Molecular Genetics and Genomics

, Volume 280, Issue 3, pp 211–221

Synthetic lethality between eIF5A and Ypt1 reveals a connection between translation and the secretory pathway in yeast

  • Mariana C. Frigieri
  • Marcus V. S. João Luiz
  • Luciano H. Apponi
  • Cleslei F. Zanelli
  • Sandro R. Valentini
Original Paper


The putative translation initiation factor 5A (eIF5A) is a small protein, highly conserved and essential in all organisms from archaea to mammals. Although the involvement of eIF5A in translation initiation has been questioned, new evidence reestablished the connection between eIF5A and this cellular process. In order to better understand the function of elF5A, a screen for synthetic lethal gene using the tif51A-3 mutant was carried out and a new mutation (G80D) was found in the essential gene YPT1, encoding a protein involved in vesicular trafficking. The precursor form of the vacuolar protein CPY is accumulated in the ypt1-G80D mutant at the nonpermissive temperature, but this defect in vesicular trafficking did not occur in the tif51A mutants tested. Overexpression of eIF5A suppresses the growth defect of a series of ypt1 mutants, but this suppression does not restore correct CPY sorting. On the other hand, overexpression of YPT1 does not suppress the growth defect of tif51A mutants. Further, it was revealed that eIF-5A is present in both soluble and membrane fractions, and its membrane association is ribosome-dependent. Finally, we demonstrated that the ypt1 and other secretion pathway mutants are sensitive to paromomycin. These results confirm the link between translation and vesicular trafficking and reinforce the implication of eIF5A in protein synthesis.


eIF5A Ypt1 Synthetic lethality Genetic interaction Vesicular trafficking Protein synthesis 


