486
(BHUM576),
TEC1Δ258-486 (pME2083), or TEC1AxY
(BHUM1306) as described above. (C) Functionality of different TEC1 variants during
adhesive growth. Yeast strain RH2500 carrying one of the plasmids YCplac33 (tec1Δ),
pME2068 (TEC1), BHUM570 (GFP-TEC1), or
BHUM1306 (TEC1AxY) was patched on
SC 2Ura medium. After 4 days the plate
was photographed before (total growth)
and after (adhesive growth), washing nonadhesive cells off the surface. (D) Rapamycin-induced destruction of GFP–Tec1 protein
levels was determined in yeast strain
RH2500 carrying GFP-TEC1 (BHUM570). (E)
Localization of GFP–Tec1. Representative
cells of yeast strains YHUM1104 (RSP5) and
YHUM1341 (rsp5Δ) carrying BHUM570 are
shown. Living cells were visualized under
a microscope by using fluorescence microscopy or Nomarski optics 0, 50, and 75 min
after addition of rapamycin to exponentially
growing cultures. Bar, 5 mm.
Tec1 might be involved in rapamycin-induced cell cycle arrest. However, we could not detect a significant difference in
rapamycin sensitivity between yeast strains lacking or overexpressing TEC1 (data not shown). We next tested whether
Tec1 might affect yeast CLS, because previous studies have
shown that this process is under control of TORC1 (Pedruzzi
et al. 2003; Powers et al. 2006). In accordance with previous
studies, low doses of rapamycin led to an extension of CLS,
as shown for the TEC1 expressing control strain (Figure 9).
However, rapamycin-induced CLS extension was not observed before prolonged stay of yeast cells in stationary
phase. Interestingly, CLS was also significantly reduced in
yeast strains that lack Tec1. As found for rapamycin treatment, the influence of Tec1 became obvious only after a prolonged stay of cells in stationary phase. However, absence of
Tec1 did not fully suppress rapamycin-induced CLS extension. Finally, CLS was also reduced in strains expressing the
TEC1AxY allele, but not as pronounced as in strains completely lacking the transcription factor. Interestingly, rapamycin-induced CLS extension was almost completely
blocked in the presence of TEC1AxY. In summary, these data
indicate that Tec1 might be a positive regulator of CLS.
Discussion
In this study, we have presented evidence that in S. cerevisiae
(i) the TORC1 and Fus3/Kss1 MAPK pathways are intercon-
nected by the TEA domain transcription factor Tec1, (ii)
both pathways control this transcription factor by affecting
its stability through independent mechanisms, (iii) the connection between TORC1 and MAPK signaling is important to
coordinate G1 cyclin and vegetative adhesin gene expression, and (iv) Tec1 affects chronological lifespan. These
findings provide several novel insights into the mechanisms
that enable integration of TOR and MAPK kinase signaling
in eukaryotic cells and ensure coordination of cell division
and development. Importantly, our study pinpoints the connection between these two pathways to a transcriptional
regulator of the TEA domain family, whose members are
known to control development in many eukaryotes (Andrianopoulos and Timberlake 1991; Anbanandam et al. 2006).
Previous studies have described other connections between
TOR and MAPK pathways. In S. cerevisiae, the TORC1 pathway is connected to the PKC-Mpk1 MAPK pathway (Angeles
De La Torre-Ruiz et al. 2002; Kuranda et al. 2006), and TOR
and stress MAPK pathways are linked in Schizosaccharomyces pombe (Petersen and Nurse 2007). In both cases, however, the connecting mechanisms have not been uncovered.
With Tec1, we provide a precise molecular link between the
TORC1 and Fus3/Kss1 MAPK pathways in S. cerevisiae. Our
study further opens the possibility that in higher eukaryotes,
TEAD transcription factors are not only regulated by PKA
(Gupta et al. 2000), PKC (Jiang et al. 2001), and MAPK
signaling pathways (Ambrosino et al. 2006), but also by
Tec1 Links TOR and MAPK Pathways
489
Figure 8 Regulation of Tec1-target genes by rapamycin. (A) FLO11 and
CLN1 transcript levels were determined in yeast strains RH2754 (TEC1)
and RH2500 (tec1Δ) 70 min after addition of rapamycin (+) or drug
vehicle (2) to exponentially growing cultures by Northern blot analysis.
FLO11 norm and CLN1 norm indicate relative transcript levels obtained by
normalization to ACT1 levels. (B) Effect of rapamycin on FLO11 and CLN1
transcript levels was analyzed in yeast strain RH2500 carrying TEC1
(pME2068), TEC1T273M (pME2102), or TEC1AxY (BHUM1306) on plasmid.
