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. 1994 May;14(5):3208–3222. doi: 10.1128/mcb.14.5.3208

Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation.

C R Vazquez de Aldana 1, A G Hinnebusch 1
PMCID: PMC358688  PMID: 8164676

Abstract

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Saccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in addition to reducing general protein synthesis. We isolated mutations in several unlinked genes that suppress the growth-inhibitory effect of eIF-2 alpha phosphorylation catalyzed by mutationally activated forms of GCN2. These suppressor mutations, affecting eIF-2 alpha and the essential subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the level of eIF-2 alpha phosphorylation in cells expressing the activated GCN2c kinase. Four GCD7 suppressors were shown to reduce the derepression of GCN4 translation in cells containing wild-type GCN2 under starvation conditions or in GCN2c strains. A fifth GCD7 allele, constructed in vitro by combining two of the GCD7 suppressors mutations, completely impaired the derepression of GCN4 translation, a phenotype characteristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammalian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the translational machinery had been rendered completely insensitive to phosphorylated eIF-2. None of the GCD7 mutations had any detrimental effect on cell growth under nonstarvation conditions, suggesting that recycling of eIF-2 occurs efficiently in the suppressor strains. We propose that GCD7 and GCD2 play important roles in the regulatory interaction between eIF-2 and eIF-2B and that the suppressor mutations we isolated in these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential catalytic function of eIF-2B in translation initiation.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abastado J. P., Miller P. F., Jackson B. M., Hinnebusch A. G. Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control. Mol Cell Biol. 1991 Jan;11(1):486–496. doi: 10.1128/mcb.11.1.486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  3. Bushman J. L., Asuru A. I., Matts R. L., Hinnebusch A. G. Evidence that GCD6 and GCD7, translational regulators of GCN4, are subunits of the guanine nucleotide exchange factor for eIF-2 in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Mar;13(3):1920–1932. doi: 10.1128/mcb.13.3.1920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bushman J. L., Foiani M., Cigan A. M., Paddon C. J., Hinnebusch A. G. Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3. Mol Cell Biol. 1993 Aug;13(8):4618–4631. doi: 10.1128/mcb.13.8.4618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chong K. L., Feng L., Schappert K., Meurs E., Donahue T. F., Friesen J. D., Hovanessian A. G., Williams B. R. Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2. EMBO J. 1992 Apr;11(4):1553–1562. doi: 10.1002/j.1460-2075.1992.tb05200.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cigan A. M., Bushman J. L., Boal T. R., Hinnebusch A. G. A protein complex of translational regulators of GCN4 mRNA is the guanine nucleotide-exchange factor for translation initiation factor 2 in yeast. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5350–5354. doi: 10.1073/pnas.90.11.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cigan A. M., Foiani M., Hannig E. M., Hinnebusch A. G. Complex formation by positive and negative translational regulators of GCN4. Mol Cell Biol. 1991 Jun;11(6):3217–3228. doi: 10.1128/mcb.11.6.3217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cigan A. M., Pabich E. K., Feng L., Donahue T. F. Yeast translation initiation suppressor sui2 encodes the alpha subunit of eukaryotic initiation factor 2 and shares sequence identity with the human alpha subunit. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2784–2788. doi: 10.1073/pnas.86.8.2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dever T. E., Chen J. J., Barber G. N., Cigan A. M., Feng L., Donahue T. F., London I. M., Katze M. G., Hinnebusch A. G. Mammalian eukaryotic initiation factor 2 alpha kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4616–4620. doi: 10.1073/pnas.90.10.4616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dever T. E., Feng L., Wek R. C., Cigan A. M., Donahue T. F., Hinnebusch A. G. 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. doi: 10.1016/0092-8674(92)90193-g. [DOI] [PubMed] [Google Scholar]
