The Polypeptide Chain-releasing Factor GSPT1/eRF3 Is Proteolytically Processed into an IAP-binding Protein (2003)

Smac/Diablo and HtrA2/Omi are inhibitors of apoptosis (IAP)-binding proteins released from the mitochondria of human cells during apoptosis and regulate apoptosis by liberating caspases from IAP inhibition. Here we describe the identification of a proteolytically processed isoform of the polypeptide chain-releasing factor GSPT1/eRF3 protein, which functions in translation, as a new IAP-binding protein. In common with other IAP-binding proteins, the processed GSPT1 protein harbors a conserved N-terminal IAP-binding motif (AKPF). Additionally, processed GSPT1 interacts biochemically with IAPs and could promote caspase activation, IAP ubiquitination and apoptosis. The IAP-binding motif of the processed GSPT1 is absolutely required for these activities. Our findings are consistent with a model whereby processing of GSPT1 into the IAP-binding isoform could potentiate apoptosis by liberating caspases from IAP inhibition, or target IAPs and the processed GSPT1 for proteasome-mediated degradation. Smac/Diablo and HtrA2/Omi are inhibitors of apoptosis (IAP)-binding proteins released from the mitochondria of human cells during apoptosis and regulate apoptosis by liberating caspases from IAP inhibition. Here we describe the identification of a proteolytically processed isoform of the polypeptide chain-releasing factor GSPT1/eRF3 protein, which functions in translation, as a new IAP-binding protein. In common with other IAP-binding proteins, the processed GSPT1 protein harbors a conserved N-terminal IAP-binding motif (AKPF). Additionally, processed GSPT1 interacts biochemically with IAPs and could promote caspase activation, IAP ubiquitination and apoptosis. The IAP-binding motif of the processed GSPT1 is absolutely required for these activities. Our findings are consistent with a model whereby processing of GSPT1 into the IAP-binding isoform could potentiate apoptosis by liberating caspases from IAP inhibition, or target IAPs and the processed GSPT1 for proteasome-mediated degradation. The inhibitors of apoptosis proteins (IAPs) 1The abbreviations used are: IAP, inhibitor of apoptosis protein; IBM, IAP-binding motif; Ub, ubiquitin; DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; GST, glutathione S-transferase; TRAIL, TNF-related apoptosis-inducing ligand. constitute a family of proteins that interact with active caspases, inhibit their protease activity and prevent programmed cell death or apoptosis (1Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1579) Google Scholar). The first member of the IAP family was identified in baculoviruses by genetic screens for modulators of baculovirus-induced apoptosis (2Crook N.E. Clem R.J. Miller L.K. J. Virol. 1993; 67: 2168-2174Crossref PubMed Google Scholar). Additional IAP family members were later found in mammals, insects, nematodes, and yeast (1Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1579) Google Scholar, 3Miller L.K. Trends Cell Biol. 1999; 9: 323-328Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 4Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2281) Google Scholar, 5Reed J.C. Bischoff J.R. Cell. 2000; 102: 545-548Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In humans there are eight family members identified so far (1Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1579) Google Scholar). All IAPs share one or more signature domains, referred to as BIRs, that are essential for the antiapoptotic activity associated with these proteins (6Duckett C.S. Li F. Wang Y. Tomaselli K.J. Thompson C.B. Armstrong R.C. Mol. Cell. Biol. 1998; 18: 608-615Crossref PubMed Scopus (193) Google Scholar). The BIR domains are zinc-binding domains that have been shown to bind directly to caspases and inhibit their activity (3Miller L.K. Trends Cell Biol. 1999; 9: 323-328Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 7Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J.C. