Structural Characterization of the Human Eukaryotic Initiation Factor 3 Protein Complex by Mass Spectrometry

Abstract

Protein synthesis in mammalian cells requires initiation factor eIF3, an ∼800-kDa protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40 S ribosomal subunit to form the 48 S initiation complex. The eIF3 complex also prevents premature association of the 40 and 60 S ribosomal subunits and interacts with other initiation factors involved in start codon selection. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. Since its initial characterization in the 1970s, the exact size, composition, and post-translational modifications of mammalian eIF3 have not been rigorously determined. Two powerful mass spectrometric approaches were used in the present study to determine post-translational modifications that may regulate the activity of eIF3 during the translation initiation process and to characterize the molecular structure of the human eIF3 protein complex purified from HeLa cells. In the first approach, the bottom-up analysis of eIF3 allowed for the identification of a total of 13 protein components (eIF3a–m) with a sequence coverage of ∼79%. Furthermore 29 phosphorylation sites and several other post-translational modifications were unambiguously identified within the eIF3 complex. The second mass spectrometric approach, involving analysis of intact eIF3, allowed the detection of a complex with each of the 13 subunits present in stoichiometric amounts. Using tandem mass spectrometry four eIF3 subunits (h, i, k, and m) were found to be most easily dissociated and therefore likely to be on the periphery of the complex. It is noteworthy that none of these four subunits were found to be phosphorylated. These data raise interesting questions about the function of phosphorylation as it relates to the core subunits of the complex. Protein synthesis in mammalian cells requires initiation factor eIF3, an ∼800-kDa protein complex that plays a central role in binding of initiator methionyl-tRNA and mRNA to the 40 S ribosomal subunit to form the 48 S initiation complex. The eIF3 complex also prevents premature association of the 40 and 60 S ribosomal subunits and interacts with other initiation factors involved in start codon selection. The molecular mechanisms by which eIF3 exerts these functions are poorly understood. Since its initial characterization in the 1970s, the exact size, composition, and post-translational modifications of mammalian eIF3 have not been rigorously determined. Two powerful mass spectrometric approaches were used in the present study to determine post-translational modifications that may regulate the activity of eIF3 during the translation initiation process and to characterize the molecular structure of the human eIF3 protein complex purified from HeLa cells. In the first approach, the bottom-up analysis of eIF3 allowed for the identification of a total of 13 protein components (eIF3a–m) with a sequence coverage of ∼79%. Furthermore 29 phosphorylation sites and several other post-translational modifications were unambiguously identified within the eIF3 complex. The second mass spectrometric approach, involving analysis of intact eIF3, allowed the detection of a complex with each of the 13 subunits present in stoichiometric amounts. Using tandem mass spectrometry four eIF3 subunits (h, i, k, and m) were found to be most easily dissociated and therefore likely to be on the periphery of the complex. It is noteworthy that none of these four subunits were found to be phosphorylated. These data raise interesting questions about the function of phosphorylation as it relates to the core subunits of the complex. The initiation phase of eukaryotic protein synthesis involves formation of an 80 S ribosomal complex containing the initiator methionyl-tRNAi bound to the initiation codon in the mRNA. This is a multistep process promoted by proteins called eukaryotic initiation factors (eIFs). 1The abbreviations used are: eIF, eukaryotic initiation factor; nano-LC, nanospray LC; IAA, iodoacetamide; DMEM, Dulbecco's modified Eagle's medium; PTM, post-translational modification; LTQ, linear quadrupole ion trap. 1The abbreviations used are: eIF, eukaryotic initiation factor; nano-LC, nanospray LC; IAA, iodoacetamide; DMEM, Dulbecco's modified Eagle's medium; PTM, post-translational modification; LTQ, linear quadrupole ion trap. Currently at least 12 eIFs, composed of at least 29 distinct subunits, have been identified (1Hershey J.W. Merrick W.C. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 33-38Google Scholar). Mammalian eIF3, the largest initiation factor, is a multisubunit complex with an apparent molecular mass of ∼800 kDa. This protein complex plays an essential role in translation by binding directly to the 40 S ribosomal subunit and promoting formation of the 43 S preinitiation complex consisting of the Met-tRNAi·eIF2·GTP ternary complex, eIF1, eIF1A, and the 40 S ribosomal subunit (2Chaudhuri J. Chowdhury D. Maitra U. Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex.J. Biol. Chem. 1999; 274: 17975-17980Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 3Hinnebusch A.G. eIF3: a versatile scaffold for translation initiation complexes.Trends Biochem. Sci. 2006; 31: 553-562Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). The ability of eIF3 to bind to 40 S subunits in the absence of other initiation factors is enhanced by the presence of its loosely associated eIF3j subunit (4Fraser C.S. Lee J.Y. Mayeur G.L. Bushell M. Doudna J.A. Hershey J.W. The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits in vitro.J. Biol. Chem. 2004; 279: 8946-8956Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The eIF3 complex also promotes binding of 5′-m7G-capped mRNA through its interaction with eIF4G, the largest member of the eIF4F cap-binding complex (5Lamphear B.J. Kirchweger R. Skern T. Rhoads R.E. Mapping of functional domains in eukaryotic protein synthesis initiation factor 4G (eIF4G) with picornaviral proteases. Implications for cap-dependent and cap-independent translational initiation.J. Biol. Chem. 1995; 270: 21975-21983Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 6Korneeva N.L. Lamphear B.J. Hennigan F.L. Rhoads R.E. Mutually cooperative binding of eukaryotic translation initiation factor (eIF) 3 and eIF4A to human eIF4G-1.J. Biol. Chem. 2000; 275: 41369-41376Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The 43 S preinitiation complex then scans the mRNA (together forming the 48 S complex) in a 5′ to 3′ direction until the initiation codon AUG is selected. Upon recognition of the initiation codon, eIF5 stimulates hydrolysis of the GTP bound to eIF2, and the eIFs are ejected from the ribosome. The 60 S ribosomal subunit then joins the 40 S initiation complex aided by eIF5B (7Pestova T.V. Lomakin I.B. Lee J.H. Choi S.K. Dever T.E. Hellen C.U. The joining of ribosomal subunits in eukaryotes requires eIF5B.Nature. 2000; 403: 332-335Crossref PubMed Scopus (301) Google Scholar). Mammalian eIF3 consists of 13 non-identical subunits that range in size from 170 to 25 kDa and are named eIF3a through eIF3m (8Browning K.S. Gallie D.R. Hershey J.W. Hinnebusch A.G. Maitra U. Merrick W.C. Norbury C. Unified nomenclature for the subunits of eukaryotic initiation factor 3.Trends Biochem. Sci. 2001; 26: 284-290Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 9Unbehaun A. Borukhov S.I. Hellen C.U. Pestova T.V. Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP.Genes Dev. 2004; 18: 3078-3093Crossref PubMed Scopus (173) Google Scholar). Characterization of eIF3 in mammalian cells has been hampered by its large size and complexity, and only recently have the cDNAs been cloned and sequenced for all of the subunits (9Unbehaun A. Borukhov S.I. Hellen C.U. Pestova T.V. Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP.Genes Dev. 2004; 18: 3078-3093Crossref PubMed Scopus (173) Google Scholar, 10Mayeur G.L. Fraser C.S. Peiretti F. Block K.L. Hershey J.W. A newly discovered subunit of mammalian translation initiation factor eIF3.Eur. J. Biochem. 