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TOR signaling pathway plays cascades
in associated with six nutrient sensing pathways
in Saccharomyces cerevisiae
Little eukaryotes make big contributions
Bingyan Wang
5/11/2010
Background / Tor, Rapamycin and Nutrients
• The yeast Saccharomyces cerevisiae senses and responds to nutrients by
adapting growth rate and morphogenic transitions
• TOR pathway is a major integrator of nutrient-derived signals in cell growth
• TOR = Target of Rapamycin, originally identified by mutations in yeast that
confer resistance to rapamycin
• Cells treated with rapamycin results in dramatic physiological changes
– G1 cell cycle arrest
– Protein synthesis inhibition
– Glycogen accumulation
– Autophagy
• Using rapamycin treatment to mimic nutrients starvation becomes a
convenient way in closely resembling cells with nutrients limit/starvation
Ref: Rohde 2008, review
• Introduction
– Tor protein structure and function
– Protein localization
– Two complexes of Tor
• Nutrient sensing pathways in associated with Tor
– 6 pathways in associate with Tor pathway cascade
– Amino acid, nitrogen, glucose, glutamine
• Summary / Conclusion
Introduction - TOR structure and functions
• TOR contains > = 20 tandem HEAT repeats, a motif to mediate proteinprotein interactions
• FATC domain: essential to couple intracellular redox potential to TOR stability
• FAT domain: FKBP12-rapamycin-binding domain (FRB)
• Kinase domain: phosphorylation
Ref: Virgilio 2006, review
Introduction - Localization
• FM4-64:
Vacuolar membrane marker
 Tor1: vacuolar membrane
• Sec7: trans-Golgi marker
• FYVE: early endosome marker
 Tor 2: plasma membrane
Ref: Sturgill 2008
Introduction: TOR Complexes
• Two complexes TORC1 and TORC2:
– TORC1: activated by nutrient cues and inhibited by rapamycin
– TORC2: insensitive to rapamycin, regulates actin polarization
• LST8 associated with both TORC1 and TORC2
• KOG1 contains 4x internal HEAT repeats
 Each complex mediates distinct physiological processes in response to
nutrient cues.
Assay: silver stain
Ref: Loewith 2002, Rohde 2008
TORC response to rapamycin
• Target of Rapamycin: TORC1 and/or TORC2?
• Rapamycin does not affect TORC integrity
(data not shown)
• FPR1-TAP pulled down TOR1, TOR2, KOG1, LST8,
but failure in AVO1, AVO2, AVO3
TORC1
FPR1: codes for rapamycin intracellular receptor
 Rapamycin binds TORC1 (model A and B) but not TORC2
Assay: IP
Ref: Loewith 2002
TORC1 is rapamycin sensitive
• Rap+, kog1 (tor1), tor1tor2 :
– swollen and expaned vacuole
– decreases in 35S met intake
• avo1 (tor2) :
– no significant change
• Rap+ and kog1 inhibits protein synthesis
 TORC1 but not TORC2 indeed mediates the rapamycin sensitive signallng.
