Isolation of a Protein Complex Containing Translation Initiation Factor Prt1 from Saccharomyces cerevisiae
1995; Elsevier BV; Volume: 270; Issue: 9 Linguagem: Inglês
10.1074/jbc.270.9.4288
ISSN1083-351X
AutoresParisa Danaie, B. Wittmer, Michael Altmann, Hans Trachsel,
Tópico(s)RNA Research and Splicing
ResumoTranslation initiation factor Prt1 was purified from a ribosomal salt wash fraction of Saccharomyces cerevisiae cells by ammonium sulfate precipitation, DEAE chromatography, phosphocellulose chromatography, sucrose density gradient centrifugation, and non-denaturing polyacrylamide gel electrophoresis. Prt1 protein cofractionates with four other polypeptides during all steps of purification suggesting that it is part of a protein complex containing polypeptide subunits with apparent molecular masses of 130, 80, 75 (Prt1), 40, and 32 kDa. Deletion of the first AUG codon in the published sequence of the PRT1 gene results in the synthesis of functional Prt1 protein indicating that the actual molecular mass of the Prt1 subunit is 82.7 kDa. This is in agreement with results from primer extension experiments reported earlier by Keierleber et al. (Keierleber, C., Wittekind, M., Qin, S., and McLaughlin, C. S.(1986) Mol. Cell. Biol. 6, 4419-4424). The Prt1-containing protein complex is an active translation factor as shown by its ability to restore translation in a cell-free system derived from a temperature-sensitive prt1 mutant strain in which endogenous Prt1 activity is inactivated by heating the extract to 37°C. The question of whether the Prt1-containing protein complex represents the yeast homologue of mammalian translation initiation factor eIF-3 is discussed. Translation initiation factor Prt1 was purified from a ribosomal salt wash fraction of Saccharomyces cerevisiae cells by ammonium sulfate precipitation, DEAE chromatography, phosphocellulose chromatography, sucrose density gradient centrifugation, and non-denaturing polyacrylamide gel electrophoresis. Prt1 protein cofractionates with four other polypeptides during all steps of purification suggesting that it is part of a protein complex containing polypeptide subunits with apparent molecular masses of 130, 80, 75 (Prt1), 40, and 32 kDa. Deletion of the first AUG codon in the published sequence of the PRT1 gene results in the synthesis of functional Prt1 protein indicating that the actual molecular mass of the Prt1 subunit is 82.7 kDa. This is in agreement with results from primer extension experiments reported earlier by Keierleber et al. (Keierleber, C., Wittekind, M., Qin, S., and McLaughlin, C. S.(1986) Mol. Cell. Biol. 6, 4419-4424). The Prt1-containing protein complex is an active translation factor as shown by its ability to restore translation in a cell-free system derived from a temperature-sensitive prt1 mutant strain in which endogenous Prt1 activity is inactivated by heating the extract to 37°C. The question of whether the Prt1-containing protein complex represents the yeast homologue of mammalian translation initiation factor eIF-3 is discussed. INTRODUCTIONTranslation initiation is a multistep pathway, which positions an 80 S ribosome with bound initiator Met-tRNAi at the initiator AUG codon of an open reading frame on mRNA(1Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar, 2Merrick W.C. Microbiol. Rev. 1992; 56: 291-315Crossref PubMed Google Scholar). In eukaryotes, the main initiation pathway is cap-dependent; ribosomes bind near the cap structure m7GpppN at the 5′ end of mRNA and then scan the mRNA in the 5′ to 3′ direction until they encounter an initiator AUG codon(3Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2794) Google Scholar). These reactions are catalyzed by a large number of polypeptides, the eukaryotic initiation factors (eIFs) 1The abbreviations used are: eIFeukaryotic initiation factorPAGEpolyacrylamide gel electrophoresis. (1Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar, 2Merrick W.C. Microbiol. Rev. 1992; 56: 291-315Crossref PubMed Google Scholar).Experiments with the yeast Saccharomyces cerevisiae revealed that translation initiation in this lower eukaryote strongly resembles cap-dependent initiation in mammals(4Müller P.P. Trachsel H. Eur. J. Biochem. 