Figure 7 Degradation of Tec1 and Tec1 variants in response to rapamycin or pheromone. (A) Rapamycin-induced degradation of different Tec1
variants was analyzed in yeast strain RH2500 carrying TEC1 (pME2068),
TEC1T273M (pME2102), TEC1P274S (pME2096), TEC1K54A (BHUM1122), or
TEC1AxY (BHUM1306) on plasmids. (B) Pheromone-induced degradation
of different Tec1 variants was analyzed in yeast strain RH2500 carrying
TEC1, TEC1AxY, or TEC1T273M grown for 70 min in the absence (2) or
presence (+) of yeast alpha factor. Numbers indicate relative Tec1 levels
(Tec1 norm) and were obtained by normalizing Tec1 to Cdc28 signals. (C)
Pheromone-induced degradation of Tec1 in yeast strains YHUM1104
(control) and YHUM1341 (rsp5Δ).
TORC1. This would hook up TEAD-controlled eukaryotic development to metabolic stimuli such as amino acids, which
regulate mTORC1 in mammals (Polak and Hall 2009).
Our data further provide insights into the physiological
relevance of the TORC1-Tec1-MAPK connection in S. cerevisiae. Previous studies have shown that Tec1 is a positive regulator of the G1 cyclin CLN1 and the vegetative adhesin
FLO11 (Madhani et al. 1999), that mating pheromone inhibits
CLN1 and FLO11 expression (Brückner et al. 2004), and that
rapamycin inhibits CLN1 expression and FLO11-dependent
yeast filamentous growth (Barbet et al. 1996; Cutler et al.
2001; Zinzalla et al. 2007). Moreover, Tec1 protein stability
is reduced by the MAPK Fus3 in response to mating pheromone (Bao et al. 2004; Brückner et al. 2004; Chou et al.
2004). Thus, our finding that rapamycin negatively regulates Tec1 protein stability implies that active TORC1, in
contrast to the pheromone-activated MAPK, positively controls cell division and vegetative adhesion by targeting Tec1
(Figure 10). Our data also show that TORC1 seems to regulate the Tec1-target genes CLN1 and FLO11 by different
mechanisms. In the case of CLN1, expression is activated
by TORC1 and correlates with Tec1 protein levels. This indicates that TORC1 regulates the promoter by simply varying
Tec1 amounts. In contrast to CLN1, the much more complex
FLO11 promoter is repressed by TORC1. In addition, FLO11
expression does not correlate to varying Tec1 levels, but is
490
S. Brückner et al.
strongly reduced in the complete absence of the transcription factor. More than one model might explain these observations. (i) The Tec1 protein amounts required for efficient
FLO11 activation might be significantly lower than in the
case of CLN1, explaining why rapamycin-induced reduction
of Tec1 is not sufficient to block FLO11 expression. (ii)
TORC1 might control not only Tec1 stability, but also its
transcriptional activity. In this case, rapamycin treatment
would lead to lower Tec1 protein amounts, but also induce
its activity at the FLO11, but not at the CLN1 promoter. (iii)
TORC1 might also regulate the FLO11 promoter by activating a repressor, whose function requires only minimal
amounts of Tec1. Via this repressor, TORC1 would be able
to control FLO11 at low and high Tec1 amounts. In the
complete absence of Tec1, however, efficient FLO11 expression and its regulation by TORC1 would be lost, due to low
repressor concentrations.
We have also uncovered that Tec1 affects yeast chronological lifespan, a process that is regulated by rapamycin and
TORC1 (Powers et al. 2006). Here we found that absence of
Tec1 causes a significantly shortenend CLS, but does not
block CLS extension by rapamycin. This indicates that
TORC1 is able to control CLS by Tec1-independent mechanisms, which might involve other factors that positively control yeast lifespan (Wei et al. 2008). It will be interesting to
see how Tec1 affects CLS and whether Tec1 is required
before and/or after entry into stationary phase. Remarkably,
both the complete absence of Tec1 and the presence of
a rapamycin-resistant variant of the transcription factor result in a similar CLS phenotype. This indicates that Tec1
might act at several stages and that its activity must be
accurately regulated to achieve maximal CLS.
Our study shows that rapamycin-induced degradation of
Tec1 is independent of the known mechanisms that confer
pheromone-induced destabilization, but depends on (i)
specific elements of the TORC1 signaling pathway, namely
Sit4, Tip41, Sap190, and Npr1; (ii) the HECT-ubiquitin
ligase Rsp5; and (iii) three conserved PxY motives in the
Figure 9 Requirement of TEC1
for rapamycin-induced extension
of yeast chronological lifespan.
