Little eukaryotes make big contributions Bingyan Wang 5/11/2010

1. 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

2. Background / Tor, Rapamycin and Nutrients • The yeast Saccharomycescerevisiaesenses 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

3. 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

4. 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

5. 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

6. 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

7. 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: IP Ref: Loewith 2002

8. 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

9. TOR signaling in yeast – the big picture Ref: Virgilio 2006, review

10. Signaling branches downstream of Torc1 • Nutrient sources • Amino Acid, Glutamine • Nitrogen • Glucose F Glutamine Ref: Ashe 2000, Crespo2002

11. 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 • 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 SAP Ref: Ashe 2000

12. 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

13. 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 GCN2 p-eIF2 rap translation TOR SAP Assay: Serial dilution Ref: Rodhe 2004

14. B. Tap42/Sit4 complex • Good nutrient conditions: • TOR interact with TAP42 • TAP42 binds to SIT4, inactivated • NPR1 maintains phosphorylated • 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 Ref: Ashe 2000, Bonenfant 2002

15. Tap42 associates with TORC1 E • Tor1 and Tor2 associate with Tap42 with the membrane fraction F • 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 Assay: IB, IP Ref: Yan 2006 S100: soluble fraction P100: membrane fraction

16. 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) YD  H2O TORC1, model B Assay: IB, Co-IP Ref: Yan 2006

17. 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

18. 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 Ref: Ashe 2000

19. 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

20. 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. Rap TOR P-Snf1 Assay: IB Ref: Orlova 2006

21. 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

22. 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 Rap TOR HXT1 Glu  TOR pathway is actively involved in the induction of expression of HXT1 by glucose Ref: Tomas-Cobos 2005

23. 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. Ref: Ashe 2000

24. 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 TOR P-Sch9 Ref: Urban 2007

25. 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

26. 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. Objective Tor signaling pathway responses to glutamine by regulating Gln3 activity F Glutamine Ref: Crespo 2002

27. Intracellular Glutamine Inhibits GLN3 via TOR Pathway • MSX causes glutamine depletion • Rap and MSX causes dephosphorylation of Gln3 • 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 MSX Rap MEP2 GLN1 GLN3 GAT1 TOR (Glutamine starvation)  Glutamine inhibits activation of GLN3 through TOR pathway Assay: IB, RT-PCR Ref: Crespo 2002

28. 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]

29. Tor pathway cascades F Glutamine TOR

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