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1719) Google Scholar). In addition to the BIR domains, some IAPs, such as human XIAP, c-IAP1, and c-IAP2, contain C-terminal RING domains (8Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Abstract Full Text Full Text PDF PubMed Scopus (1047) Google Scholar). Recent studies suggest that the RING domain functions in the regulation of ubiquitination and proteasome degradation of IAPs and IAP-associated proteins (9Holley C.L. Olson M.R. Colon-Ramos D.A. Kornbluth S. Nat. Cell Biol. 2002; 4: 439-444Crossref PubMed Scopus (181) Google Scholar, 10Hays R. Wickline L. Cagan R. Nat. Cell Biol. 2002; 4: 425-431Crossref PubMed Scopus (109) Google Scholar, 11Wing J.P. Schreader B.A. Yokokura T. Wang Y. Andrews P.S. Huseinovic N. Dong C.K. Ogdahl J.L. Schwartz L.M. White K. Nambu J.R. Nat. Cell Biol. 2002; 4: 451-456Crossref PubMed Scopus (116) Google Scholar, 12Yoo S.J. Huh J.R. Muro I. Yu H. Wang L. Wang S.L. Feldman R.M. Clem R.J. Muller H.A. Hay B.A. Nat. Cell Biol. 2002; 4: 416-424Crossref PubMed Scopus (319) Google Scholar, 13Ryoo H.D. Bergmann A. Gonen H. Ciechanover A. Steller H. Nat. Cell Biol. 2002; 4: 432-438Crossref PubMed Scopus (245) Google Scholar, 14MacFarlane M. Merrison W. Bratton S.B. Cohen G.M. J. Biol. Chem. 2002; 277: 36611-36616Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 15Li X. Yang Y. Ashwell J.D. Nature. 2002; 416: 345-347Crossref PubMed Scopus (394) Google Scholar, 16Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (869) Google Scholar, 17Wilson R. Goyal L. Ditzel M. Zachariou A. Baker D.A. Agapite J. Steller H. Meier P. Nat. Cell Biol. 2002; 4: 445-450Crossref PubMed Scopus (253) Google Scholar, 18Suzuki Y. Nakabayashi Y. Takahashi R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8662-8667Crossref PubMed Scopus (546) Google Scholar). The activity of the IAP proteins is regulated by a group of IAP-regulatory proteins that bind to IAPs and inhibit their anti-apoptotic activity (reviewed in Refs. 1Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1579) Google Scholar and 19Shi Y. Cell Death Differ. 2002; 9: 93-95Crossref PubMed Scopus (75) Google Scholar). This group of proteins is characterized by the presence of a conserved 4-residue IAP-binding motif (IBM) at their N termini that allows them to bind to the BIR domain of IAPs. This association prevents IAPs from binding to caspases and in some cases stimulates their auto-ubiquitination and degradation (9Holley C.L. Olson M.R. Colon-Ramos D.A. Kornbluth S. Nat. Cell Biol. 2002; 4: 439-444Crossref PubMed Scopus (181) Google Scholar, 12Yoo S.J. Huh J.R. Muro I. Yu H. Wang L. Wang S.L. Feldman R.M. Clem R.J. Muller H.A. Hay B.A. Nat. Cell Biol. 2002; 4: 416-424Crossref PubMed Scopus (319) Google Scholar, 13Ryoo H.D. Bergmann A. Gonen H. Ciechanover A. Steller H. Nat. Cell Biol. 2002; 4: 432-438Crossref PubMed Scopus (245) Google Scholar, 20Srinivasula S.M. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (860) Google Scholar, 21Chai J. Shiozaki E. Srinivasula S.M. Wu Q. Dataa P. Alnemri E.S. Shi Y. Cell. 2001; 104: 769-780Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). In Drosophila melanogaster, five IBM-containing proteins known as Reaper, Hid, Grim, Sickle and Jafrac2 have been identified as direct IAP-binding proteins that promote caspase activation by binding to the BIR domains of IAPs (22Abrams J.M. Trends Cell Biol. 1999; 9: 435-440Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 23Vucic D. Kaiser W.J. Miller L.K. Mol. Cell. Biol. 1998; 18: 3300-3309Crossref PubMed Scopus (188) Google Scholar, 24Goyal L. McCall K. Agapite J. Hartwieg E. Steller H. EMBO J. 2000; 19: 589-597Crossref PubMed Scopus (380) Google Scholar, 25Srinivasula S.M. Datta P. Kobayashi M. Wu J.W. Fujioka M. Hegde R. Zhang Z. Mukattash R. Fernandes-Alnemri T. Shi Y. Jaynes J.B. Alnemri E.S. Curr. Biol. 2002; 12: 125-130Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 26Christich A. Kauppila S. Chen P. Sogame N. Ho S.I. Abrams J.M. Curr. Biol. 2002; 12: 137-140Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 27Wing J.P. Karres J.S. Ogdahl J.L. Zhou L. Schwartz L.M. Nambu J.R. Curr. Biol. 2002; 12: 131-135Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 28Tenev T. Zachariou A. Wilson R. Paul A. Meier P. EMBO J. 2002; 21: 5118-5129Crossref PubMed Scopus (78) Google Scholar). Recent studies revealed that binding of some of these protein to IAPs could stimulate their auto-ubiquitination and degradation, thus preventing IAPs from inhibiting caspases (9Holley C.L. Olson M.R. Colon-Ramos D.A. Kornbluth S. Nat. Cell Biol. 2002; 4: 439-444Crossref PubMed Scopus (181) Google Scholar, 12Yoo S.J. Huh J.R. Muro I. Yu H. Wang L. Wang S.L. Feldman R.M. Clem R.J. Muller H.A. Hay B.A. Nat. Cell Biol. 2002; 4: 416-424Crossref PubMed Scopus (319) Google Scholar, 13Ryoo H.D. Bergmann A. Gonen H. Ciechanover A. Steller H. Nat. Cell Biol. 2002; 4: 432-438Crossref PubMed Scopus (245) Google Scholar). In humans two IBM-containing proteins known as Smac/DIABLO (29Du C. Fang M. Li Y. Li L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar, 30Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1972) Google Scholar) and Omi/HtrA2 (31Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, 32Verhagen A.M. Silke J. Ekert