Ref: Loewith 2002
TOR signaling in yeast – the big picture
Ref: Virgilio 2006, review
Signaling branches downstream of Torc1
• Nutrient sources
– Amino Acid, Glutamine
– Nitrogen
– Glucose
F
Glutamine
Ref: Ashe 2000, Crespo 2002
A. Gcn2 and eIF2 under General amino acid control (GAAC)
Hypothesis
Under amino acid starvation, deletion of Gcn2
will activate translation and restore cell growth
by phosphorylating eIF2α
•
General amino acid control (GAAC) is a major effector of
the TOR pathway
SAP
•
In yeast, Gcn2 is activated at amino acid starvation, which
in turn phosphorylates eIF2α and inhibit translation
•
Sit4, a key phosphatase in Tor pathway
•
Target genes:
Gcn2, Sit4, Sap, eIF2α
Ref: Ashe 2000
Gcn2 is required for eIF2 phosphorylation
• In response to amino acid
starvation, Gcn2 kinase is to
phosphorylate eIF2α and inhibit
translation
• Under rapa+
– all Sap increases p- eIF2α
– Gcn2 blocks phosphorylation
– Sit4 shows no effects
GCN2
p-eIF2
rap
translation
TOR
SAP
Assay: WB
Ref: Rodhe 2004
Gcn2 inhibits S.c. growth at nutrient starvation
• At good nutrient condition:
– Gcn2 has no effect
(gcn2 only activated at nutrient starvation)
– Sap185 sap190 inhibit growth
(Sap activate amino acid synthesis)
• At rapamycin (starvation):
GCN2
p-eIF2
rap
– Gcn2 increases rapamycnin resistance in
comparison to WT
(gcn2 blocks translation at nutrient
starvation)
– Sap185 sap190 inhibit growth
– Sap185 sap190 can be rescued by gcn2
translation
TOR
SAP
Assay: Serial dilution
Ref: Rodhe 2004
B. Tap42/Sit4 complex
• Good nutrient conditions:
– TOR interact with TAP42
– TAP42 binds to SIT4,
inactivated
– NPR1 maintains
phosphorylated
• Nutrient limitation or
rapamycin inhibition
Objective
To demonstrate TOR is required for TAP42/SIT4
association
– TOR – TAP42 interaction
is inhibited
– SIT4 released from
TAP42, activated
– dephosphorylates NRP1
– Regulating gene
expression
Ref: Ashe 2000, Bonenfant 2002
Tap42 associates with TORC1
E
F
• Tor1 and Tor2 associate
with Tap42 with the
membrane fraction
• Tap42 physically
• At rap+, Tap42associated with TORC1
TORC1 is association
but not TORC2
disrupted
 Tap42 physically interact with TORC1, with a rapamycin sensitive manner
S100: soluble fraction
P100: membrane fraction
Assay: IB, IP
Ref: Yan 2006
Nutrient starvation disassembles the complex
YD  H2O
TORC1, model B
•
•
Tor2, Sit4 and Pph21 interaction with Tap42 was disrupted in response to nutrient
shift
Tap42-Sit4 complexes disassemble after their release from TORC1
 Nutrient starvation causes a rapid release of the TAP42 phosphatase complex
from TORC1 (shown with Tor2)
Assay: IB, Co-IP
Ref: Yan 2006
Nutrient starvation disassembles the complex
E: Exponentially
S: Stationary
YP: Rich
Glu: YP-glucose
SC: Synthetic complete
SD: Minimal
GE: Glycerol/Ethanol
• Tap42/Sit4 complex forms in
exponentially growing cells
• Tap42/Sit4 complex is glucose
dependent
• Stationary cells refed by nutrients
• Tap42/Sit4 can be restored at good
nutrient condition but not rap+
 Tor signally pathway is required for Tap42 to associate with Sit4.
Assay: IP
Ref: Di Como 1996
C. Snf1 kinase complex
Hypothesis
Snf1 is activated at glucose/nitrogen limit
conditions, therefore Snf1 is negatively
regulated by Tor
• Snf1 plays a direct role in glucose signaling, for
transcriptional and metabolic adaptation to
glucose starvation
• Snf1 is required for transcription of glucoserepressed genes
Ref: Ashe 2000
SNF1 phosphorylation is required
•
•
•
SLAD plates: Solid synthetic low-ammonia
HA-Snf1 restored PH development
HA-Snf1 mutant showed no phenotypic
improvement (similar the deletion vector)
 PH differentiation requires Snf1-Thr210
phorphorylation.
• Glucose abundant (2%)
• Nitrogen rich or limit
• P-Snf1 increase when
Nitrogen-limit
• T210A mutant showed no
phosphorylation
 Nitrogen limitation improves
Thr210 phosphorylation
Assay: IB
Ref: Orlova 2006
TOR negatively regulates Snf1
Rap
TOR
P-Snf1
•
Rapamycin treatment resulted in a significant improvement of T210
phosphorylation (TOR inhibited)
•
•
RR – Rapamycin resistant (Tor1-S1972R mutant)
Detectable increase within 30min to rap treatment
 Rapamycin-sensitive TOR negatively regulates Snf1.