2003; 270: 4133-4139Crossref PubMed Scopus (48) Google Scholar). The functions of the individual subunits are not yet well established. In the yeast Saccharomyces cerevisiae, eIF3 appears to consist of a core complex of only five stoichiometric subunits (eIF3a, -b, -c, -g, and -i), plus a non-stoichiometric subunit, eIF3j. In mammalian cells, the corresponding homologs also may constitute a “core” complex to which the other mammalian subunits bind and regulate eIF3 activity. Interestingly of eIF3 in the yeast the five core subunits plus the and eIF3m subunits, the other the and subunits C. F. A. J. proteins and eIF3m distinct translation initiation factor 3 Biol. PubMed Scopus Google Scholar). It is not yet mammalian eIF3 as containing of eIF3 interacts with eIF1, and eIF5 to form a complex Hinnebusch A.G. A of eIF3 subunits and eIF5 and stimulates binding of mRNA and J. 2001; PubMed Scopus Google Scholar, J. Hinnebusch A.G. formation by all five of mammalian translation initiation factor 3 subunits from yeast Saccharomyces Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Mammalian eIF3 also and eIF5 T. Maitra U. interaction of eukaryotic translation initiation factor with the of Biol. Chem. Full Text Full Text PDF PubMed Scopus (81) Google Scholar, A. Maitra U. and characterization of the subunit of mammalian translation initiation factor 3 that eIF3 interacts with eIF5 in mammalian 1999; PubMed Scopus Google Scholar, R. A. Maitra U. Mammalian translation initiation factor functions with eIF1A and eIF3 in the formation of a stable 40 S preinitiation complex.J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google in A in and eukaryotic initiation factor and interaction with Biol. PubMed Scopus Google and eukaryotic translation initiation factor 4G (eIF4G) and binding sites for Biol. PubMed Scopus Google Scholar). of these have been to individual eIF3 In for eIF3a and J. A. Hinnebusch A.G. A complex of eukaryotic initiation eIF1, eIF2, eIF3, and initiator is an translation initiation in Dev. 2000; PubMed Scopus Google Scholar, Hinnebusch A.G. in the complex is for translation initiation in J. PubMed Scopus Google eIF5 J. A. Hinnebusch A.G. A complex of eukaryotic initiation eIF1, eIF2, eIF3, and initiator is an translation initiation in Dev. 2000; PubMed Scopus Google and eIF3a Hinnebusch A.G. in the complex is for translation initiation in J. PubMed Scopus Google Scholar). In Pestova T.V. Hellen C.U. and of the translation initiation factor J. 1999; 18: PubMed Scopus Google and eIF5 interacts with C. M. A. U. The structure of the of human translation initiation factor Biol. 2006; PubMed Scopus Google Scholar). eIF3a in A in and eukaryotic initiation factor and interaction with Biol. PubMed Scopus Google and in C. A. Hinnebusch A.G. of the in with promotes binding of eukaryotic initiation factor 3 in Biol. 2006; 26: PubMed Scopus Google and K.S. T. initiation factor ribosomal of J. 2004; PubMed Scopus Google to in N.L. M. U. Hershey J.W. Rhoads R.E. initiation factor to eIF3 through the Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). with its binding activity and its ability to bind to the 40 S mammalian eIF3 plays a central role in the initiation as an of other initiation factors on the of the 40 S ribosomal the function of human eIF3 from a structure by that eIF3 as an structure with five domains named to Fraser C.S. Doudna J.A. for human translation factor eIF3 in initiation of protein PubMed Scopus Google Scholar). the structure on the 40 S ribosomal subunit eIF3 on the of the to the by A. J. initiation factor 3 not association through of the ribosomal Biol. PubMed Scopus Google with of the to the binding and initiation codon the of each of the five is not yet and the molecular mechanisms by which eIF3 exerts its functions are poorly understood. The that eIF3 activity is in mammalian cells is on the that a of its subunits are and that phosphorylation of eIF3 is by of cells with other initiation factors are J.W. Protein phosphorylation translation Biol. Chem. Full Text PDF PubMed Google Scholar, T.E. by translation Full Text Full Text PDF PubMed Scopus Google and phosphorylation has been to function as a for the of initiation T.E. by translation Full Text Full Text PDF PubMed Scopus Google Scholar). These have a mass study to characterize the molecular structure of the eIF3 protein complex. The study the identification of post-translational modifications that may its activity during the translation initiation the first data the mass of the intact 13 subunit complex. other proteins may be associated with the eIF3 complex, only 13 proteins the complex are in The of has the for the molecular characterization of by mass has a powerful molecular for protein structure J. Protein by tandem mass Sci. U. A. PubMed Scopus Google Scholar, A. J. D.R. analysis of protein complexes mass 1999; PubMed