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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