Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity
Autor(a) principal: | |
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Data de Publicação: | 2011 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | http://repositorio.unifesp.br/handle/11600/34151 http://dx.doi.org/10.1074/jbc.M111.248898 |
Resumo: | The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. the ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. the purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2 Delta cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2 alpha, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis. |
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Visweswaraiah, JyothsnaLageix, SebastienCastilho, Beatriz Amaral de [UNIFESP]Izotova, LaraKinzy, Terri GossHinnebusch, Alan G.Sattlegger, EvelynMassey UnivNICHDUniversidade Federal de São Paulo (UNIFESP)Univ Med & Dent New Jersey2016-01-24T14:17:20Z2016-01-24T14:17:20Z2011-10-21Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 286, n. 42, p. 36568-36579, 2011.0021-9258http://repositorio.unifesp.br/handle/11600/34151http://dx.doi.org/10.1074/jbc.M111.24889810.1074/jbc.M111.248898WOS:000296538300041The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. the ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. the purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2 Delta cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2 alpha, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.National Institutes of HealthFundacao de Apoio a Pesquisa no Estado de São PauloMarsden Fund CouncilMassey UniversityRoyal Society of New ZealandMassey Univ, Inst Nat Sci, N Shore Mail Ctr, Albany 0745, New ZealandNICHD, Lab Gene Regulat & Dev, NIH, Bethesda, MD 20892 USAUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniv Med & Dent New Jersey, Robert Wood Johnson Med Sch, Dept Mol Genet Microbiol & Immunol, Piscataway, NJ 08854 USAUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilRoyal Society of New Zealand: MAU0607: RO1 GM57483Web of Science36568-36579engAmer Soc Biochemistry Molecular Biology IncJournal of Biological ChemistryEvidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activityinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP11600/341512023-01-12 22:12:02.588metadata only accessoai:repositorio.unifesp.br:11600/34151Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestopendoar:34652023-01-13T01:12:02Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.en.fl_str_mv |
Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity |
title |
Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity |
spellingShingle |
Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity Visweswaraiah, Jyothsna |
title_short |
Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity |
title_full |
Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity |
title_fullStr |
Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity |
title_full_unstemmed |
Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity |
title_sort |
Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity |
author |
Visweswaraiah, Jyothsna |
author_facet |
Visweswaraiah, Jyothsna Lageix, Sebastien Castilho, Beatriz Amaral de [UNIFESP] Izotova, Lara Kinzy, Terri Goss Hinnebusch, Alan G. Sattlegger, Evelyn |
author_role |
author |
author2 |
Lageix, Sebastien Castilho, Beatriz Amaral de [UNIFESP] Izotova, Lara Kinzy, Terri Goss Hinnebusch, Alan G. Sattlegger, Evelyn |
author2_role |
author author author author author author |
dc.contributor.institution.none.fl_str_mv |
Massey Univ NICHD Universidade Federal de São Paulo (UNIFESP) Univ Med & Dent New Jersey |
dc.contributor.author.fl_str_mv |
Visweswaraiah, Jyothsna Lageix, Sebastien Castilho, Beatriz Amaral de [UNIFESP] Izotova, Lara Kinzy, Terri Goss Hinnebusch, Alan G. Sattlegger, Evelyn |
description |
The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. the ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. the purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2 Delta cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2 alpha, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis. |
publishDate |
2011 |
dc.date.issued.fl_str_mv |
2011-10-21 |
dc.date.accessioned.fl_str_mv |
2016-01-24T14:17:20Z |
dc.date.available.fl_str_mv |
2016-01-24T14:17:20Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 286, n. 42, p. 36568-36579, 2011. |
dc.identifier.uri.fl_str_mv |
http://repositorio.unifesp.br/handle/11600/34151 http://dx.doi.org/10.1074/jbc.M111.248898 |
dc.identifier.issn.none.fl_str_mv |
0021-9258 |
dc.identifier.doi.none.fl_str_mv |
10.1074/jbc.M111.248898 |
dc.identifier.wos.none.fl_str_mv |
WOS:000296538300041 |
identifier_str_mv |
Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 286, n. 42, p. 36568-36579, 2011. 0021-9258 10.1074/jbc.M111.248898 WOS:000296538300041 |
url |
http://repositorio.unifesp.br/handle/11600/34151 http://dx.doi.org/10.1074/jbc.M111.248898 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartof.none.fl_str_mv |
Journal of Biological Chemistry |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
36568-36579 |
dc.publisher.none.fl_str_mv |
Amer Soc Biochemistry Molecular Biology Inc |
publisher.none.fl_str_mv |
Amer Soc Biochemistry Molecular Biology Inc |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UNIFESP instname:Universidade Federal de São Paulo (UNIFESP) instacron:UNIFESP |
instname_str |
Universidade Federal de São Paulo (UNIFESP) |
instacron_str |
UNIFESP |
institution |
UNIFESP |
reponame_str |
Repositório Institucional da UNIFESP |
collection |
Repositório Institucional da UNIFESP |
repository.name.fl_str_mv |
Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP) |
repository.mail.fl_str_mv |
|
_version_ |
1802764188797370368 |