Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1995 Jul 3;14(13):3184–3199. doi: 10.1002/j.1460-2075.1995.tb07321.x

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.

C R Vazquez de Aldana 1, M J Marton 1, A G Hinnebusch 1
PMCID: PMC394380  PMID: 7621831

Abstract

GCN2 is a protein kinase that phosphorylates the alpha-subunit of translation initiation factor 2 (eIF-2) and thereby stimulates translation of GCN4 mRNA in amino acid-starved cells. We isolated a null mutation in a previously unidentified gene, GCN20, that suppresses the growth-inhibitory effect of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2. The deletion of GCN20 in otherwise wild-type strains impairs derepression of GCN4 translation and reduces the level of eIF-2 alpha phosphorylation in vivo, showing that GCN20 is a positive effector of GCN2 kinase function. In accordance with this conclusion, GCN20 was co-immunoprecipitated from cell extracts with GCN1, another factor required to activate GCN2, and the two proteins interacted in the yeast two-hybrid system. We conclude that GCN1 and GCN20 are components of a protein complex that couples the kinase activity of GCN2 to the availability of amino acids. GCN20 is a member of the ATP binding cassette (ABC) family of proteins and is closely related to ABC proteins identified in Caenorhabditis elegans, rice and humans, suggesting that the function of GCN20 may be conserved among diverse eukaryotic organisms.

Full text

PDF
3184

Images in this article

3185Image
on p.3185
3186Image
on p.3186
3187Image
on p.3187
3188Image
on p.3188
3189Image
on p.3189
3190Image
on p.3190
3192Image
on p.3192

