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Mol Cell Biol. 1997 August; 17(8): 4474–4489.
PMCID: PMC232301

Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2.


In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.

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Selected References

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  • Alani E, Cao L, Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987 Aug;116(4):541–545. [PubMed]
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed]
  • Anderson JT, Paddy MR, Swanson MS. PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Oct;13(10):6102–6113. [PMC free article] [PubMed]
  • Belfield GP, Ross-Smith NJ, Tuite MF. Translation elongation factor-3 (EF-3): an evolving eukaryotic ribosomal protein? J Mol Evol. 1995 Sep;41(3):376–387. [PubMed]
  • Berkower C, Michaelis S. Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily. EMBO J. 1991 Dec;10(12):3777–3785. [PubMed]
  • Boeke JD, LaCroute F, Fink GR. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984;197(2):345–346. [PubMed]
  • Boguski MS, McCormick F. Proteins regulating Ras and its relatives. Nature. 1993 Dec 16;366(6456):643–654. [PubMed]
  • Cutting GR, Kasch LM, Rosenstein BJ, Zielenski J, Tsui LC, Antonarakis SE, Kazazian HH., Jr A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein. Nature. 1990 Jul 26;346(6282):366–369. [PubMed]
  • Dasmahapatra B, Chakraburtty K. Protein synthesis in yeast. I. Purification and properties of elongation factor 3 from Saccharomyces cerevisiae. J Biol Chem. 1981 Oct 10;256(19):9999–10004. [PubMed]
  • Dever TE, Feng L, Wek RC, Cigan AM, Donahue TF, Hinnebusch AG. Phosphorylation of initiation factor 2 alpha by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell. 1992 Feb 7;68(3):585–596. [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Donahue TF, Daves RS, Lucchini G, Fink GR. A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast. Cell. 1983 Jan;32(1):89–98. [PubMed]
  • Durfee T, Becherer K, Chen PL, Yeh SH, Yang Y, Kilburn AE, Lee WH, Elledge SJ. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. [PubMed]
  • Fath MJ, Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. [PMC free article] [PubMed]
  • Feilotter HE, Hannon GJ, Ruddell CJ, Beach D. Construction of an improved host strain for two hybrid screening. Nucleic Acids Res. 1994 Apr 25;22(8):1502–1503. [PMC free article] [PubMed]
  • Feinberg B, McLaughlin CS, Moldave K. Analysis of temperature-sensitive mutant ts 187 of Saccharomyces cerevisiae altered in a component required for the initiation of protein synthesis. J Biol Chem. 1982 Sep 25;257(18):10846–10851. [PubMed]
  • Ames GF. Bacterial periplasmic permeases as model systems for the superfamily of traffic ATPases, including the multidrug resistance protein and the cystic fibrosis transmembrane conductance regulator. Int Rev Cytol. 1992;137:1–35. [PubMed]
  • Foiani M, Cigan AM, Paddon CJ, Harashima S, Hinnebusch AG. GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3203–3216. [PMC free article] [PubMed]
  • Gietz RD, Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. [PubMed]
  • Hannig EM, Williams NP, Wek RC, Hinnebusch AG. The translational activator GCN3 functions downstream from GCN1 and GCN2 in the regulatory pathway that couples GCN4 expression to amino acid availability in Saccharomyces cerevisiae. Genetics. 1990 Nov;126(3):549–562. [PubMed]
  • Hershey JW. Translational control in mammalian cells. Annu Rev Biochem. 1991;60:717–755. [PubMed]
  • Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. [PubMed]
  • Kamath A, Chakraburtty K. Role of yeast elongation factor 3 in the elongation cycle. J Biol Chem. 1989 Sep 15;264(26):15423–15428. [PubMed]
  • Kane PM, Kuehn MC, Howald-Stevenson I, Stevens TH. Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase. J Biol Chem. 1992 Jan 5;267(1):447–454. [PubMed]
  • Klionsky DJ, Herman PK, Emr SD. The fungal vacuole: composition, function, and biogenesis. Microbiol Rev. 1990 Sep;54(3):266–292. [PMC free article] [PubMed]
  • Koerner TJ, Hill JE, Myers AM, Tzagoloff A. High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. Methods Enzymol. 1991;194:477–490. [PubMed]
  • Lanker S, Bushman JL, Hinnebusch AG, Trachsel H, Mueller PP. Autoregulation of the yeast lysyl-tRNA synthetase gene GCD5/KRS1 by translational and transcriptional control mechanisms. Cell. 1992 Aug 21;70(4):647–657. [PubMed]