Survival (CFU ml21) was assessed
in the absence (open bars) or
presence (solid bars) of rapamycin after 8, 17, and 20 days in
yeast strains expressing TEC1,
no TEC1 (tec1Δ), or TEC1AxY. Bars
depict relative values expressed in
percentage of the TEC1-expressing
strain.
C-terminal part of Tec1 that are required for physical interaction with Rsp5. An important question is how these components could work together to confer degradation of Tec1
in response to rapamycin. Previous studies have shown that
Tip41 controls activity of Sit4 via interaction with Tap42
(Jacinto et al. 2001), that Sit4 can associate with Sap190
(Luke et al. 1996), and that regulation of Npr1 by TORC1
depends on Sit4 (Schmidt et al. 1998). Thus, our data are
compatible with a model, in which TORC1 regulates Tec1
protein stability via control of Sit4/Sap190 and Npr1 (Figure 10). However, Sit4 and Npr1 might not act in a strict
linear way, because we found that Tec1 degradation is only
partially blocked in npr1Δ mutants, but is fully inhibited in
the absence of SIT4. In addition, regulation of Tec1 by
TORC1 and nutrients might be more complex, because we
found that nitrogen deprivation leads to increased levels of
the transcription factor, even in the absence of Sit4 and
Rsp5. Here, future detailed studies are required to determine how different nutrient signals and responsive pathways control Tec1 stability.
Although we cannot exclude an indirect effect of Rsp5 on
Tec1 stability, our finding that the transcription factor physically interacts with the ligase via three PxY motives suggests that Rsp5 acts by directly ubiquitylating Tec1.
Similarly, single PxY motifs located in the C terminus of
the homologous S. cerevisiae transcription factors Spt23
and Mga2 have been shown to be sufficient to mediate physical and functional interaction with Rsp5 (Shcherbik et al.
2004). Surprisingly, however, we found that the interaction
between Rsp5 and Tec1 appears to be constitutive rather
than regulated by rapamycin. In addition, the mechanism
of Tec1 degradation remains to be established. Rsp5 is
known for monoubiquitylation as well as polyubiquitylation
via K63-linked chains and contributes to the modification of
membrane-associated as well as soluble nuclear proteins
(Rotin and Kumar 2009). Downstream processing of its
targets may involve the 26S proteasome, but proteasomeindependent degradation pathways are also well known.
Hence, given the variety of mechanisms by which Rsp5 controls protein stability, regulation of Tec1 by Rsp5 might be
much more complicated than anticipated and will have to be
addressed in detail in the future.
In summary, our study has identified a member of the
highly conserved TEA domain family of eukaryotic develop-
mental regulators as a common target for MAPK and TOR
signaling pathways, which control this type of transcription
factor at the level of protein stability by independent
mechanisms. Complex regulation of transcription factor
stabilization by post-translational modifications is an important theme in eukaryotic cell growth and development with
implications for disease control (Desterro et al. 2000; Hay
2006). A prominent example is the tumor suppressor p53,
a transcription factor whose stability is regulated by multiple
proteins including several ubiquitin ligases (Lavin and
Gueven 2006). Modulation of p53 degradation is now becoming a promising therapeutic approach (Dey et al. 2008).
Thus, future studies directed at the stability control of the
TEA domain regulator Tec1 in S. cerevisiae may contribute to
a more precise understanding of the molecular mechanisms
underlying the complex regulation of transcription factors
during cellular development.
Acknowledgments
We thank Bruno Andre, Kim Arndt, Gerhard Braus, Roger
Brent, Claudio DeVirgilio, Erica Golemis, Satoshi Harashima, Stefan Irniger, Yona Kassir, Hiten Madhani, and
Raffael Schaffrath for generous gifts of plasmids and strains.
We are grateful to Diana Kruhl for technical assistance. This
Figure 10 Model for the control of different yeast developmental programs by the Fus3/Kss1 MAPK cascade, the TORC1 pathway, and by
Tec1. For details see text.
Tec1 Links TOR and MAPK Pathways
491
work was supported by grants from the Deutsche Forschungsgemeinschaft, DFG MO 825/1-4 and GRK 1216, by
the International Max Planck Research School for Environmental, Cellular and Molecular Microbiology, and by the
Marburg Center for Synthetic Microbiology.
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Communicating editor: M. Hampsey
GENETICS
Supporting Information
http://www.genetics.org/content/suppl/2011/08/12/genetics.111.133629.DC1
The TEA Transcription Factor Tec1 Links TOR
and MAPK Pathways to Coordinate Yeast Development
Stefan Brückner, Sandra Kern, Raphael Birke, Irene Saugar,
Helle D. Ulrich, and Hans-Ulrich Mösch
Copyright © 2011 by the Genetics Society of America
DOI: 10.1534/genetics.111.133629
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