Inhibition of TOR signalling in lea1 mutant induces apoptosis in Saccharomyces cerevisiae

  • Pavan Kumar
  • Debasree Kundu
  • Alok K. Mondal
  • Vikrant Nain
  • Rekha PuriaEmail author
Original Article


The target of rapamycin, TOR, maintains cell growth and proliferation under vivid environmental conditions by orchestrating wide array of growth-related process. In addition to environmental conditions, e.g., nutrient and stress, TOR also governs cellular response to varied intracellular cues including perturbed intracellular mRNA levels which may arise due to altered regulation of mRNA processing at splicing or turnover levels. The purpose of this study is to explore the role of TOR signalling in growth of cells with accumulated unprocessed RNA. Growth analysis of lea1∆ (splicing deficient) was carried out under varied conditions leading to nitrogen starvation. The expression of TORC1 and TORC2 marker genes was examined in this delete strain. Sensitivity of the lea1∆ towards oxidative agents was observed. Apoptosis was analyzed in caffeine-treated lea1∆ cells. The hypersensitivity of lea1∆ cells towards caffeine is outcome of highly perturbed TOR signalling. The growth defect is independent of PKC pathway. Cells with accumulated unprocessed RNA experience high oxidative stress that induces apoptosis. An inadequate TOR signalling in lea1∆ cells substantiates the effect of oxidative stress induced by accumulated RNA to the extent of inducing cell death via apoptosis.


RNA accumulation Oxidative stress Reactive oxygen species Caffeine Splicing 



Initial work contribution of Shubhi Sahni is highly acknowledged. We are highly thankful to Dr. Maria E. Cardenas for providing us Jk9-3d strain.

Author contribution

This study was designed by PK and RP. Experiments were performed by PK and analyzed along with VN, DK, AKM, and RP. PK and RP wrote the manuscript. PK completed all the figures. All the results and final version of manuscript were reviewed by all the authors.


This work was supported by research grant to RP from SERB, Department of Science and Technology, Govt. of India (grant no. SR/FT/LS-93/2010). Pavan is thankful to SERB for fellowship.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals (if applicable)


Informed consent



  1. Adami A, García-Alvarez B, Arias-Palomo E et al (2007) Structure of TOR and its complex with KOG1. Mol Cell 27:509–516. CrossRefPubMedGoogle Scholar
  2. Albig AR, Decker CJ (2001) The target of rapamycin signaling pathway regulates mRNA turnover in the yeast Saccharomyces cerevisiae. Mol Biol Cell 12:3428–34383CrossRefGoogle Scholar
  3. Almeida B, Ohlmeier S, Almeida AJ et al (2009) Yeast protein expression profile during acetic acid-induced apoptosis indicates causal involvement of the TOR pathway. Proteomics 9:720–732. CrossRefPubMedGoogle Scholar
  4. Beck T, Schmidt A, Hall MN (1999) Starvation induces vacuolar targeting and degradation of the tryptophan permease in yeast. J Cell Biol 146:1227–1238CrossRefGoogle Scholar
  5. Bergkessel M, Whitworth GB, Guthrie C (2011) Diverse environmental stresses elicit distinct responses at the level of pre-mRNA processing in yeast. RNA 17:1461–1478. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bonawitz ND, Chatenay-Lapointe M, Pan Y, Shadel GS (2007) Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression. Cell Metab 5:265–277. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cardenas ME, Cutler NS, Lorenz MC et al (1999) The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev 13:3271–3279. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Caspary F, Séraphin B (1998) The yeast U2A’/U2B complex is required for pre-spliceosome formation. EMBO J 17:6348–6358. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Crespo JL, Powers T, Fowler B, Hall MN (2002) The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine. Proc Natl Acad Sci U S A 99:6784–6789. CrossRefPubMedPubMedCentralGoogle Scholar
  10. De Virgilio C, Loewith R (2006) The TOR signalling network from yeast to man. Int J Biochem Cell Biol 38:1476–1481. CrossRefPubMedGoogle Scholar
  11. Dreumont N, Séraphin B (2013) Rapid screening of yeast mutants with reporters identifies new splicing phenotypes. FEBS J 280:2712–2726. CrossRefPubMedGoogle Scholar
  12. Fadri M, Daquinag A, Wang S et al (2005) The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2. Mol Biol Cell 16:1883–1900. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Farrugia G, Balzan R (2012) Oxidative stress and programmed cell death in yeast. Front Oncol 2:64. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Fukai T, Ushio-Fukai M (2011) Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal 15:1583–1606. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Gaubitz C, Oliveira T, Prouteau M et al (2015) Molecular basis of the rapamycin insensitivity of target of rapamycin complex 2. Mol Cell 58:977–988. CrossRefPubMedGoogle Scholar
  16. Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360. CrossRefPubMedGoogle Scholar
  17. He Y, Li D, Cook SL et al (2013) Mammalian target of rapamycin and Rictor control neutrophil chemotaxis by regulating Rac/Cdc42 activity and the actin cytoskeleton. Mol Biol Cell 24:3369–3380. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Heintz C, Doktor TK, Lanjuin A et al (2016) Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans. Nature 541:102–106. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Heitman J, Movva NR, Hall MN (1991a) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253:905–909CrossRefGoogle Scholar
  20. Heitman J, Movva NR, Hiestand PC, Hall MN (1991b) FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 88:1948–1952CrossRefGoogle Scholar
  21. Inoki K, Ouyang H, Li Y, Guan K-L (2005) Signaling by target of rapamycin proteins in cell growth control. Microbiol Mol Biol Rev 69:79–100. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Jacinto E, Loewith R, Schmidt A et al (2004) Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 6:1122–1128. CrossRefPubMedGoogle Scholar
  23. Kamada Y, Fujioka Y, Suzuki NN et al (2005) Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization. Mol Cell Biol 25:7239–7248. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Kingsbury JM, Cardenas ME (2016) Vesicular trafficking systems impact TORC1-controlled transcriptional programs in Saccharomyces cerevisiae. G3(Bethesda) 6:641–652. CrossRefGoogle Scholar
  25. Kuranda K, Leberre V, Sokol S et al (2006) Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. Mol Microbiol 61:1147–1166. CrossRefPubMedGoogle Scholar
  26. Lempiäinen H, Uotila A, Urban J et al (2009) Sfp1 interaction with TORC1 and Mrs6 reveals feedback regulation on TOR signaling. Mol Cell 33:704–716. CrossRefPubMedGoogle Scholar
  27. Liang Q, Li W, Zhou B (2008) Caspase-independent apoptosis in yeast. Biochim Biophys Acta Mol Cell Res 1783:1311–1319. CrossRefGoogle Scholar
  28. Loewith R, Hall MN (2011) Target of rapamycin (TOR) in nutrient signaling and growth control. Genetics 189:1177–1201. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Loewith R, Jacinto E, Wullschleger S et al (2002) Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell.