Selected References

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

  1. 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. doi: 10.1534/genetics.112.541.test. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [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., 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]
  8. Colthurst D. R., Santos M., Grant C. M., Tuite M. F. Candida albicans and three other Candida species contain an elongation factor structurally and functionally analogous to elongation factor 3. FEMS Microbiol Lett. 1991 May 1;64(1):45–49. doi: 10.1016/0378-1097(91)90207-q. [DOI] [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. 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. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Donahue T. F., Daves R. S., Lucchini G., Fink G. R. A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast. Cell. 1983 Jan;32(1):89–98. doi: 10.1016/0092-8674(83)90499-3. [DOI] [PubMed] [Google Scholar]
  13. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  14. Fath M. J., Kolter R. ABC transporters: bacterial exporters. Microbiol Rev. 1993 Dec;57(4):995–1017. doi: 10.1128/mr.57.4.995-1017.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  16. Gaber R. F., Mathison L., Edelman I., Culbertson M. R. Frameshift Suppression in SACCHAROMYCES CEREVISIAE VI. Complete Genetic Map of Twenty-Five Suppressor Genes. Genetics. 1983 Mar;103(3):389–407. doi: 10.1093/genetics/103.3.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Higgins C. F. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. doi: 10.1146/annurev.cb.08.110192.000435. [DOI] [PubMed] [Google Scholar]
  21. Higgins C. F., Hiles I. D., Salmond G. P., Gill D. R., Downie J. A., Evans I. J., Holland I. B., Gray L., Buckel S. D., Bell A. W. A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature. 1986 Oct 2;323(6087):448–450. doi: 10.1038/323448a0. [DOI] [PubMed] [Google Scholar]
  22. Higgins C. F., Hiles I. D., Whalley K., Jamieson D. J. Nucleotide binding by membrane components of bacterial periplasmic binding protein-dependent transport systems. EMBO J. 1985 Apr;4(4):1033–1039. doi: 10.1002/j.1460-2075.1985.tb03735.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Kato J., Chu L., Kitano K., DeVault J. D., Kimbara K., Chakrabarty A. M., Misra T. K. Nucleotide sequence of a regulatory region controlling alginate synthesis in Pseudomonas aeruginosa: characterization of the algR2 gene. Gene. 1989 Dec 7;84(1):31–38. doi: 10.1016/0378-1119(89)90136-4. [DOI] [PubMed] [Google Scholar]
  25. Koerner T. J., Hill J. E., Myers A. M., Tzagoloff A. High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. Methods Enzymol. 1991;194:477–490. doi: 10.1016/0076-6879(91)94036-c. [DOI] [PubMed] [Google Scholar]
  26. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  27. Kölling R., Hollenberg C. P. The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J. 1994 Jul 15;13(14):3261–3271. doi: 10.1002/j.1460-2075.1994.tb06627.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Maicas E., Pluthero F. G., Friesen J. D. The accumulation of three yeast ribosomal proteins under conditions of excess mRNA is determined primarily by fast protein decay. Mol Cell Biol. 1988 Jan;8(1):169–175. doi: 10.1128/mcb.8.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Marton M. J., Crouch D., Hinnebusch A. G. 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. doi: 10.1128/mcb.13.6.3541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mendenhall M. D., Leeds P., Fen H., Mathison L., Zwick M., Sleiziz C., Culbertson M. R. Frameshift suppressor mutations affecting the major glycine transfer RNAs of Saccharomyces cerevisiae. J Mol Biol. 1987 Mar 5;194(1):41–58. doi: 10.1016/0022-2836(87)90714-5. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Messenguy F., Delforge J. Role of transfer ribonucleic acids in the regulation of several biosyntheses in Saccharomyces cerevisiae. Eur J Biochem. 1976 Aug 16;67(2):335–339. doi: 10.1111/j.1432-1033.1976.tb10696.x. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Mueller P. P., Hinnebusch A. G. Multiple upstream AUG codons mediate translational control of GCN4. Cell. 1986 Apr 25;45(2):201–207. doi: 10.1016/0092-8674(86)90384-3. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Parkinson J. S., Kofoid E. C. Communication modules in bacterial signaling proteins. Annu Rev Genet. 1992;26:71–112. doi: 10.1146/annurev.ge.26.120192.000443. [DOI] [PubMed] [Google Scholar]
  40. Qin S. L., Xie A. G., Bonato M. C., McLaughlin C. S. Sequence analysis of the translational elongation factor 3 from Saccharomyces cerevisiae. J Biol Chem. 1990 Feb 5;265(4):1903–1912. [PubMed] [Google Scholar]
  41. Ramirez M., Wek R. C., Hinnebusch A. G. 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. doi: 10.1128/mcb.11.6.3027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Riles L., Dutchik J. E., Baktha A., McCauley B. K., Thayer E. C., Leckie M. P., Braden V. V., Depke J. E., Olson M. V. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. doi: 10.1093/genetics/134.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  46. Sandbaken M., Lupisella J. A., 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. doi: 10.1016/0167-4781(90)90172-x. [DOI] [PubMed] [Google Scholar]
  47. 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] [Google Scholar]
  48. 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]
  49. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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. doi: 10.1093/oxfordjournals.jbchem.a122405. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Vazquez de Aldana C. R., Hinnebusch A. G. Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation. Mol Cell Biol. 1994 May;14(5):3208–3222. doi: 10.1128/mcb.14.5.3208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vazquez de Aldana C. R., Wek R. C., Segundo P. S., Truesdell A. G., Hinnebusch A. G. Multicopy tRNA genes functionally suppress mutations in yeast eIF-2 alpha kinase GCN2: evidence for separate pathways coupling GCN4 expression to unchanged tRNA. Mol Cell Biol. 1994 Dec;14(12):7920–7932. doi: 10.1128/mcb.14.12.7920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wanner B. L. Gene regulation by phosphate in enteric bacteria. J Cell Biochem. 1993 Jan;51(1):47–54. doi: 10.1002/jcb.240510110. [DOI] [PubMed] [Google Scholar]
  57. Wek R. C., Jackson B. M., Hinnebusch A. G. 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. doi: 10.1073/pnas.86.12.4579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  60. Ypma-Wong M. F., Fonzi W. A., Sypherd P. S. Fungus-specific translation elongation factor 3 gene present in Pneumocystis carinii. Infect Immun. 1992 Oct;60(10):4140–4145. doi: 10.1128/iai.60.10.4140-4145.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES