YIH1 Is an Actin-binding Protein That Inhibits Protein Kinase GCN2 and Impairs General Amino Acid Control When Overexpressed

The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the α subunit of translation initiation factor 2 (eIF2α) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn– phenotype) by suppressing eIF2α phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn– phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2α phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn– phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin. The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the α subunit of translation initiation factor 2 (eIF2α) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn– phenotype) by suppressing eIF2α phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn– phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2α phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn– phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin. Phosphorylation of the α subunit of translation initiation factor 2 (eIF2α) 1The abbreviations used are: eIF2α, α subunit of translation initiation factor 2; GAAC, general amino acid control; NTD, N-terminal domain; 3AT, 3-aminotriazole; 5FT, 5-fluoro-dl-tryptophan; TRA, triazolealanine; SM, sulfometuron; WCE, whole cell extract; GST, glutathione S-transferase. is a key regulatory mechanism for down-regulating protein synthesis in response to starvation or stress in eukaryotic cells. Four different eIF2α kinases have been identified in mammalian cells that are activated by different stimuli: HRI by hemin deprivation (1Chen J.-J. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 529-546Google Scholar), PKR by double-stranded RNA (2Kaufman R. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 503-527Google Scholar), PEK or PERK by unfolded proteins in the endoplasmic reticulum (3Ron D. Harding H.P. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 547-560Google Scholar), and GCN2 by amino acid starvation (4Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 185-243Google Scholar, 5Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2431) Google Scholar). eIF2 is necessary for delivery of charged initiator methionyl-tRNA (Met-tRNAMeti) to the 40 S ribosomal subunits in an eIF2-GTP-Met-tRNAMeti ternary complex and is released from the initiation complex in the inactive GDP-bound form. Phosphorylation of eIF2α at serine 51 by eIF2α kinases converts eIF2 from a substrate to an inhibitor of its guanine nucleotide exchange factor, eIF2B. Since only eIF2-GTP can bind Met-tRNAMeti, the inhibition of eIF2B evoked by eIF2 phosphorylation leads to a decrease in ternary complex formation and a general reduction in protein synthesis (4Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 185-243Google Scholar). GCN2 is the sole eIF2α kinase in the yeast Saccharomyces cerevisiae, where it was first identified as being required for growth under amino acid starvation conditions. Phosphorylation of eIF2α by GCN2 induces the translation of GCN4 mRNA, encoding a transcriptional activator of amino acid biosynthetic enzymes, while dampening the translation of most other messages. Four short open reading frames in the GCN4 mRNA leader underlie a specialized reinitiation mechanism that allows efficient translation of this mRNA when the ternary complex level drops (4Hinnebusch A.G. Sonenberg N. Hershey J.W.B. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 185-243Google Scholar). The increased expression of GCN4 and its amino acid biosynthetic target genes in starved cells is known as general amino acid control (GAAC) (6Hinnebusch A.G. Broach J.R. Jones E.W. Pringle J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 319-414Google Scholar). GCN2 is present as a latent kinase under nonstarvation conditions and is activated by uncharged tRNAs that accumulate in amino acid-starved cells. The uncharged tRNA binds to a regulatory domain in GCN2 that resembles histidyl-tRNA synthetase, and this interaction is believed to induce a