P.G. Pakusch M. Kaufmann H. Connolly L.M. Day C.L. Tikoo A. Burke R. Wrobel C. Moritz R.L. Simpson R.J. Vaux D.L. J. Biol. Chem. 2002; 277: 445-454Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 33Martins L.M. Iaccarino I. Tenev T. Gschmeissner S. Totty N.F. Lemoine N.R. Savopoulos J. Gray C.W. Creasy C.L. Dingwall C. Downward J. J. Biol. Chem. 2002; 277: 439-444Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 34Suzuki Y. Imai Y. Nakayama H. Takahashi K. Takio K. Takahashi R. Mol Cell. 2001; 8: 613-621Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 35van Loo G. van Gurp M. Depuydt B. Srinivasula S.M. Rodriguez I. Alnemri E.S. Gevaert K. Vandekerckhove J. Declercq W. Vandenabeele P. Cell Death Differ. 2002; 9: 20-26Crossref PubMed Scopus (276) Google Scholar) can bind to IAPs and prevent them from inhibiting active caspases. Interestingly, the two proteins are localized in the intermembrane space of the mitochondria and released into the cytoplasm during apoptosis. Both proteins are synthesized as precursor proteins with N-terminal mitochondrial localization signal peptides that are removed during maturation to expose the N-terminal IBM. In this report we describe the characterization of a third human IBM-containing protein. This protein is a proteolytically processed form of a previously identified protein known as GSPT1 (G1 to S phase transition protein), which functions in translation through interaction with poly(A)-binding protein PABP and as a polypeptide chain release factor 3 (eRF3) (36Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 37Uchida N. Hoshino S. Imataka H. Sonenberg N. Katada T. J. Biol. Chem. 2002; 277: 50286-50292Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Like Smac and HtrA2, the processed GSPT1 contains a conserved IBM (AKPF) at its N terminus, which is exposed after proteolytic cleavage of a 69-residue leader sequence. Our results suggest that the processed GSPT1 could participate in the apoptotic pathways by binding to IAPs and inhibiting their activity or target them for proteasome-mediated degradation. Affinity Purification of IAP-binding Proteins from 293 Cells—IAP binding proteins in 293 cellular extracts were purified on GST-BIR3-bound beads as described in detail previously (31Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). cDNA Cloning and Expression of Recombinant Proteins—The full-length human GSPT1 cDNA clone was obtained from the IMAGE Consortium (GenBank™ accession number AL516539). Constructs encoding full-length GSPT1 or truncated mutants were generated by PCR using modified complementary PCR adapter-primers. C-terminal FLAG-epitope tagging was done by cloning the PCR generated GSPT1 cDNAs into the C-terminal GSPT1 were generated by cloning the PCR generated GSPT1 cDNAs in the The was by the first of processed GSPT1 to the N of This was done by PCR using encoding the first of processed GSPT1 by the first of and a The PCR was into the The was by PCR generated human cDNA to the of cDNA using The PCR was into the The encoding C-terminal Drosophila protein was by the cDNA to the cDNA using and its domain were in as proteins using a The was a of The was in XIAP, c-IAP1, and were in in the presence of in using Smac or and its mutants were in E. as or proteins using a or In in were as described previously S.M. Hegde R. Saleh A. Datta P. Shiozaki E. Chai J. Lee R.A. Robbins P.D. Fernandes-Alnemri T. Shi Y. Alnemri E.S. Nature. 2001; 410: 112-116Crossref PubMed Scopus (860) Google Scholar, S.M. Datta P. Fernandes-Alnemri T. Z. Alnemri E.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, G. Chai J. Wu J.W. C. Wang X. Shi Y. Nature. 2000; PubMed Scopus Google Scholar). In of by IAP-binding were as described previously (31Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, S.M. Datta P. Fernandes-Alnemri T. Z. Alnemri E.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Cell Death and of processed GSPT1 to potentiate or apoptosis was by human cells in with of of or encoding human or a using the were with or for were with and and apoptotic cells were using The of apoptotic cells in was as the of apoptotic cells as a of the number of The of processed GSPT1 to the caspase activity of cells was after 293 cells with or encoding human or a using the after the cells were in and extracts were by the at The caspase activity in the extracts was after with and a of using the caspase The release of from the was by In ubiquitination were as described or were with or for the beads were to the for The beads were with ubiquitination and of proteins was in ubiquitination of with or of of of Ub, and of at for The were by The were a and for protein