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Little eukaryotes make big contributions
Bingyan Wang5/11/2010
TOR signaling pathway plays cascades in associated with six nutrient sensing pathways in Saccharomyces cerevisiae
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 protein-
protein 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
Assay: silver stainRef: Loewith 2002, Rohde 2008
• 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.
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
FPR1: codes for rapamycin intracellular receptor
Rapamycin binds TORC1 (model A and B) but not TORC2
TORC1
Assay: IPRef: Loewith 2002
Ref: Loewith 2002
• 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.
TORC1 is rapamycin sensitive
TOR signaling in yeast – the big picture
Ref: Virgilio 2006, review
Signaling branches downstream of Torc1
• Nutrient sources– Amino Acid, Glutamine– Nitrogen– Glucose
FGlutamine
Ref: Ashe 2000, Crespo 2002
A. Gcn2 and eIF2 under General amino acid control (GAAC)
• General amino acid control (GAAC) is a major effector of
the TOR pathway
• 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
Hypothesis
Under amino acid starvation, deletion of Gcn2 will activate translation and restore cell growth by phosphorylating eIF2α
Hypothesis
Under amino acid starvation, deletion of Gcn2 will activate translation and restore cell growth by phosphorylating eIF2α
Ref: Ashe 2000
SAP
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
Assay: WBRef: Rodhe 2004
GCN2GCN2 p-eIF2p-eIF2
translationtranslation
SAPSAPTORTOR
raprap
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 increases rapamycnin resistance in
comparison to WT(gcn2 blocks translation at nutrient starvation)
– Sap185 sap190 inhibit growth– Sap185 sap190 can be rescued by gcn2
Assay: Serial dilutionRef: Rodhe 2004
GCN2GCN2 p-eIF2p-eIF2
translationtranslation
SAPSAPTORTOR
raprap
B. Tap42/Sit4 complex
• Good nutrient conditions:– TOR interact with TAP42– TAP42 binds to SIT4,
inactivated– NPR1 maintains
phosphorylated
Ref: Ashe 2000, Bonenfant 2002
• Nutrient limitation or rapamycin inhibition– TOR – TAP42 interaction
is inhibited– SIT4 released from
TAP42, activated– dephosphorylates NRP1– Regulating gene
expression
Objective
To demonstrate TOR is required for TAP42/SIT4 association
Objective
To demonstrate TOR is required for TAP42/SIT4 association
Tap42 associates with TORC1
• Tor1 and Tor2 associate with Tap42 with the membrane fraction
E
Assay: IB, IPRef: Yan 2006
• Tap42 physically associated with TORC1 but not TORC2
• At rap+, Tap42-TORC1 is association disrupted
Tap42 physically interact with TORC1, with a rapamycin sensitive manner
S100: soluble fractionP100: membrane fraction
F
Nutrient starvation disassembles the complex
• 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
TORC1, model B
YD H2O
Assay: IPRef: Di Como 1996
E: ExponentiallyS: Stationary
YP: Rich Glu: YP-glucoseSC: Synthetic completeSD: MinimalGE: 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.
Nutrient starvation disassembles the complex
C. Snf1 kinase complex
• Snf1 plays a direct role in glucose signaling, for transcriptional and metabolic adaptation to glucose starvation
• Snf1 is required for transcription of glucose-repressed genes
Hypothesis
Snf1 is activated at glucose/nitrogen limit conditions, therefore Snf1 is negatively regulated by Tor
Hypothesis
Snf1 is activated at glucose/nitrogen limit conditions, therefore Snf1 is negatively regulated by Tor
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.
Assay: IBRef: Orlova 2006
• Glucose abundant (2%)• Nitrogen rich or limit• P-Snf1 increase when
Nitrogen-limit • T210A mutant showed no
phosphorylation
Nitrogen limitation improves Thr210 phosphorylation
TOR negatively regulates 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
P-Snf1P-Snf1TORTORRapRap
D, Hexose transporter (HXT)
• 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
Hypothesis
HXT1 is activated at high glucose, therefore TOR pathway up-regulates HXT1 expression
Hypothesis
HXT1 is activated at high glucose, therefore TOR pathway up-regulates HXT1 expression
TOR1 regulates HXT1 expression
• Rapamycin treatment at glucose pulse• HXT1 induction inhibited by Rap+• Tor1-1 mutant can partially restore
HXT1 induction
• Control: GAP1, promoter of LacZ• Greatly induced by rapamycin • HXT1 induction by glucose is specific,
not related to a general rapamycin Txp/Tsl defect
Ref: Tomas-Cobos 2005
TOR pathway is actively involved in the induction of expression of HXT1 by glucose
HXT1HXT1TORTORRapRap
GluGlu
E, Sch9 is a target of Torc1
• 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
Hypothesis
Torc1 and its kinase activity is required for Sch9 activity.
Hypothesis
Torc1 and its kinase activity is required for Sch9 activity.
Ref: Ashe 2000
Sch9 is a major target of TORC1
• TORC1 phosphorylates Sch9• Phosphorylation is strongly diminished in Tor1 mutant, rap+, and sch9 mutants
TORC1 is required for Sch9 phosphorylation
Ref: Urban 2007
P-Sch9P-Sch9TORTOR
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
Assay: Serial dilutionRef: Urban 2007
P-Sch9P-Sch9TORTORRapRap GrowthGrowth
Sch9 function depends on Torc1 mediated phosphorylation TORC1 is required for Sch9 phosphorylation
F, Regulation of TORC1 by glutamine, Gln3
• 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
FGlutamine
Objective
Tor signaling pathway responses to glutamine by regulating Gln3 activity
Objective
Tor signaling pathway responses to glutamine by regulating Gln3 activity
Intracellular Glutamine Inhibits GLN3 via TOR Pathway
• MSX causes glutamine depletion
• Rap and MSX causes dephosphorylation of Gln3
Assay: IB, RT-PCRRef: Crespo 2002
GLN3GAT1GLN3GAT1
TORTORMSXRapMSXRap
MEP2GLN1MEP2GLN1
(Glutamine starvation)
• Gln1 and Mep2: Gln3 target genes
• Induced in MSX treatment but not in gln3 knock-out cells
• Gat1: another Tor controlled transcription factor
• Growth is only inhibited in gln3 knock-outs
Glutamine inhibits activation of GLN3 through TOR pathway
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
FGlutamine
TORTOR
References
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