Abstract
To understand how phosphorylation of eukaryotic translation initiation factor (eIF)-2 alpha in Saccharomyces cerevisiae stimulates GCN4 mRNA translation while at the same time inhibiting general translation initiation, we examined the effects of altering the gene dosage of initiator tRNA(Met), eIF-2, and the guanine nucleotide exchange factor for eIF-2, eIF-2B. Overexpression of all three subunits of eIF-2 or all five subunits of eIF-2B suppressed the effects of eIF-2 alpha hyperphosphorylation on both GCN4-specific and general translation initiation. Consistent with eIF-2 functioning in translation as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA(iMet), reduced gene dosage of initiator tRNA(Met) mimicked phosphorylation of eIF-2 alpha and stimulated GCN4 translation. In addition, overexpression of a combination of eIF-2 and tRNA(iMet) suppressed the growth-inhibitory effects of eIF-2 hyperphosphorylation more effectively than an increase in the level of either component of the ternary complex alone. These results provide in vivo evidence that phosphorylation of eIF-2 alpha reduces the activities of both eIF-2 and eIF-2B and that the eIF-2.GTP. Met-tRNA(iMet) ternary complex is the principal component limiting translation in cells when eIF-2 alpha is phosphorylated on serine 51. Analysis of eIF-2 alpha phosphorylation in the eIF-2-overexpressing strain also provides in vivo evidence that phosphorylated eIF-2 acts as a competitive inhibitor of eIF-2B rather than forming an excessively stable inactive complex. Finally, our results demonstrate that the concentration of eIF-2-GTP. Met-tRNA(iMet) ternary complexes is the cardinal parameter determining the site of reinitiation on GCN4 mRNA and support the idea that reinitiation at GCN4 is inversely related to the concentration of ternary complexes in the cell.
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- 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]
- 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]
- 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]
- 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]
- Byström A. S., Fink G. R. A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast. Mol Gen Genet. 1989 Apr;216(2-3):276–286. doi: 10.1007/BF00334366. [DOI] [PubMed] [Google Scholar]
- Christianson T. W., Sikorski R. S., Dante M., Shero J. H., Hieter P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 1992 Jan 2;110(1):119–122. doi: 10.1016/0378-1119(92)90454-w. [DOI] [PubMed] [Google Scholar]
- 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]
- Cigan A. M., Donahue T. F. The methionine initiator tRNA genes of yeast. Gene. 1986;41(2-3):343–348. doi: 10.1016/0378-1119(86)90118-6. [DOI] [PubMed] [Google Scholar]
- Cigan A. M., Feng L., Donahue T. F. tRNAi(met) functions in directing the scanning ribosome to the start site of translation. Science. 1988 Oct 7;242(4875):93–97. doi: 10.1126/science.3051379. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- Foiani M., Cigan A. M., Paddon C. J., Harashima S., Hinnebusch A. G. 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. doi: 10.1128/mcb.11.6.3203. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
- 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]
- Hinnebusch A. G. Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2. Mol Microbiol. 1993 Oct;10(2):215–223. doi: 10.1111/j.1365-2958.1993.tb01947.x. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Krupitza G., Thireos G. Translational activation of GCN4 mRNA in a cell-free system is triggered by uncharged tRNAs. Mol Cell Biol. 1990 Aug;10(8):4375–4378. doi: 10.1128/mcb.10.8.4375. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Moehle C. M., Hinnebusch A. G. Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1991 May;11(5):2723–2735. doi: 10.1128/mcb.11.5.2723. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Niederberger P., Aebi M., Hütter R. Identification and characterization of four new GCD genes in Saccharomyces cerevisiae. Curr Genet. 1986;10(9):657–664. doi: 10.1007/BF00410913. [DOI] [PubMed] [Google Scholar]
- Paddon C. J., Hannig E. M., Hinnebusch A. G. Amino acid sequence similarity between GCN3 and GCD2, positive and negative translational regulators of GCN4: evidence for antagonism by competition. Genetics. 1989 Jul;122(3):551–559. doi: 10.1093/genetics/122.3.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- Wek R. C., Cannon J. F., Dever T. E., Hinnebusch A. G. Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2 alpha kinase GCN2. Mol Cell Biol. 1992 Dec;12(12):5700–5710. doi: 10.1128/mcb.12.12.5700. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wolfner M., Yep D., Messenguy F., Fink G. R. Integration of amino acid biosynthesis into the cell cycle of Saccharomyces cerevisiae. J Mol Biol. 1975 Aug 5;96(2):273–290. doi: 10.1016/0022-2836(75)90348-4. [DOI] [PubMed] [Google Scholar]
- von Pawel-Rammingen U., Aström S., Byström A. S. Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Apr;12(4):1432–1442. doi: 10.1128/mcb.12.4.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]