  1. Alory C, Balch WE (2001) Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease. Traffic 2:532–543PubMedCrossRefGoogle Scholar
  2. Appling DR (1999) Genetic approaches to the study of protein–protein interactions. Methods 19:338–349PubMedCrossRefGoogle Scholar
  3. Bacon RA, Salminen A, Ruohola H, Novick P, Ferro-Novick S (1989) The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants. J Cell Biol 109:1015–1022PubMedCrossRefGoogle Scholar
  4. Benne R, Brown-Luedi ML, Hershey JW (1978) Purification and characterization of protein synthesis initiation factors eIF-1, eIF-4C, eIF-4D, and eIF-5 from rabbit reticulocytes. J Biol Chem 253:3070–3077PubMedGoogle Scholar
  5. Benne R, Hershey JW (1978) The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes. J Biol Chem 253:3078–3087PubMedGoogle Scholar
  6. Bevec D, Hauber J (1997) Eukaryotic initiation factor 5A activity and HIV-1 Rev function. Biol Signals 6:124–133PubMedCrossRefGoogle Scholar
  7. Bevec D et al (1996) Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A. Science 271:1858–1860PubMedCrossRefGoogle Scholar
  8. Cano VS et al (2008) Mutational analyses of human eIF5A-1—identification of amino acid residues critical for eIF5A activity and hypusine modification. FEBS J 275:44–58PubMedGoogle Scholar
  9. Chen KY, Liu AY (1997) Biochemistry and function of hypusine formation on eukaryotic initiation factor 5A. Biol Signals 6:105–109PubMedCrossRefGoogle Scholar
  10. Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P (1992) Multifunctional yeast high-copy-number shuttle vectors. Gene 110:119–122PubMedCrossRefGoogle Scholar
  11. De Antoni A, Schmitzova J, Trepte HH, Gallwitz D, Albert S (2002) Significance of GTP hydrolysis in Ypt1p-regulated endoplasmic reticulum to Golgi transport revealed by the analysis of two novel Ypt1-GAPs. J Biol Chem 277:41023–41031PubMedCrossRefGoogle Scholar
  12. Dias CA et al (2008) Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis. Febs JGoogle Scholar
  13. Du LL, Novick P (2001) Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p. Mol Biol Cell 12:1215–1226PubMedGoogle Scholar
  14. Finger FP, Novick P (2000) Synthetic interactions of the post-Golgi sec mutations of Saccharomyces cerevisiae. Genetics 156:943–951PubMedGoogle Scholar
  15. Frey S, Pool M, Seedorf M (2001) Scp160p, an RNA-binding, polysome-associated protein, localizes to the endoplasmic reticulum of Saccharomyces cerevisiae in a microtubule-dependent manner. J Biol Chem 276:15905–15912PubMedCrossRefGoogle Scholar
  16. Frigieri MC, Thompson GM, Pandolfi JR, Zanelli CF, Valentini SR (2007) Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae. Genet Mol Res 6:152–165PubMedGoogle Scholar
  17. Gilmore R (1991) The protein translocation apparatus of the rough endoplasmic reticulum, its associated proteins, and the mechanism of translocation. Curr Opin Cell Biol 3:580–584PubMedCrossRefGoogle Scholar
  18. Gonzalez A, Jimenez A, Vazquez D, Davies JE, Schindler D (1978) Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes. Biochim Biophys Acta 521:459–469PubMedGoogle Scholar
  19. Grigull J, Mnaimneh S, Pootoolal J, Robinson MD, Hughes TR (2004) Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors. Mol Cell Biol 24:5534–5547PubMedCrossRefGoogle Scholar
  20. Guthrie C, Fink GR (1991) Guide to yeast genetics and molecular biology. Academic Press, New York, p 194Google Scholar
  21. Hansen JL, Moore PB, Steitz TA (2003) Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit. J Mol Biol 330:1061–1075PubMedCrossRefGoogle Scholar
  22. Hausner TP, Geigenmuller U, Nierhaus KH (1988) The allosteric three-site model for the ribosomal elongation cycle. New insights into the inhibition mechanisms of aminoglycosides, thiostrepton, and viomycin. J Biol Chem 263:13103–13111PubMedGoogle Scholar
  23. Henderson BR, Percipalle P (1997) Interactions between HIV Rev and nuclear import and export factors: the Rev nuclear localisation signal mediates specific binding to human importin-beta. J Mol Biol 274:693–707PubMedCrossRefGoogle Scholar
  24. Hughes TR et al (2000) Functional discovery via a compendium of expression profiles. Cell 102:109–126PubMedCrossRefGoogle Scholar
  25. Jao DL, Chen KY (2006) Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex. J Cell Biochem 97:583–598PubMedCrossRefGoogle Scholar
  26. Jao DL, Yu Chen K (2002) Subcellular localization of the hypusine-containing eukaryotic initiation factor 5A by immunofluorescent staining and green fluorescent protein tagging. J Cell Biochem 86:590–600PubMedCrossRefGoogle Scholar
  27. Jedd G, Richardson C, Litt R, Segev N (1995) The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway. J Cell Biol 131:583–590PubMedCrossRefGoogle Scholar
  28. Jin BF et al (2003) Proteomic analysis of ubiquitin–proteasome effects: insight into the function of eukaryotic initiation factor 5A. Oncogene 22:4819–4830PubMedCrossRefGoogle Scholar
  29. Kang HA, Hershey JW (1994) Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae. J Biol Chem 269:3934–3940PubMedGoogle Scholar
  30. Kovac L, Nelson BD, Ernster L (1986) A method for determining the intracellular distribution of enzymes in yeast provides no evidence for the association of hexokinase with mitochondria. Biochem Biophys Res Commun 134:285–291PubMedCrossRefGoogle Scholar
  31. Levin DE (2005) Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 69:262–291PubMedCrossRefGoogle Scholar
  32. Lipowsky G et al (2000) Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes. EMBO J 19:4362–4371PubMedCrossRefGoogle Scholar
  33. Martinez O, Goud B (1998) Rab proteins. Biochim Biophys Acta 1404:101–112PubMedCrossRefGoogle Scholar
  34. Masurekar M, Palmer E, Ono BI, Wilhelm JM, Sherman F (1981) Misreading of the ribosomal suppressor SUP46 due to an altered 40S subunit in yeast. J Mol Biol 147:381–390PubMedCrossRefGoogle Scholar
  35. Obrig TG, Culp WJ, McKeehan WL, Hardesty B (1971) The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J Biol Chem 246:174–181PubMedGoogle Scholar
  36. Pruyne D, Legesse-Miller A, Gao L, Dong Y, Bretscher A (2004) Mechanisms of polarized growth and organelle segregation in yeast. Annu Rev Cell Dev Biol 20:559–591PubMedCrossRefGoogle Scholar
  37. Pylypenko O et al (2006) Structure of doubly prenylated Ypt1:GDI complex and the mechanism of GDI-mediated Rab recycling. EMBO J 25:13–23PubMedCrossRefGoogle Scholar
  38. Rao SS, Grollman AP (1967) Cycloheximide resistance in yeast: a property of the 60s ribosomal subunit. Biochem Biophys Res Commun 29:696–704PubMedCrossRefGoogle Scholar
  39. Ruhl M et al (1993) Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activation. J Cell Biol 123:1309–1320PubMedCrossRefGoogle Scholar
  40. Sandbaken MG, Culbertson MR (1988) Mutations in elongation factor EF-1 alpha affect the frequency of frameshifting and amino acid misincorporation in Saccharomyces cerevisiae. Genetics 120:923–934PubMedGoogle Scholar
  41. Sasaki K, Abid MR, Miyazaki M (1996) Deoxyhypusine synthase gene is essential for cell viability in the yeast Saccharomyces cerevisiae. FEBS Lett 384:151–154PubMedCrossRefGoogle Scholar
  42. Schnier J, Schwelberger HG, Smit-McBride Z, Kang HA, Hershey JW (1991) Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae. Mol Cell Biol 11:3105–3114PubMedGoogle Scholar
  43. Segev N (2001a) Ypt and Rab GTPases: insight into functions through novel interactions. Curr Opin Cell Biol 13:500–511PubMedCrossRefGoogle Scholar
  44. Segev N (2001b) Ypt/rab gtpases: regulators of protein trafficking. Sci STKE 2001:RE11Google Scholar
  45. Segev N, Botstein D (1987) The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response. Mol Cell Biol 7:2367–2377PubMedGoogle Scholar
  46. Segev N, Mulholland J, Botstein D (1988) The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell 52:915–924PubMedCrossRefGoogle Scholar
  47. Shi XP, Yin KC, Waxman L (1997) Effects of inhibitors of RNA and protein synthesis on the subcellular distribution of the eukaryotic translation initiation factor, eIF-5A, and the HIV-1 Rev protein. Biol Signals 6:143–149PubMedCrossRefGoogle Scholar
  48. Shi XP, Yin KC, Zimolo ZA, Stern AM, Waxman L (1996) The subcellular distribution of eukaryotic translation initiation factor, eIF-5A, in cultured cells. Exp Cell Res 225:348–356PubMedCrossRefGoogle Scholar
  49. Singh A, Ursic D, Davies J (1979) Phenotypic suppression and misreading Saccharomyces cerevisiae. Nature 277:146–148PubMedCrossRefGoogle Scholar
  50. Taylor CA et al (2007) Eukaryotic translation initiation factor 5A induces apoptosis in colon cancer cells and associates with the nucleus in response to tumour necrosis factor alpha signalling. Exp Cell Res 313:437–449PubMedCrossRefGoogle Scholar
  51. Valentini SR, Casolari JM, Oliveira CC, Silver PA, McBride AE (2002) Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling. Genetics 160:393–405PubMedGoogle Scholar
  52. Vida TA, Graham TR, Emr SD (1990) In vitro reconstitution of intercompartmental protein transport to the yeast vacuole. J Cell Biol 111:2871–2884PubMedCrossRefGoogle Scholar
  53. Wolff EC, Kang KR, Kim YS, Park MH (2007) Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification. Amino Acids 33:341–350PubMedCrossRefGoogle Scholar
  54. Zanelli CF et al (2006) eIF5A binds to translational machinery components and affects translation in yeast. Biochem Biophys Res Commun 348:1358–1366PubMedCrossRefGoogle Scholar
  55. Zanelli CF, Valentini S (2007) Is there a role for eIF5A in translation? Amino Acids 33:351–358PubMedCrossRefGoogle Scholar
  56. Zanelli CF, Valentini SR (2005) Pkc1 acts through Zds1 and Gic1 to suppress growth and cell polarity defects of a yeast eIF5A mutant. Genetics 171:1571–1581PubMedCrossRefGoogle Scholar
  57. Zuk D, Jacobson A (1998) A single amino acid substitution in yeast eIF-5A results in mRNA stabilization. EMBO J 17:2914–2925PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Mariana C. Frigieri
    • 1
  • Marcus V. S. João Luiz
    • 1
  • Luciano H. Apponi
    • 1
  • Cleslei F. Zanelli
    • 1
  • Sandro R. Valentini
    • 1
  1. 1.Department of Biological Sciences, School of Pharmaceutical SciencesSão Paulo State University, UNESPAraraquaraBrazil

Personalised recommendations