  11. Donahue T. F., Cigan A. M., Pabich E. K., Valavicius B. C. Mutations at a Zn(II) finger motif in the yeast eIF-2 beta gene alter ribosomal start-site selection during the scanning process. Cell. 1988 Aug 26;54(5):621–632. doi: 10.1016/s0092-8674(88)80006-0. [DOI] [PubMed] [Google Scholar]
  12. Ernst H., Duncan R. F., Hershey J. W. Cloning and sequencing of complementary DNAs encoding the alpha-subunit of translational initiation factor eIF-2. Characterization of the protein and its messenger RNA. J Biol Chem. 1987 Jan 25;262(3):1206–1212. [PubMed] [Google Scholar]
  13. Hannig E. M., Cigan A. M., Freeman B. A., Kinzy T. G. GCD11, a negative regulator of GCN4 expression, encodes the gamma subunit of eIF-2 in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Jan;13(1):506–520. doi: 10.1128/mcb.13.1.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hannig E. M., Hinnebusch A. G. Molecular analysis of GCN3, a translational activator of GCN4: evidence for posttranslational control of GCN3 regulatory function. Mol Cell Biol. 1988 Nov;8(11):4808–4820. doi: 10.1128/mcb.8.11.4808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hannig E. M., Williams N. P., Wek R. C., Hinnebusch A. G. 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. doi: 10.1093/genetics/126.3.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harashima S., Hannig E. M., Hinnebusch A. G. Interactions between positive and negative regulators of GCN4 controlling gene expression and entry into the yeast cell cycle. Genetics. 1987 Nov;117(3):409–419. doi: 10.1093/genetics/117.3.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harashima S., Hinnebusch A. G. Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Nov;6(11):3990–3998. doi: 10.1128/mcb.6.11.3990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hershey J. W. Translational control in mammalian cells. Annu Rev Biochem. 1991;60:717–755. doi: 10.1146/annurev.bi.60.070191.003441. [DOI] [PubMed] [Google Scholar]
  19. Hill D. E., Struhl K. Molecular characterization of GCD1, a yeast gene required for general control of amino acid biosynthesis and cell-cycle initiation. Nucleic Acids Res. 1988 Oct 11;16(19):9253–9265. doi: 10.1093/nar/16.19.9253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hinnebusch A. G., Fink G. R. Positive regulation in the general amino acid control of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5374–5378. doi: 10.1073/pnas.80.17.5374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hinnebusch A. G. Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol Rev. 1988 Jun;52(2):248–273. doi: 10.1128/mr.52.2.248-273.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Konieczny A., Safer B. Purification of the eukaryotic initiation factor 2-eukaryotic initiation factor 2B complex and characterization of its guanine nucleotide exchange activity during protein synthesis initiation. J Biol Chem. 1983 Mar 10;258(5):3402–3408. [PubMed] [Google Scholar]
  24. Lanker S., Bushman J. L., Hinnebusch A. G., Trachsel H., Mueller P. P. Autoregulation of the yeast lysyl-tRNA synthetase gene GCD5/KRS1 by translational and transcriptional control mechanisms. Cell. 1992 Aug 21;70(4):647–657. doi: 10.1016/0092-8674(92)90433-d. [DOI] [PubMed] [Google Scholar]
  25. Lucchini G., Hinnebusch A. G., Chen C., Fink G. R. Positive regulatory interactions of the HIS4 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jul;4(7):1326–1333. doi: 10.1128/mcb.4.7.1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matts R. L., Levin D. H., London I. M. Effect of phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 on the function of reversing factor in the initiation of protein synthesis. Proc Natl Acad Sci U S A. 1983 May;80(9):2559–2563. doi: 10.1073/pnas.80.9.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Merrick W. C. Mechanism and regulation of eukaryotic protein synthesis. Microbiol Rev. 1992 Jun;56(2):291–315. doi: 10.1128/mr.56.2.291-315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Meurs E., Chong K., Galabru J., Thomas N. S., Kerr I. M., Williams B. R., Hovanessian A. G. Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell. 1990 Jul 27;62(2):379–390. doi: 10.1016/0092-8674(90)90374-n. [DOI] [PubMed] [Google Scholar]