Scopus Google the study of and A. of protein by mass PubMed Scopus Google the characterization of complexes of intact protein complexes by mass 2004; PubMed Scopus Google Scholar, J.A. for binding to mass Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and the analysis of complex by with and Lee protein identification a on an ion mass PubMed Scopus Google Scholar, D. A. R. of proteins by mass spectrometry of an A. PubMed Scopus Google Scholar). has been used to of size and spectrometry of and Biol. 2006; PubMed Scopus Google Scholar, A. T. of complexes directly from 2006; PubMed Scopus Google Scholar). In the present study mass spectrometric approaches were used to on the molecular structure of the human eIF3 protein complex purified from HeLa cells and to determine post-translational modifications that may regulate its activity during the translation initiation analysis of an eIF3 a linear ion mass used for the identification of eIF3 protein subunits and corresponding post-translational to the and of protein of from eIF3 and at the of from and Chem. 2004; PubMed Scopus Google used to characterization to data on the and sites of In the second approach, a mass F. A tandem mass for and analysis of large Chem. PubMed Scopus Google Scholar, of large in mass Chem. 2004; PubMed Scopus Google Scholar, and post-translational modifications in the yeast protein 2006; Full Text Full Text PDF PubMed Scopus Google used to the intact eIF3 complex to as well as identification of the subunits within the eIF3 complex. and were from from containing were from with were from other were from and used HeLa cells were in Dulbecco's modified Eagle's with were with and in with for to were with for to by the of the of cells were on and in and in containing and Protein were by the protein of protein were with by protein The were by by to HeLa were a from of each of HeLa of in a and with the The by at for at by of to a of The then in a at for at The of the and in a at with the of to a of at for the by at for at The in of A containing and in of the for at the through a and a in A containing The with a from to in A at and were The containing eIF3 were identified by A containing for at and a S in the The with a in A at and were The containing eIF3 were an to and a in A containing that and were The at and were The containing eIF3 were and an to a of between and A of the eIF3 is in the used to from the eIF3 The purified eIF3 protein complex at for by of to the eIF3 a at the and the eIF3 allowed to eIF3 of first at for in a of and and then in the at for by a of to the protein in a of and the at for The by the with The eIF3 then containing were used for the of eIF3 as by the of the eIF3 in of The then to the and with of by with of in and with with of The then in of of in and of in were and in a of in to with were from and used to the of the eIF3 in 25 of a of containing and that been with the were by the with of containing and then with of were with 3 25 of were and in a of in to tandem mass spectrometric analysis on an linear ion ion mass with a nanospray ion a and a The eIF3 in in and on a in with The to a of 12 with a of size, a with The for at in and A in at a of by a linear from to in at a of The mass in the the and the the to were in the mass with at to a of in the linear ion and were by with the a of on the most in the The and the for were for the to were by with at to a of in the linear ion and were by of the five most in the linear ion a of The in the for each were to an in the linear ion a of a of for the and the five most The data for each were to The were sequence and for sequence the were with four and a on the mass of in and for structure modifications allowed were of of of and phosphorylation of and and protein The for data were only the the were of the data used for to sites in were all by from and eIF3 subunit with of sequence 2003; 31: PubMed Scopus Google for with and all the are in the and and 3 with the of the protein of the This identified on the by and the is in the of phosphorylation sites and from the of the subunit by The the ion of the with the mass of This ion and in the linear ion to the The sequence identified by and by from the data is in the ion for the intact protein complexes were on a mass modified for mass detection F. A tandem mass for and analysis of large