using to or and on were with and with human GSPT1 and a and were used as In some and protein were the were on and using were as described previously (31Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). of a of GSPT1 as a a protein as to new IAP-binding proteins from extracts of human cells and we previously 3 proteins of and (31Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). The and proteins were identified as mitochondrial Smac and proteins, the third protein, a of purified proteins from 293 cells was by and were and to and as described (31Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). N-terminal of the revealed N-terminal of the protein revealed that this is found in human GSPT1 and of a of the that is a processed form of the human GSPT1 protein, the first (36Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). the N-terminal the processed purified GSPT1 protein at with N-terminal which a conserved IBM and of the revealed that the motif is conserved in and GSPT1 that the protein is a processed form of we extracts from 293 cells to or the which bind to the IBM, and the proteins by with a shown in the in the in the The results were by of a C-terminal full-length GSPT1 protein in 293 cells of by the cells in two to the full-length and the processed GSPT1 The of the processed GSPT1 isoform in the cellular were to of the of the full-length GSPT1 protein. the processed GSPT1 isoform was to bind to that GSPT1 proteolytic processing in 293 cells to IAP-binding isoform of on the of the full-length GSPT1 protein, the IBM is at that the IAP-binding isoform of GSPT1 is generated by proteolytic processing of the full-length GSPT1 at a GSPT1 the first could interact with the protein, we this protein with a C-terminal in and purified to Smac and proteins were in and used as Like Smac and HtrA2, the processed GSPT1 was to interact with the protein with the GSPT1 interact with protein, Smac and with their interaction with of the motif in GSPT1 binding to proteins, that the motif is for binding of GSPT1 to the BIR GSPT1 with processed GSPT1 could interact with human full-length IAPs, we in interaction with processed GSPT1 proteins and in human XIAP, and shown in the processed GSPT1 was to interact with XIAP, and of the motif the interaction of GSPT1 with these IAPs. the processed GSPT1 is in a with the in 293 cellular we using for that the processed GSPT1 isoform can with these that processed GSPT1 interacts with cellular IAPs. GSPT1 studies that the IAP-binding proteins such as Smac and HtrA2, could potentiate caspase activation by preventing binding of caspases to IAPs or we in caspase activation to the processed GSPT1 protein could promote activity in extracts in the presence of of the activity in these we to the extracts and the extracts with and we have shown that Smac and could potentiate caspase cleavage these R. Wickline L. Cagan R. Nat. Cell Biol. 2002; 4: 425-431Crossref PubMed Scopus (109) Google Scholar). shown in the processed GSPT1 protein was to the of the caspase activity in the at a Smac or This activity was on IAP-binding motif as of this motif the activity of the first N-terminal of GSPT1 to was to to the as the processed GSPT1 at This that the IBM of GSPT1 is for its IAP-binding and caspase activities. The of the caspase activity of GSPT1 was by the activity of extracts with and as The that the activity in the extracts with HtrA2, or the GSPT1 proteins, with the of cleavage shown in the Expression of GSPT1 in and the processed IAP-binding form of GSPT1 we a encoding N-terminal protein, which the first The protein contains a C-terminal to by The protein is on the that the and other proteins can by which a and at the H.D. Bergmann A. Gonen H. Ciechanover A. Steller H. Nat. Cell Biol. 2002; 4: 432-438Crossref PubMed Scopus (245) Google Scholar, 14MacFarlane M. Merrison W. Bratton S.B. Cohen G.M. J. Biol. Chem. 2002; 277: 36611-36616Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 15Li X. Yang Y. Ashwell J.D. Nature. 