conformational change that overcomes an intrinsic defect in the adjacent kinase domain (7Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar, 8Zhu S. Wek R.C. J. Biol. Chem. 1998; 273: 1808-1814Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 9Dong J. Qiu H. Garcia-Barrio M. Anderson J. Hinnebusch A.G. Mol. Cell. 2000; 6: 269-279Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 10Qiu H. Hu C. Dong J. Hinnebusch A.G. Genes Dev. 2002; 16: 1271-1280Crossref PubMed Scopus (42) Google Scholar). The products of GCN1 and GCN20 are also necessary for the activation of GCN2 by uncharged tRNA in starved cells (11Marton M.J. Crouch D. Hinnebusch A.G. Mol. Cell. Biol. 1993; 13: 3541-3556Crossref PubMed Scopus (96) Google Scholar, 12Vazquez de Aldana C.R. Marton M.J. Hinnebusch A.G. J. 1995; PubMed Scopus Google Scholar). GCN1 and GCN20 a protein complex de Aldana C.R. Marton M.J. Hinnebusch A.G. J. 1995; PubMed Scopus Google that binds to the N-terminal domain of GCN2, which is GCN2 The GCN2 is required for kinase in vivo, and its overexpression complex formation native GCN1 and GCN2 and a Gcn– control phenotype M. Dong J. S. Hinnebusch A.G. J. 2000; PubMed Google Scholar, H. Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (53) Google Scholar). in GCN1 is necessary and for complex formation with the GCN2 and, when overexpressed in vivo, with full-length GCN1 for GCN2 and a Gcn– phenotype H. Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (53) Google Scholar, Hinnebusch A.G. J. 2000; PubMed Scopus Google Scholar). in the domain of GCN1 was to be for in vivo, and the Gcn– phenotype produced by this was suppressed by GCN2 Hinnebusch A.G. 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This mechanism with that in for activation of protein by uncharged tRNA in the which the response to amino acid starvation M. M. and Cellular and Molecular for D. Scholar, H. Mol. PubMed Scopus Google Scholar). YIH1, a yeast of the of a domain in to the GCN2 and was to in the yeast with the same of GCN1 that binds to GCN2 H. Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (53) Google Scholar). YIH1 and GCN2 for the same GCN1 in vivo, YIH1 GCN2 from GCN1 and the activation of GCN2 and of GCN4 a Gcn– it was found that overexpression of YIH1 a Gcn– to an inhibitor of H. Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (53) Google Scholar). However, was that YIH1 by of the GCN1-GCN2 complex or that it as an of GCN2 when at native levels in yeast cells. this we that YIH1 the GAAC response by with GCN2 for GCN1 the of GCN1-GCN2 in the cell and phosphorylation of However, deletion of YIH1 does not have a GCN2 kinase under nonstarvation or starvation that YIH1 at native levels is not a general inhibitor of GCN2. we found that YIH1 with monomeric actin and that a reduction in actin levels a Gcn– phenotype that is at the that YIH1 GCN2 only under growth conditions or in restricted in the where YIH1 is displaced from and and used in this are in and of are as of and the was from PubMed Google as an fragment and used to to The of to was by of in Yeast Cold Spring Harbor Laboratory Press, Cold Spring to the fragment and to the of this fragment used in this M. S. Hinnebusch A.G. Mol. Cell. Biol. PubMed Scopus Google R. de Aldana and Wek R.C. Hinnebusch A.G. PubMed Google and M. Wek R.C. de Aldana C.R. Hinnebusch A.G. Mol. Cell. Biol. PubMed Google and and and and and encoding of and and and and and and and and and in a used in this in amino by the genes for expression in yeast 2 A.G. S. PubMed Scopus Google GCN2 M. Wek R.C. de Aldana C.R. Hinnebusch A.G. Mol. Cell. Biol. PubMed Google at the of the open reading Marton and at the of the open reading at the of the open reading YIH1 in at the of the open reading Hinnebusch A.G. J. 2000; PubMed Scopus Google at the of the open reading at the of the open reading Hinnebusch A.G. J. 2000; PubMed Scopus Google at the of the open reading Hinnebusch A.G. 