1990; 191: 257-261Crossref PubMed Scopus (14) Google Scholar, 5Linder P. Antonie Leeuwenhoek. 1992; 62: 47-62Crossref PubMed Scopus (14) Google Scholar, 6Feng L. Donahue T.F. Ilan J. Translational Regulation of Gene Expression 2. Plenum Press, New York1993: 69-86Crossref Google Scholar). This is perhaps most convincingly demonstrated by the finding that some mammalian initiation factors can substitute for yeast factors in vivo(7Altmann M. Müller P.P. Pelletier J. Sonenberg N. Trachsel H. J. Biol. Chem. 1989; 264: 12145-12147Abstract Full Text PDF PubMed Google Scholar, 8Schwelberger H.G. Kang H.A. Hershey J.W.B. J. Biol. Chem. 1993; 268: 14018-14025Abstract Full Text PDF PubMed Google Scholar). The availability of yeast cell-free translation systems (9Gasior E. Herrera F. Sadnik I. McLaughlin C.S. Moldave K. J. Biol. Chem. 1979; 254: 3965-3969Abstract Full Text PDF PubMed Google Scholar, 10Tuite M.F. Plesset J. Yeast. 1986; 2: 35-52Crossref PubMed Scopus (31) Google Scholar) and powerful genetic approaches make this system an attractive model system to study the mechanism and regulation of translation in eukaryotes.Most of the translation initiation factors identified earlier in the mammalian system have also been isolated and their genes cloned from S. cerevisiae(4Müller P.P. Trachsel H. Eur. J. Biochem. 1990; 191: 257-261Crossref PubMed Scopus (14) Google Scholar, 5Linder P. Antonie Leeuwenhoek. 1992; 62: 47-62Crossref PubMed Scopus (14) Google Scholar, 6Feng L. Donahue T.F. Ilan J. Translational Regulation of Gene Expression 2. Plenum Press, New York1993: 69-86Crossref Google Scholar). In addition, yeast initiation factors were identified and their genes cloned whose mammalian homologues are not yet known(6Feng L. Donahue T.F. Ilan J. Translational Regulation of Gene Expression 2. Plenum Press, New York1993: 69-86Crossref Google Scholar, 11Hinnebusch A.G. Wek R.C. Dever T.E. Cigan A.M. Feng L. Donahue T.F. Ilan J. Translational Regulation of Gene Expression 2. Plenum Press, New York1993: 87-115Crossref Google Scholar).Among the few initiation factors that have not yet been isolated from yeast is the factor eIF-3. Mammalian eIF-3 is composed of eight polypeptide chains with a total mass of about 550 kDa(1Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar, 2Merrick W.C. Microbiol. Rev. 1992; 56: 291-315Crossref PubMed Google Scholar). Reconstitution experiments showed that this factor is involved in several consecutive steps in the initiation pathway including ribosome dissociation, Met-tRNAi binding to 40 S ribosomes, and mRNA binding to 40 S•Met-tRNAi complexes(1Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar).The analysis of the yeast mutant strain prt1-1 originally isolated by Hartwell (12Hartwell L.H. J. Bacteriol. 1967; 93: 1662-1670Crossref PubMed Google Scholar) showed that Prt1 protein stimulates the interaction of the ternary complex eIF-2•GTP•Met-tRNAi with the 40 S ribosomal subunit(13Feinberg B. McLaughlin C.S. Moldave K. J. Biol. Chem. 1982; 257: 10846-10851Abstract Full Text PDF PubMed Google Scholar). Based on these findings and unpublished data, 2K. Moldave and C. S. McLaughlin, unpublished data. Moldave and McLaughlin (14Moldave K. McLaughlin C.S. NATO ASI Ser. 1988; 14: 271-281Google Scholar) suggested that the PRT1 gene may encode a subunit of eIF-3. The cloning of the PRT1 gene (15Keierleber C. Wittekind M. Qin S. McLaughlin C.S. Mol. Cell. Biol. 1986; 6: 