Chem. PubMed Scopus Google Scholar). The eIF3 complex and and were in The within the mass were to The mass at a of and a of 40 of the intact eIF3 complex by tandem mass spectrometry of a at with of phosphorylation of initiation factors has been as a to regulate has been to eIF3 in identified as a J.A. of phosphorylation of initiation factors and ribosomal protein by and Biol. Chem. Full Text PDF PubMed Google it not that the eIF3 subunits, and characterize the phosphorylation of eIF3, HeLa cells were with of and then with were to with and analysis by The in corresponding to and eIF3j with a for These are about that eIF3 phosphorylation with of protein that eIF3 is on subunits and may be by post-translational It therefore to the sites of phosphorylation to study the activity of the factor be This the mass spectrometry The human eIF3 protein complex purified from HeLa cells with and then with with IAA, and with The eIF3 a to a linear ion mass and the and analysis as were and the and 13 protein components of human eIF3 complex were unambiguously The subunit is identified as a protein that a of a scaffold for and eIF3 PubMed Scopus Google a found in five other eIF3 subunits k, and and in subunits of the and This protein to be of the eIF3 from HeLa cells and on the identification of only of the protein sequence (9Unbehaun A. Borukhov S.I. Hellen C.U. Pestova T.V. Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP.Genes Dev. 2004; 18: 3078-3093Crossref PubMed Scopus (173) Google Scholar). In the present a sequence coverage of for the eIF3m subunit the and its found to have the with formation of a as an sequence coverage of the human eIF3 complex the mass and of the mass in with The sequence coverage of each of the 13 eIF3 subunits is in the A total of 29 phosphorylation sites were identified within the and eIF3j subunits of which phosphorylation sites were identified only of by and and the 29 eIF3 phosphorylation sites identified in were and be found in and The phosphorylation sites and from and from and from the were identified in the HeLa by and M. D. J. J. characterization of HeLa Sci. U. A. 2004; PubMed Scopus Google Scholar). Two other phosphorylation sites were analysis of human A. J. M. J. analysis on human PubMed Scopus Google and human cells J. F. of human PubMed Scopus Google Scholar). other post-translational modifications of human eIF3 protein subunits as of protein were also identified in and all are in and of eIF3 protein subunits and corresponding from the mass of the corresponding eIF3 protein subunit plus post-translational modifications found only found only of phosphorylation phosphorylation of of phosphorylation of phosphorylation of phosphorylation of of of from the mass of the corresponding eIF3 protein subunit plus post-translational modifications found only found only in a and corresponding phosphorylation sites identified in eIF3 in a the identification of eIF3 and corresponding phosphorylation the of of the used in to the of the the is of with the of and to and as by and M. D. J. J. characterization of HeLa Sci. U. A. 2004; PubMed Scopus Google were of The in a from which the sequence be the modified and A. M. to the yeast Full Text Full Text PDF PubMed Scopus Google have the on a linear ion mass that has the of mass of and of The is that the are in and to In the large range of the of in the the for the identification of the from the is in an of a ion in the with a and a mass at of This ion in the linear ion and by to the from which the most ion corresponding to the ion and to an The unambiguously identified by the with a of a and identified as on the and present in the The of to as by a of and present in the The from the of the subunit also identified by the with a of and all phosphorylation sites and were unambiguously as by data The the ion with the mass of A total of and from the of the were found to be and the for phosphorylation were by to be the This may be involved in the binding of to through of the of to the eIF5 through its and C. M. A. U. The structure of the of human translation initiation factor Biol. 2006; PubMed Scopus Google Scholar). This sequence of to each of the proteins and a large of phosphorylation sites within the subunits It that phosphorylation sites be found in protein not by the in that have the during eIF3 were not the