2002; 416: 345-347Crossref PubMed Scopus (394) Google Scholar). of the protein in 293 cells in of the processed which was to bind of the processed GSPT1 in 293 cells caspase we extracts from the 293 cells with shown in the activity in the extracts from the processed cells was the activity in the extracts from the or that binding of the processed GSPT1 to cellular IAPs can caspase activity in these The of the processed GSPT1 to bind to IAPs and caspase activation in human cells that could potentiate apoptosis. this we cells with the encoding full-length or processed GSPT1 and their to apoptotic such as and shown in of the processed GSPT1 protein, the full-length the apoptotic of and suggest that GSPT1 could potentiate cell death by IAPs. GSPT1 the of studies that binding of the Drosophila IAP-binding proteins and to stimulates its ubiquitination activity (9Holley C.L. Olson M.R. Colon-Ramos D.A. Kornbluth S. Nat. Cell Biol. 2002; 4: 439-444Crossref PubMed Scopus (181) Google Scholar, 12Yoo S.J. Huh J.R. Muro I. Yu H. Wang L. Wang S.L. Feldman R.M. Clem R.J. Muller H.A. Hay B.A. Nat. Cell Biol. 2002; 4: 416-424Crossref PubMed Scopus (319) Google Scholar, 13Ryoo H.D. Bergmann A. Gonen H. Ciechanover A. Steller H. Nat. Cell Biol. 2002; 4: 432-438Crossref PubMed Scopus (245) Google which could to degradation of the by the binding of the processed GSPT1 to cellular in could stimulate its ubiquitination we processed GSPT1 to or RING human proteins and the in the presence or of and shown in the processed GSPT1 was to stimulate the ubiquitination activity of the RING the of processed GSPT1 to stimulate ubiquitination activity was on its as of this motif ubiquitination ubiquitination was the first N-terminal of processed GSPT1 to was that other of GSPT1 are required for of ubiquitination processed GSPT1 was to the auto-ubiquitination activity of or GSPT1 with the the localization of we cells with a with its in translation and its association with the of the cells revealed that the GSPT1 protein of endoplasmic localization the localization of we the cells with which is a known shown in the GSPT1 and was the localization of we cells with a encoding C-terminal full-length GSPT1 protein and the cells with and protein which is known The full-length GSPT1 protein which with the protein The localization of GSPT1 was by and using This the presence of a in the and protein in the mitochondrial or The presence of GSPT1 in the that GSPT1 is associated with the and its association with could of cells in a the localization of the processed we cells with a encoding C-terminal processed GSPT1 protein, and the cells with Interestingly, of the first in of GSPT1 in the cytoplasm and that its association with the is by of its N suggest that proteolytic processing of GSPT1 at could in release of the processed GSPT1 from the into the The of apoptotic in the cell are the death and the which is to proteins that regulate apoptosis and the third is the endoplasmic which the to apoptotic G. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). The first two IAP Smac and HtrA2, in the mitochondrial intermembrane space and are released with which the apoptotic (31Hegde R. Srinivasula S.M. Zhang Z. Wassell R. Mukattash R. Cilenti L. DuBois G. Lazebnik Y. Zervos A.S. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2002; 277: 432-438Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). In this we that the associated GSPT1 protein could participate in the death pathways of cells after proteolytic processing into IAP-binding protein. Our that processed GSPT1 cells to apoptotic that processed GSPT1 could with the other proteins, Smac and HtrA2, to regulate apoptosis. The that processed GSPT1 apoptosis by binding to IAPs and their interaction with the and the caspases 3 and the activation of these caspases. the processed GSPT1 protein could promote apoptosis by ubiquitination and degradation of the of the family of proteins is at these proteins and to of the yeast known as (36Hoshino S. Imai M. Mizutani M. Kikuchi Y. Hanaoka F. Ui M. Katada T. J. Biol. Chem. 1998; 273: 22254-22259Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The yeast is a protein to polypeptide factor and functions as a polypeptide factor 3 (eRF3) in in the were shown to the of E.S. PubMed Scopus Google Scholar, J.M. F. J. Mol. Biol. PubMed Scopus Google Scholar, Google Scholar). The human GSPT1 was shown to of the of the yeast S. H. T. Hanaoka F. Kikuchi Y. Kikuchi A. Ui M. EMBO J. 8: PubMed Scopus Google that human GSPT1 is a of the yeast and could as Recent results suggest that proteins could in translation through association with PABP N. Hoshino S. Imataka H. Sonenberg N. Katada T. J. Biol. Chem. 2002; 277: 50286-50292Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). processing of GSPT1 into IAP-binding protein to during cellular by as of GSPT1 by or other cellular the that the or the GSPT1 protein was processed to the IAP-binding form the that processing of GSPT1 could a to the protein of GSPT1 during the cell by for degradation the IAP This is consistent with the that processed can the activity of c-IAP1, and that of GSPT1 is cell and is at the to S phase of the cell S. H. T. Hanaoka F. Kikuchi Y. Kikuchi A. Ui M. EMBO J. 8: PubMed Scopus Google Scholar). Duckett for the and Ashwell for the