Gcn1 and actin binding to Yih1: implications for activation of the eIF2 kinase GCN2.
Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68-258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68-258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation.
Institute of Natural Sciences, Massey University, Auckland 0745, New Zealand. e.sattlegger@massey.ac.nz
Evelyn
E
Sattlegger
João A R G
JA
Barbosa
Maria Carolina S
MC
Moraes
Rafael M
RM
Martins
Alan G
AG
Hinnebusch
Beatriz A
BA
Castilho
D000199:Actins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D000199:Actins; C501389:GCN1 protein, S cerevisiae; D008840:Microfilament Proteins; D009097:Multienzyme Complexes; D010445:Peptide Elongation Factors; D029701:Saccharomyces cerevisiae Proteins; C488521:YIH1 protein, S cerevisiae; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases
Science. 1991 Jul 12;253(5016):164-70
1853201
J Immunol. 2006 Jun 1;176(11):6752-61
16709834
J Biol Chem. 2009 Feb 13;284(7):4739-47
19095653
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
Science. 2005 Mar 18;307(5716):1776-8
15774759
EMBO J. 1995 Jul 3;14(13):3184-99
7621831
Cell. 2002 Feb 22;108(4):545-56
11909525
Proteins. 1994 Jul;19(3):165-73
7937731
J Comp Neurol. 2008 Apr 10;507(5):1811-30
18260151
J Mol Biol. 1995 Apr 7;247(4):536-40
7723011
Mol Genet Genomics. 2002 Sep;268(1):10-8
12242494
Mol Cell Biol. 2007 Mar;27(5):1974-89
17178834
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2256-68
15572779
Nature. 2005 Aug 25;436(7054):1166-73
16121183
J Cell Biol. 1994 Apr;125(2):381-91
8163554
EMBO J. 2000 Dec 1;19(23):6622-33
11101534
J Biol Chem. 2000 Jul 7;275(27):20243-6
10801780
J Biol Chem. 2004 Jul 16;279(29):29952-62
15126500
Nat Struct Mol Biol. 2005 Sep;12(9):772-8
16116436
J Mol Biol. 1993 Dec 5;234(3):779-815
8254673
J Biol Chem. 2005 Aug 5;280(31):28316-23
15937339
Mol Syst Biol. 2006;2:2006.0008
16738554
Genetics. 2001 Apr;157(4):1425-36
11290701
Proteins. 2004 May 15;55(3):775-7
15103642
Mol Cell Biol. 1993 Jun;13(6):3541-56
8497269
Proteins. 1997 Jul;28(3):405-20
9223186
Nature. 2003 Oct 16;425(6959):737-41
14562106
Nucleic Acids Res. 2000 Jan 1;28(1):235-42
10592235
Cell Mol Immunol. 2008 Oct;5(5):333-9
18954556
Nature. 2000 Feb 10;403(6770):623-7
10688190
J Biol Chem. 2001 May 18;276(20):17591-6
11350982
Biochemistry. 1972 Oct 24;11(22):4120-31
4343790
Cell Metab. 2005 Apr;1(4):273-7
16054071
Mol Cell Biol. 1997 Aug;17(8):4474-89
9234705
Mol Cell Biol. 1991 Jun;11(6):3203-16
2038326
Cell. 1992 Feb 7;68(3):585-96
1739968
Nucleic Acids Res. 1996 Jul 1;24(13):2519-24
8692690
Nature. 2002 Jan 10;415(6868):141-7
11805826
J Biol Chem. 2005 Apr 22;280(16):16514-21
15722345
Genome Res. 2002 Jan;12(1):47-56
11779830
Eukaryot Cell. 2009 Jul;8(7):1061-71
19448108
Methods Mol Biol. 1996;59:1-16
8798181
Protein Sci. 2004 Aug;13(8):2089-100
15273307
Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9249-54
9256468
Structure. 1997 Aug 15;5(8):1093-108
9309224
Nature. 1992 Jul 2;358(6381):86-9
1614539
Biochem Biophys Res Commun. 2005 Jul 1;332(2):450-5
15896712
Nucleic Acids Res. 1997 Dec 15;25(24):4876-82
9396791
J Mol Biol. 1990 Oct 5;215(3):403-10
2231712