  29. Paddon C. J., Hinnebusch A. G. gcd12 mutations are gcn3-dependent alleles of GCD2, a negative regulator of GCN4 in the general amino acid control of Saccharomyces cerevisiae. Genetics. 1989 Jul;122(3):543–550. doi: 10.1093/genetics/122.3.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pain V. M. Initiation of protein synthesis in mammalian cells. Biochem J. 1986 May 1;235(3):625–637. doi: 10.1042/bj2350625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Parent S. A., Fenimore C. M., Bostian K. A. Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast. 1985 Dec;1(2):83–138. doi: 10.1002/yea.320010202. [DOI] [PubMed] [Google Scholar]
  32. Ramirez M., Wek R. C., Vazquez de Aldana C. R., Jackson B. M., Freeman B., Hinnebusch A. G. Mutations activating the yeast eIF-2 alpha kinase GCN2: isolation of alleles altering the domain related to histidyl-tRNA synthetases. Mol Cell Biol. 1992 Dec;12(12):5801–5815. doi: 10.1128/mcb.12.12.5801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rolfes R. J., Hinnebusch A. G. Translation of the yeast transcriptional activator GCN4 is stimulated by purine limitation: implications for activation of the protein kinase GCN2. Mol Cell Biol. 1993 Aug;13(8):5099–5111. doi: 10.1128/mcb.13.8.5099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rose M. D., Broach J. R. Cloning genes by complementation in yeast. Methods Enzymol. 1991;194:195–230. doi: 10.1016/0076-6879(91)94017-7. [DOI] [PubMed] [Google Scholar]
  35. Rowlands A. G., Montine K. S., Henshaw E. C., Panniers R. Physiological stresses inhibit guanine-nucleotide-exchange factor in Ehrlich cells. Eur J Biochem. 1988 Jul 15;175(1):93–99. doi: 10.1111/j.1432-1033.1988.tb14170.x. [DOI] [PubMed] [Google Scholar]
  36. Rowlands A. G., Panniers R., Henshaw E. C. The catalytic mechanism of guanine nucleotide exchange factor action and competitive inhibition by phosphorylated eukaryotic initiation factor 2. J Biol Chem. 1988 Apr 25;263(12):5526–5533. [PubMed] [Google Scholar]
  37. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schürch A., Miozzari J., Hütter R. Regulation of tryptophan biosynthesis in Saccharomyces cerevisiae: mode of action of 5-methyl-tryptophan and 5-methyl-tryptophan-sensitive mutants. J Bacteriol. 1974 Mar;117(3):1131–1140. doi: 10.1128/jb.117.3.1131-1140.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Siekierka J., Manne V., Ochoa S. Mechanism of translational control by partial phosphorylation of the alpha subunit of eukaryotic initiation factor 2. Proc Natl Acad Sci U S A. 1984 Jan;81(2):352–356. doi: 10.1073/pnas.81.2.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Siekierka J., Mauser L., Ochoa S. Mechanism of polypeptide chain initiation in eukaryotes and its control by phosphorylation of the alpha subunit of initiation factor 2. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2537–2540. doi: 10.1073/pnas.79.8.2537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sikorski R. S., 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. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vazquez de Aldana C. R., Dever T. E., Hinnebusch A. G. Mutations in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) that overcome the inhibitory effect of eIF-2 alpha phosphorylation on translation initiation. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7215–7219. doi: 10.1073/pnas.90.15.7215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wek R. C., Ramirez M., Jackson B. M., Hinnebusch A. G. Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression. Mol Cell Biol. 1990 Jun;10(6):2820–2831. doi: 10.1128/mcb.10.6.2820. [DOI] [PMC free article] [PubMed] [Google Scholar]

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