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The deletion was as fragment of YIH1 was from yeast and of an and to of YIH1 of a and to the of YIH1 a fragment of YIH1 was and of a to the of YIH1, and are to of of a and of YIH1 a as and and the and of YIH1 and by only a The was with and and the The was from with and in of from Yeast for from of yeast in of of and of and the for 2 The was with of and used as a in vitro and in and proteins and and as Hinnebusch A.G. J. 2000; PubMed Scopus Google Scholar). by or in by with in and and with and proteins to to the and proteins the with S in to J. Molecular Laboratory Cold Spring Harbor Cold Spring Harbor, Scholar). by the GCN1 de Aldana C.R. Marton M.J. Hinnebusch A.G. J. 1995; PubMed Scopus Google GCN2 Garcia-Barrio Zhang Zhang C. Mathews M.B. J. Hinnebusch A.G. Mol. Cell. Biol. 1998; PubMed Google eIF2α Wek R.C. Hinnebusch A.G. Cell. Full Text PDF PubMed Scopus Google eIF2α actin J. D. D. D. J. Biol. PubMed Scopus Google and to to of or to protein of actin of the in to in a in inhibitor to of and a was and the was by at for in a of the with of the protein with to the of the was to a and in a of the was to a and by and the same a was and a of and with and the the with The with and the as M. M. Chem. PubMed Scopus Google Scholar). The a in a of by at a of by an the S. open reading frames the J.R. J. PubMed Scopus Google Scholar). of the identified the proteins in the as J. J.R. PubMed Scopus Google Scholar). YIH1 with GCN2 for GCN1 Binding in of the GCN2 to amino of GCN1 is for activation of GCN2 under amino acid starvation conditions Hinnebusch A.G. J. 2000; PubMed Scopus Google Scholar). YIH1 can bind to the same of GCN1 in the yeast and overexpression of YIH1 produced a Gcn– by to H. Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (53) Google Scholar). that YIH1, overexpressed, can with GCN2 for GCN1 and activation of GCN2 in cells starved for with overexpression of GCN2 overcome this and the Gcn– phenotype of this we a or at the of the YIH1 open reading and it under a overexpression of the YIH1 in an and overexpression of GCN2 from a suppressed the Gcn– phenotype with overexpression consistent with the that YIH1 GAAC by with GCN2 for the same for this we of the that a GCN2 protein by M. Wek R.C. de Aldana C.R. Hinnebusch A.G. Mol. Cell. Biol. PubMed Google a growth phenotype from of eIF2α and the consequent general inhibition of protein The phenotype of is suppressed by deletion of GCN1 M. Wek R.C. de Aldana C.R. Hinnebusch A.G. Mol. Cell. Biol. PubMed Google Scholar), suggesting that requires GCN1 for its overexpressed YIH1 GCN1-GCN2 it the phenotype with the with this overexpression of suppressed the phenotype of genetic that overexpressed YIH1 activation of GCN2 and the GAAC response by with GCN2 for to YIH1-GCN1 interaction been by the yeast and with an in vitro GCN1 the H. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that overexpressed YIH1 native GCN1 in vivo, we GCN1 can be with from of yeast cells We found that GCN1, not GCN2, with that overexpressed binds to native GCN1 not to the GCN1-GCN2 complex in This is consistent with the that YIH1 with GCN2 for GCN1 we that YIH1 reduces GCN1-GCN2 complex formation in of YIH1 or with GCN1 and the with GCN1 and GCN2 The in that of cellular GCN2 was with GCN1 in the in with Hinnebusch A.G. J. 2000; PubMed Scopus Google Scholar). was overexpressed in the the of GCN2 with GCN1 was by of cellular GCN2 with that YIH1 reduces GCN1-GCN2 complex formation in was to that the Gcn– phenotype of YIH1 from phosphorylation of eIF2α the of phosphorylation by GCN2. with this we found that YIH1 overexpression the levels of eIF2α by under starvation conditions and by in cells the in and 2 show that overexpressed YIH1 GCN2 kinase activity by GCN1-GCN2 interaction. Arg-2259 in GCN1 for YIH1 Binding in and in that of Arg-2259 in GCN1 of native GCN1 and GCN2 in and interaction of the of GCN2 with of GCN1 in vitro Hinnebusch A.G. J. 2000; PubMed Scopus Google Scholar). The that the for YIH1 and GCN2 to the same in GCN1 H. Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (53) Google Scholar, Hinnebusch A.G. J. 2000; PubMed Scopus Google that GCN2 and YIH1 with the same in YIH1 also requires Arg-2259 for GCN1 we a protein in for interaction with which the GCN1 fragment for GCN2 Hinnebusch A.G. J. 2000; PubMed Scopus Google Scholar). We found that is for to in vitro and that this interaction was by the in the GCN1 fragment to show that Arg-2259 in native GCN1 is for YIH1 in vivo, we of and GCN1 or a in GCN1, not with from cell suggest that YIH1 and GCN2 a key in GCN1 the that for complex formation with YIH1 a of the GAAC at suggest that YIH1 is a of GCN2 be to a GAAC response in starved cells. deletion of YIH1 to activation of GCN2. this we deletion of YIH1 to the and and a phenotype by known that to induction of GCN4 and the and biosynthetic genes under GAAC phenotype) M. Wek R.C. de Aldana C.R. Hinnebusch A.G. Mol. Cell. Biol. PubMed Google Scholar). the the same to and as the of a the YIH1 produced the a phenotype deletion of YIH1 not the phenotype of the under the conditions YIH1 does not to be a of GCN2. We also the that YIH1 at native levels of expression as an activator of GCN2 and the general control response. in and the and YIH1 cells in to in the of or an inhibitor of and with in known genes to and We deletion of YIH1 increase the level of eIF2α phosphorylation in nonstarvation conditions or decrease the required for to to the level the of to cells. We increase in the level of in cells under nonstarvation conditions and or in cells starved for or by the of the or we found that levels at in and to the level in The with amino as the nonstarvation We also found that not the levels of in cells the of growth the not is that YIH1 is required to GCN2 activation in nonstarvation to the of GCN2 activation in starved or to increase the at which eIF2α phosphorylation to levels when starved cells are with the amino The yeast other proteins to the in GCN2 and YIH1, known as and We the that YIH1 is with of proteins for of GCN2. this we for genes or in with for to and The that the only or the of the YIH1 and or and the from the in to not it that of the proteins to the domain in GCN2 have an in GCN2 activity in nonstarvation at under the growth conditions YIH1 a with in the cellular of YIH1 by proteins that with YIH1 at native this we a at the of YIH1 and the protein from whole cell it from the with The was by a or by a and by and to and with a protein of with and The protein was from the and to and the The identified the protein as actin, by and we this by actin and GCN1 we not GCN1 with suggesting that under the growth conditions used for of the YIH1 complex native of YIH1 does not with GCN1, or the YIH1-GCN1 interaction is to the We that GCN1 is with overexpressed in the same we found that actin also with that YIH1 a complex with monomeric actin in that does not GCN1 as a at native levels of YIH1 of YIH1 the of the of YIH1 with monomeric actin in the that YIH1 actin or we deletion or overexpression of YIH1 the of the actin this we and cells with to actin yeast cells of actin, with cells actin the and actin the cell Pringle J.R. J. Biol. PubMed Scopus Google Scholar). The and the of actin in the the cell We a of cells that for the YIH1 cells it is this phenotype can be to a defect in the actin the cells that we change in the of actin or of the in the and in the actin and YIH1 cells. overexpression of YIH1 not the or of actin it change the of actin of the Cellular a Gcn– that YIH1 interacts with actin of GCN1, and found that overexpression of YIH1 GCN2, we to a decrease in the cellular actin YIH1 from the YIH1-actin complex and GCN2. this we of the phenotype of a growth defect at that is by the of D. PubMed Google Scholar). We by of that the a level of actin with an and also is to a that actin J. N. M. J. Biol. PubMed Scopus Google not to of actin in the at to SM, a Gcn– for the is a and at the of not at the of the was to with the of a the phenotype of the that it from actin YIH1 the inhibition of GCN2 from actin YIH1 overexpression the phenotype of the with this we found that overexpression of not from a the phenotype of the at key of the that YIH1 GCN2 in the is that the phenotype of the be suppressed by of this we YIH1 in the and the of of the YIH1, or in and the YIH1 to with that This the phenotype with in the of YIH1 that was in the of the was to than that YIH1 and than that and that YIH1 a of the Gcn– phenotype by The of the with the in a of the Gcn– defect with actin was that YIH1 interacts with the domain in GCN1 and that overexpression of YIH1 to 3AT, an inhibitor of This to the that YIH1 can GCN2 by formation of the GCN1-GCN2 complex H. Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus (53) Google Scholar). this we of that the phenotype by YIH1 overexpression from of the GCN1-GCN2 complex and consequent activation of GCN2. we that overexpression of YIH1 can the growth defect by a activated of GCN2 that general translation initiation by level phosphorylation of eIF2α M. Wek R.C. de Aldana C.R. Hinnebusch A.G. Mol. Cell. Biol. PubMed Google Scholar). This genetic that YIH1 overexpression GCN2 for this from that cells YIH1 levels of in nonstarvation conditions and also in response to that overexpressed YIH1 with GCN2 for GCN1 from in that overexpressed a complex with native GCN1, not with GCN2, and from that YIH1 overexpression reduces the level of GCN1-GCN2 in We also that of YIH1 to GCN1, in vitro and in vivo, is the same in GCN1 that is for GCN1-GCN2 interaction Hinnebusch A.G. J. 2000; PubMed Scopus Google Scholar). YIH1 and GCN2 a in the of to a for YIH1 as a of GCN2 and the GAAC response. However, we to complex formation YIH1 and GCN1 in YIH1 was we not of YIH1 the GAAC response or the levels of eIF2α phosphorylation under starvation or nonstarvation conditions. We the that YIH1 be required only to a of eIF2α when starved cells are with the amino However, we not and cells in the of eIF2α when cells with we have that YIH1 as a general inhibitor of GCN2 under growth conditions. We also the that the regulatory of YIH1 be by of the other proteins in yeast that a domain to the in GCN2 and YIH1, and However, and or in with a deletion of YIH1 the GAAC response. an to the of YIH1 in vivo, we a YIH1 from the under its and found that the protein in a complex with monomeric actin of This the that the of YIH1 to GCN2 be by its with This was by that actin in an to an inhibitor of and and that this was by YIH1 overexpression. This Gcn– phenotype was partially suppressed by deletion of YIH1, that the defect in GAAC with actin was at by the actin levels in the may the of monomeric actin, to of YIH1 from the YIH1-actin complex and consequent formation of the YIH1-GCN1 complex with inhibition of GCN2. The phenotype in the a reduction in the expression or activation of GCN4 in cells with an actin with the that actin leads to YIH1-mediated inhibition of GCN2 is that we have been to a reduction in levels in the not factor is that a of the Gcn– phenotype of the is of YIH1 and may not activation of GCN2. that actin is a of transcriptional 2002; PubMed Scopus Google Scholar), a defect in transcriptional activation by GCN4 be for the of the Gcn– phenotype of the However, is that YIH1-mediated inhibition of GCN2 be a restricted to the of where an level of eIF2 activity and translation initiation This we to an increase in the of in cells. are for of eIF2α phosphorylation and control in mammalian cells Mol. Cell. Biol. PubMed Scopus (96) Google Scholar, Y. Y. Y. R. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). This that the of YIH1-actin be at the of of the actin in yeast cells in the actin of in the is that actin are of to and the protein in The actin are proteins and regulatory and are Dev. Biol. PubMed Scopus Google Scholar). been that an actin also be the of in the to the of actin as the cell to D. 2002; PubMed Scopus Google Scholar). that the of other proteins in actin at the and at the of actin YIH1 from This YIH1-GCN1 complex formation and inhibition of GCN2 activity and a mechanism for of protein synthesis in the We for with the for actin for eIF2α and Qiu for for the for the and for in the