  • Lennon G, Auffray C, Polymeropoulos M, Soares MB. The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics. 1996 Apr 1;33(1):151–152. [PubMed]
  • Marton MJ, Crouch D, Hinnebusch AG. GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2. Mol Cell Biol. 1993 Jun;13(6):3541–3556. [PMC free article] [PubMed]
  • Naranda T, MacMillan SE, Hershey JW. Purified yeast translational initiation factor eIF-3 is an RNA-binding protein complex that contains the PRT1 protein. J Biol Chem. 1994 Dec 23;269(51):32286–32292. [PubMed]
  • Nichols CG, Shyng SL, Nestorowicz A, Glaser B, Clement JP, 4th, Gonzalez G, Aguilar-Bryan L, Permutt MA, Bryan J. Adenosine diphosphate as an intracellular regulator of insulin secretion. Science. 1996 Jun 21;272(5269):1785–1787. [PubMed]
  • Petitjean A, Bonneaud N, Lacroute F. The duplicated Saccharomyces cerevisiae gene SSM1 encodes a eucaryotic homolog of the eubacterial and archaebacterial L1 ribosomal proteins. Mol Cell Biol. 1995 Sep;15(9):5071–5081. [PMC free article] [PubMed]
  • Ramirez M, Wek RC, Hinnebusch AG. Ribosome association of GCN2 protein kinase, a translational activator of the GCN4 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):3027–3036. [PMC free article] [PubMed]
  • Richter D. Stringent factor from Escherichia coli directs ribosomal binding and release of uncharged tRNA. Proc Natl Acad Sci U S A. 1976 Mar;73(3):707–711. [PubMed]
  • Roberts CJ, Raymond CK, Yamashiro CT, Stevens TH. Methods for studying the yeast vacuole. Methods Enzymol. 1991;194:644–661. [PubMed]
  • Rolfes RJ, Zhang F, Hinnebusch AG. The transcriptional activators BAS1, BAS2, and ABF1 bind positive regulatory sites as the critical elements for adenine regulation of ADE5,7. J Biol Chem. 1997 May 16;272(20):13343–13354. [PubMed]
  • Sandbaken M, Lupisella JA, DiDomenico B, Chakraburtty K. Isolation and characterization of the structural gene encoding elongation factor 3. Biochim Biophys Acta. 1990 Aug 27;1050(1-3):230–234. [PubMed]
  • Sato T, Ohsumi Y, Anraku Y. An arginine/histidine exchange transport system in vacuolar-membrane vesicles of Saccharomyces cerevisiae. J Biol Chem. 1984 Sep 25;259(18):11509–11511. [PubMed]
  • Sato T, Ohsumi Y, Anraku Y. Substrate specificities of active transport systems for amino acids in vacuolar-membrane vesicles of Saccharomyces cerevisiae. Evidence of seven independent proton/amino acid antiport systems. J Biol Chem. 1984 Sep 25;259(18):11505–11508. [PubMed]
  • Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. [PubMed]
  • Skogerson L, Wakatama E. A ribosome-dependent GTPase from yeast distinct from elongation factor 2. Proc Natl Acad Sci U S A. 1976 Jan;73(1):73–76. [PubMed]
  • Szczypka MS, Wemmie JA, Moye-Rowley WS, Thiele DJ. A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein. J Biol Chem. 1994 Sep 9;269(36):22853–22857. [PubMed]
  • Triana-Alonso FJ, Chakraburtty K, Nierhaus KH. The elongation factor 3 unique in higher fungi and essential for protein biosynthesis is an E site factor. J Biol Chem. 1995 Sep 1;270(35):20473–20478. [PubMed]
  • Uritani M, Miyazaki M. Role of yeast peptide elongation factor 3 (EF-3) at the AA-tRNA binding step. J Biochem. 1988 Jul;104(1):118–126. [PubMed]
  • Vazquez de Aldana CR, Marton MJ, Hinnebusch AG. GCN20, a novel ATP binding cassette protein, and GCN1 reside in a complex that mediates activation of the eIF-2 alpha kinase GCN2 in amino acid-starved cells. EMBO J. 1995 Jul 3;14(13):3184–3199. [PubMed]
  • Wek RC, Jackson BM, Hinnebusch AG. Juxtaposition of domains homologous to protein kinases and histidyl-tRNA synthetases in GCN2 protein suggests a mechanism for coupling GCN4 expression to amino acid availability. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4579–4583. [PubMed]
  • Wek SA, Zhu S, Wek RC. The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids. Mol Cell Biol. 1995 Aug;15(8):4497–4506. [PMC free article] [PubMed]
  • Wemmie JA, Szczypka MS, Thiele DJ, Moye-Rowley WS. Cadmium tolerance mediated by the yeast AP-1 protein requires the presence of an ATP-binding cassette transporter-encoding gene, YCF1. J Biol Chem. 1994 Dec 23;269(51):32592–32597. [PubMed]
  • Yamashiro CT, Kane PM, Wolczyk DF, Preston RA, Stevens TH. Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase. Mol Cell Biol. 1990 Jul;10(7):3737–3749. [PMC free article] [PubMed]
  • Yon J, Fried M. Precise gene fusion by PCR. Nucleic Acids Res. 1989 Jun 26;17(12):4895–4895. [PMC free article] [PubMed]

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