  30. Madeo F, Fröhlich E, Fröhlich KU (1997) A yeast mutant showing diagnostic markers of early and late apoptosis. J Cell Biol 139:729–734. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Martin SG, Arkowitz RA (2014) Cell polarization in budding and fission yeasts. FEMS Microbiol Rev 38:228–253. CrossRefPubMedGoogle Scholar
  32. Mazzoni C, Falcone C (2011) mRNA stability and control of cell proliferation: figure 1. Biochem Soc Trans 39:1461–1465. CrossRefPubMedGoogle Scholar
  33. Mazzoni C, Herker E, Palermo V et al (2005) Yeast caspase 1 links messenger RNA stability to apoptosis in yeast. EMBO Rep 6:1076–1081. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Mazzoni C, Mancini P, Verdone L et al (2003) A truncated form of KlLsm4p and the absence of factors involved in mRNA decapping trigger apoptosis in yeast. Mol Biol Cell 14:721–729. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Morita M, Gravel S-P, Hulea L et al (2015) mTOR coordinates protein synthesis, mitochondrial activity and proliferation. Cell Cycle 14:473–480. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Müller J, Mettbach U, Menzel D, Samaj J (2007) Molecular dissection of endosomal compartments in plants. Plant Physiol 145:293–304. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Munchel SE, Shultzaberger RK, Takizawa N, Weis K (2011) Dynamic profiling of mRNA turnover reveals gene-specific and system-wide regulation of mRNA decay. Mol Biol Cell 22:2787–2795. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Niles BJ, Powers T (2014) TOR complex 2-Ypk1 signaling regulates actin polarization via reactive oxygen species. Mol Biol Cell 25:3962–3972. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Ocampo A, Barrientos A (2011) Quick and reliable assessment of chronological life span in yeast cell populations by flow cytometry. Mech Ageing Dev 132:315–323. CrossRefPubMedGoogle Scholar
  40. Pereira C, Bessa C, Saraiva L (2012) Endocytosis inhibition during H 2 O 2 -induced apoptosis in yeast. FEMS Yeast Res 12:755–760. CrossRefPubMedGoogle Scholar
  41. Powers T, Walter P (1999) Regulation of ribosome biogenesis by the rapamycin-sensitive TOR-signaling pathway in Saccharomyces cerevisiae. Mol Biol Cell 10:987–1000CrossRefGoogle Scholar
  42. Puria R, Zurita-Martinez SA, Cardenas ME (2008) Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae. Proc Natl Acad Sci 105:7194–7199. CrossRefPubMedGoogle Scholar
  43. Raju KK, Natarajan S, Kumar NS et al (2015) Role of cytoplasmic deadenylation and mRNA decay factors in yeast apoptosis. FEMS Yeast Res 15.
  44. Reinke A, Anderson S, McCaffery JM et al (2004) TOR complex 1 includes a novel component, Tco89p (YPL180w), and cooperates with Ssd1p to maintain cellular integrity in Saccharomyces cerevisiae. J Biol Chem 279:14752–14762. CrossRefPubMedGoogle Scholar
  45. Sariki SK, Sahu PK, Golla U et al (2016) Sen1, the homolog of human Senataxin, is critical for cell survival through regulation of redox homeostasis, mitochondrial function, and the TOR pathway in Saccharomyces cerevisiae. FEBS J 283:4056–4083. CrossRefPubMedGoogle Scholar
  46. Siebel CW, Feng L, Guthrie C, Fu XD (1999) Conservation in budding yeast of a kinase specific for SR splicing factors. Proc Natl Acad Sci U S A 96:5440–5445CrossRefGoogle Scholar
  47. Stanfel MN, Shamieh LS, Kaeberlein M, Kennedy BK (2009) The TOR pathway comes of age. Biochim Biophys Acta Gen Subj 1790:1067–1074. CrossRefGoogle Scholar
  48. Urban J, Soulard A, Huber A et al (2007) Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Mol Cell 26:663–674. CrossRefPubMedGoogle Scholar
  49. Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128:779–792CrossRefGoogle Scholar
  50. Wanke V, Cameroni E, Uotila A et al (2008) Caffeine extends yeast lifespan by targeting TORC1. Mol Microbiol 69:277–285. CrossRefPubMedGoogle Scholar
  51. Wullschleger S, Loewith R, Hall MN (2006) TOR signaling in growth and metabolism. Cell 124:471–484. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Zhang N, Fan Y, Li C et al (2018) Cell permeability and nuclear DNA staining by propidium iodide in basidiomycetous yeasts. Appl Microbiol Biotechnol 102:4183–4191. CrossRefPubMedGoogle Scholar
  53. Zurita-Martinez SA, Puria R, Pan X et al (2007) Efficient Tor signaling requires a functional class C Vps protein complex in Saccharomyces cerevisiae. Genetics 176:2139–2150. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Università degli studi di Milano 2019

Authors and Affiliations

  • Pavan Kumar
    • 1
  • Debasree Kundu
    • 2
  • Alok K. Mondal
    • 2
  • Vikrant Nain
    • 1
  • Rekha Puria
    • 1
    Email author
  1. 1.School of BiotechnologyGautam Buddha UniversityGreater NoidaIndia
  2. 2.School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia

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