Assay: IB
Ref: Orlova 2006
D, Hexose transporter (HXT)
Hypothesis
HXT1 is activated at high glucose, therefore
TOR pathway up-regulates HXT1 expression
• HXT1, a gene encoding a Saccharomyces cerevisiae
low-affinity glucose transporter, is regulated by
glucose availability
• HXT is activated only at high glucose, and is inhibited
at glucose starvation
TOR1 regulates HXT1 expression
• Rapamycin treatment at glucose pulse
• HXT1 induction inhibited by Rap+
• Tor1-1 mutant can partially restore
HXT1 induction
Rap
TOR
HXT1
Glu
• Control: GAP1, promoter of LacZ
• Greatly induced by rapamycin
• HXT1 induction by glucose is specific,
not related to a general rapamycin
Txp/Tsl defect
 TOR pathway is actively involved in the induction of expression of HXT1 by glucose
Ref: Tomas-Cobos 2005
E, Sch9 is a target of Torc1
Hypothesis
Torc1 and its kinase activity is required for Sch9
activity.
• Tor/Sch9 and the cAMP-PKA pathways often
function in parallel to regulate genes that are
required for entry in to cell cylce G0 phase.
• Sch9 is a substrate of yeast TORC1
• 6 amino acids in C-terminus of Sch9 are directly
phosphorylated by TORC1
• TORC1 is required for Sch9 activity
Ref: Ashe 2000
Sch9 is a major target of TORC1
TOR
•
•
P-Sch9
TORC1 phosphorylates Sch9
Phosphorylation is strongly diminished in Tor1 mutant, rap+, and sch9 mutants
 TORC1 is required for Sch9 phosphorylation
Ref: Urban 2007
Torc1 is required for Sch9 phosphorylation
•
•
•
Glucose substituted by Galactose
Sch9- or Sch9 mutant cannot grow
Simulation Sch9 3E and 2D3E
conffered a slight resistance to
rapamycin
•
Sch9 kinase activity from rap-treated
cells
Sch9 null mutants are inactivate
Simulation Sch9 highest avtivity
•
•
Rap
TOR
P-Sch9
Growth
 Sch9 function depends on Torc1 mediated phosphorylation
TORC1 is required for Sch9 phosphorylation
Assay: Serial dilution
Ref: Urban 2007
F, Regulation of TORC1 by glutamine, Gln3
F
Objective
Glutamine
Tor signaling pathway responses to glutamine
by regulating Gln3 activity
• Glutamine: a preferred nitrogen source and a key intermediate in yeast
nitrogen metabolism, possible regulator of Tor.
• TOR regulates a specific subset of proteins in response to glutamine.
• In the presence Glutamine, TOR keeps the transcription factors GLN3,
GAT1, RTG13, and MSN24 inactive.
• GLN3 is an activator of genes involved in ammonium assimilation.
Ref: Crespo 2002
Intracellular Glutamine Inhibits GLN3 via TOR Pathway
• MSX causes
• Gln1 and Mep2:
• Gat1: another Tor
glutamine depletion
Gln3 target genes
controlled transcription
• Rap and MSX causes • Induced in MSX
factor
dephosphorylation
treatment but not in gln3 • Growth is only inhibited
of Gln3
knock-out cells
in gln3 knock-outs
MSX
TOR
Rap
(Glutamine starvation)
GLN3
GAT1
MEP2
GLN1
 Glutamine inhibits activation of GLN3 through TOR pathway
Assay: IB, RT-PCR
Ref: Crespo 2002
Summary / Conclusions
•
Two complexes of Tor, TORC1 and TORC2, TORC1 is rapamycin sensitive
•
Rapamycin treatment provides a convenient tool in nutrient starvation research
•
Mechanism by which nutrient and stress signals are transmitted to Tor remains
unknown
•
TOR pathway branches in regulation of genes in associated with several nutrient
signal pathways
– Regulating Gcn2 phosphorylation of eIF2α and translation initiation [amino acid]
– Physically associating with Tap42/Sit4 protein complex [all nutrients]
– Negatively regulating Snf1 [nitrogen, glucose]
– Upregulating HXT1 induction [glucose]
– TORC1 is required for Sch9 phosphorylation [glucose]
– Inhibiting Gln3 and its downstream genes [glutamine, glucose]
Tor pathway cascades
F
Glutamine
TOR
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