4419-4424Crossref PubMed Scopus (17) Google Scholar, 16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar) and the development of a cell-free system in which Prt1 activity can be measured (17Mandel T. Trachsel H. Biochim. Biophys. Acta. 1989; 1007: 80-83Crossref PubMed Scopus (2) Google Scholar) allowed attempts to purify Prt1. Here, we show that Prt1 is a subunit of a protein complex that may be the yeast homologue of mammalian eIF-3.MATERIALS AND METHODSPurification of the Prt1-containing Protein ComplexHomogenization of CellsStrain ABYS (a,pra1,prb1,prc1,prs1,ade(18Achstetter T. Emter O. Ehmann C. Wolf D.H. J. Biol. Chem. 1984; 259: 13334-13343Abstract Full Text PDF PubMed Google Scholar) was grown in YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 30°C to an optical density of about 4 at 600 nm. About 100 g of cells (wet weight) were resuspended in 200 ml of buffer A (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 7 mM β-mercaptoethanol) and homogenized with 1 volume of glass beads in a bead beater (Biospec Products) for 2 min at 0°C. All subsequent steps were carried out at 0-4°C.Preparation of Ribosomal Salt WashThe homogenate was first centrifuged at 7,500 × g for 15 min, then at 23,000 × g for 15 min, and finally at 260,000 × g for 2 h. The resulting supernatant was frozen and the pellet (crude ribosomes) resuspended in 60 ml of buffer A containing 0.35 M KCl at a concentration of about 200 A260/ml. Ribosomes were pelleted at 260,000 × g for 2 h and proteins in the the ribosomal wash fraction (supernatant) precipitated by addition of solid (NH4)2SO4 to 70% saturation. Precipitated protein was pelleted at 7,500 × g for 15 min, dissolved in 10 ml of buffer B (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.1 mM EDTA, 7 mM β-mercaptoethanol), and dialyzed against this buffer overnight.DEAE ChromatographyThe ribosomal salt wash fraction was diluted to 25 ml with buffer B and loaded onto a DEAE column (2-cm diameter, 50-ml volume) preequilibrated with buffer B. Unbound protein was washed out with buffer B and bound protein eluted stepwise with buffer B containing 150 mM KCl and buffer B containing 300 mM KCl at a flow rate of about 13 ml/h.Phosphocellulose ChromatographyThe 300 mM KCl step eluate from the DEAE column was diluted with buffer C (30 mM phosphate buffer, pH 7.4, 0.1 mM EDTA) to a final KCl concentration of 100 mM and applied to a phosphocellulose column (1.5-cm diameter, 16-ml volume) preequilibrated with buffer C containing 100 mM KCl. Unbound protein was washed out with buffer C containing 100 mM KCl and bound protein eluted with buffer C containing 300 mM KCl. The 300 mM KCl eluate fraction was precipitated by dialysis against (NH4)2SO4 (70% saturation), and precipitated protein was collected by centrifugation for 10 min at 16,000 × g, dissolved in 0.3 ml of buffer B, and dialyzed against buffer B for 5 h.Sucrose Density Gradient Centrifugation300 μl of dialyzed 300 mM KCl eluate from the phosphocellulose column was applied to a 12-ml 5-30% sucrose gradient in 20 mM Tris-HCl, pH 7.5, 300 mM KCl, 0.1 mM EDTA. The sucrose gradient was centrifuged at 290,000 × g for 16 h. The tube was then punctured at the bottom and 0.5-ml fractions collected.SDS-PAGE and Western BlottingSDS-PAGE (19Anderson C.W. Baum P.R. Gesteland R.F. J. Virol. 1973; 12: 241-252Crossref PubMed Google Scholar) and Western blotting (20Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44704) Google Scholar) were done as described. Silver staining of SDS-polyacrylamide gels was done according to Oakley et al.(21Oakley B.R. Kirsch D.R. Morris N.R. Anal. Biochem. 1980; 105: 361-363Crossref PubMed Scopus (2436) Google Scholar).Polyclonal Rat Anti-Prt1 AntibodyA 2900-base pair EcoRI DNA fragment containing the entire PRT1 open reading frame starting at the most 5′-proximal EcoRI restriction site (16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar) was cloned into the phage λ gt11(22Young R.A. Davies R.