used to the HeLa cells and the is on by during J. Hershey J.W. The interacts with the subunit of eukaryotic initiation factor 3 during Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). on to determine of the identified as not for Mapping of eIF3 phosphorylation sites in the of the role of phosphorylation in the of eIF3 activity in is a for and for the approaches that not eIF3 phosphorylation plays a role in translational that are for eIF3 with other translational of eIF3 phosphorylation may be involved in and eIF5 bind within the yeast complex to the of J. A. Hinnebusch A.G. A complex of eukaryotic initiation eIF1, eIF2, eIF3, and initiator is an translation initiation in Dev. 2000; PubMed Scopus Google a of phosphorylation sites are phosphorylation may regulate formation of complex. eIF3j is for eIF3 to the 40 S subunit (4Fraser C.S. Lee J.Y. Mayeur G.L. Bushell M. Doudna J.A. Hershey J.W. The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits in vitro.J. Biol. Chem. 2004; 279: 8946-8956Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, C. A. Hinnebusch A.G. of the in with promotes binding of eukaryotic initiation factor 3 in Biol. 2006; 26: PubMed Scopus Google Scholar). In eIF3j is found dissociated from the eIF3 complex and bound to 40 S with eIF3 Hershey J.W. in ribosomal binding activity of eIF3 with translation during of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the association of eIF3j with eIF3 and the is by an of of it is likely that phosphorylation plays a role in these are to and to eIF3 sites are and the modifications to translational identified phosphorylation a second mass spectrometric used to the mass of the intact complex. The of a mass F. A tandem mass for and analysis of large Chem. PubMed Scopus Google Scholar, of large in mass Chem. 2004; PubMed Scopus Google allowed of the intact complex of eIF3 as in of a mass range of an at and of the the presence of with the intact eIF3 complex with each of its 13 subunits present in stoichiometric amounts. The molecular mass of the complex is as with the of all the modified proteins of The mass between and is and most likely from of of of intact Chem. 2006; PubMed Scopus Google Scholar). the molecular and complex is within of complex that all 13 subunits are of in a complex in to the distinct of eIF3 found in C. F. A. J. proteins and eIF3m distinct translation initiation factor 3 Biol. PubMed Scopus Google Scholar). of ion and tandem mass spectrometry of the subunits, and The mass of the modified subunit is in with the mass A between the and mass of the eIF3m subunit also from the analysis of the ion These data in with of the complex the of subunits and and The data that these four subunits and therefore are likely to be on the periphery of the complex M. T. of the from of intact Biol. 2006; Scopus Google Scholar). It is interesting to that the subunits that to the of the eIF3 complex are not the proteins that are phosphorylated. The proteins not and the core of the complex. The of the and of the human eIF3 complex have not been Fraser (4Fraser C.S. Lee J.Y. Mayeur G.L. Bushell M. Doudna J.A. Hershey J.W. The j-subunit of human translation initiation factor eIF3 is required for the stable binding of eIF3 and its subcomplexes to 40 S ribosomal subunits in vitro.J. Biol. Chem. 2004; 279: 8946-8956Abstract Full Text Full Text PDF PubMed Scopus (99) Google the between the human eIF3 core subunits the The subunit to be a central subunit to which not of the other eIF3 core subunits of phosphorylation sites on role of post-translational are to all of the protein mass analysis and a of first in the 1970s, on the molecular structure of the mammalian initiation factor eIF3 and of the subunits has been Two mass spectrometric approaches were used in the present study to about human eIF3 protein complex. In the first approach, the bottom-up analysis of the eIF3 allowed for the identification of a total of 13 protein components (eIF3a–m) with a sequence coverage of ∼79%. Furthermore 29 phosphorylation sites and several other post-translational modifications were unambiguously identified within the eIF3 complex. The second mass spectrometric approach, involving analysis of intact eIF3, allowed for the first of the intact complex of eIF3 with each of its 13 subunits present in stoichiometric amounts.