The beta/Gcd7 subunit of eukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor, is crucial for binding eIF2 in vivo.
Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor (GEF) for eukaryotic translation initiation factor 2, which stimulates formation of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) in a manner inhibited by phosphorylated eIF2 [eIF2(αP)]. While eIF2B contains five subunits, the ε/Gcd6 subunit is sufficient for GEF activity in vitro. The δ/Gcd2 and β/Gcd7 subunits function with α/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(αP)-GDP, but the essential functions of δ/Gcd2 and β/Gcd7 are not well understood. We show that the depletion of wild-type β/Gcd7, three lethal β/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in β/Gcd7 and eIF2α all impair eIF2 binding to eIF2B without reducing ε/Gcd6 abundance in the native eIF2B-eIF2 holocomplex. Additionally, β/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2α (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, β/Gcd7 can overcome the toxicity of eIF2(αP) and rescue native eIF2B function when overexpressed with δ/Gcd2 or γ/Gcd1. In aggregate, these findings provide compelling evidence that β/Gcd7 is crucial for binding of substrate by eIF2B in vivo, beyond its dispensable regulatory role in the inhibition of eIF2B by eIF (αP).
Dev
Kamal
K
Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA.
Qiu
Hongfang
H
Dong
Jinsheng
J
Zhang
Fan
F
Barthlme
Dominik
D
Hinnebusch
Alan G
AG
D019943:Amino Acid Substitution; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D020717:Eukaryotic Initiation Factor-2B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D021122:Protein Subunits; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D015852:Eukaryotic Initiation Factor-2; D020717:Eukaryotic Initiation Factor-2B; C068999:GCD2 protein, S cerevisiae; C079879:GCD7 protein, S cerevisiae; D021122:Protein Subunits; D012331:RNA, Fungal; D012358:RNA, Transfer, Met; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins
Mol Cell Biol. 2006 Feb;26(4):1355-72
16449648
Genetics. 2001 May;158(1):123-32
11333223
Genes Dev. 1995 Jul 15;9(14):1781-96
7542616
Mol Cell Biol. 1993 Mar;13(3):1920-32
8441423
J Mol Biol. 2004 Jan 23;335(4):923-36
14698289
Mol Cell Biol. 2000 Jun;20(11):3965-76
10805739
Gene. 1988 Dec 30;74(2):527-34
3073106
Yeast. 1995 Apr 15;11(4):355-60
7785336
Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7215-9
8102207
Science. 2000 Jun 2;288(5471):1643-7
10834843
Genes Dev. 1998 Feb 15;12(4):514-26
9472020
J Mol Biol. 2007 Jul 6;370(2):315-30
17512538
Biochim Biophys Acta. 2000 Jun 21;1492(1):56-62
10858531
Genetics. 1989 May;122(1):19-27
2659436
Methods Enzymol. 1987;154:164-75
3323810
Mol Cell Biol. 1991 Jun;11(6):3217-28
2038327
EMBO J. 2002 Oct 1;21(19):5292-301
12356745
Mol Cell Biol. 1996 Nov;16(11):6603-16
8887689
Yeast. 1998 Jul;14(10):953-61
9717241
Gene. 1987;57(2-3):267-72
3319781
Science. 1988 Oct 7;242(4875):93-7
3051379
Annu Rev Microbiol. 2005;59:407-50
16153175
J Mol Biol. 2009 Sep 25;392(3):701-22
19616556
EMBO J. 1996 Nov 15;15(22):6311-20
8947054
Mol Cell Biol. 2001 Aug;21(15):5018-30
11438658
J Biol Chem. 1982 Nov 10;257(21):13056-61
6290491
Mol Cell Biol. 1995 Nov;15(11):6351-63
7565788
Mol Cell Biol. 1991 Jun;11(6):3203-16
2038326
Mol Cell Biol. 1992 Dec;12(12):5801-15
1448107
Genetics. 1990 Nov;126(3):549-62
2249755
Mol Cell Biol. 2005 Apr;25(8):3063-75
15798194
Mol Cell Biol. 1994 Dec;14(12):7920-32
7969132
Cell. 2005 Sep 23;122(6):887-900
16179258
J Biol Chem. 1997 May 9;272(19):12359-65
9139680
J Biol Chem. 2001 Jul 6;276(27):24697-703
11323413
Yeast. 1997 Jun 15;13(7):647-53
9200814
EMBO J. 1999 Mar 15;18(6):1673-88
10075937
Mol Cell Biol. 1994 May;14(5):3208-22
8164676
Science. 1994 Mar 4;263(5151):1273-6
8122109
Mol Cell Biol. 1997 Mar;17(3):1298-313
9032257
Curr Top Cell Regul. 1992;32:243-369
1318183
Biochem Biophys Res Commun. 1996 Mar 27;220(3):843-7
8607853
Mol Cell Biol. 2007 Jul;27(14):5225-34
17526738
J Biol Chem. 1999 Nov 5;274(45):32198-203
10542257
Mol Cell Biol. 2008 Nov;28(22):6877-88
18794367
Mol Cell Biol. 2004 Mar;24(6):2352-63
14993275
J Biol Chem. 1998 May 22;273(21):12841-5
9582312
Mol Cell Biol. 1997 Jul;17(7):4146-58
9199350