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1194-1198Crossref PubMed Scopus (1284) Google Scholar). Escherichia coli cells were infected with recombinant phage, and the synthesis of the β-galactosidase-Prt1 hybrid protein was induced on agar plates under conditions of cell lysis as described(22Young R.A. Davies R.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1194-1198Crossref PubMed Scopus (1284) Google Scholar). Cell lysates were collected from agar plates and fractionated by SDS-PAGE, and the hybrid protein was excised and eluted from gel slices with an Electro-Eluter (model 422, Bio-Rad). Polyclonal antibodies against Prt1 were obtained by injecting rats intraperitoneally with 12 μg of β-galactosidase-Prt1 hybrid protein in 50% complete Freund's adjuvant. The injection was repeated two times with incomplete Freund's adjuvant at intervals of 10 days. The rat was killed by cervical dislocation, and blood was collected from the heart and centrifuged; serum was stored in aliquots at −70°C.In Vitro TranslationThe strain P501-1 (a,his6,ura3,prt1-1) was used for the preparation of the cell-free translation system. Preparation of the extract, cell-free translation, and the preparation of total yeast RNA were as described earlier(23Altmann M. Edery I. Sonenberg N. Trachsel H. Biochemistry. 1985; 24: 6085-6089Crossref PubMed Scopus (51) Google Scholar).Expression of Prt1 Protein from pGAL Promoter in VivoThe plasmid pGEM1 (Promega) containing the PRT1 gene as a 3000-base pair long HindIII restriction fragment (16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar) was cleaved with the restriction enzymes SacI and KpnI, and the SacI/KpnI restriction fragment was separated from the vector by agarose gel electrophoresis. The vector was then ligated to a SacI/KpnI DNA fragment on which the PRT1 open reading frame starts with the AUG at position 490 (numbering of (16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar). This fragment was generated by polymerase chain reaction using the 5′ primer 5′-CCGAGCTCAGATCTAAGGAGATAATTCAAATGACT-3′ (SacI site underlined, initiator codon in boldface) and the 3′ primer 5′-AAATAGGTACCCTTTGGGG-3′ (KpnI site underlined). From this plasmid the entire PRT1 open reading frame was excised by digestion with SacI and HindIII and inserted behind the yeast GAL1/10 promoter in the shuttle vector K10. This plasmid was amplified in E. coli and transformed into the yeast strain T92A (a,leu2,ura3,prt1-1).RESULTSPurification of the Prt1-containing Protein ComplexWe used a polyclonal rat anti-Prt1 antibody to identify and quantitate Prt1 protein by Western blotting during purification (see "Materials and Methods"). As starting material for the purification we chose the ribosomal salt wash fraction because pilot experiments had indicated that this fraction contained about 50% of the Prt1 protein but only a minor part of total cellular protein. Separation of the ribosomal wash fraction by DEAE-cellulose chromatography, phosphocellulose chromatography, and by sucrose density gradient centrifugation (see "Materials and Methods") resulted in a roughly 20-fold purification and the recovery of about 5% of Prt1 protein (Table 1). Substantial loss of Prt1 protein occurred during phosphocellulose chromatography. We don't know the reason for this.Tabled 1The optical density profile of the sucrose gradient showed a major peak of fast sedimenting material in fractions 7-9 followed by multiple peaks of more slowly migrating components (Fig. 1A). Individual fractions of the sucrose gradient were analyzed by SDS-PAGE and Western blotting. The Coomassie Brilliant Blue-stained gel revealed the copurification of five major polypeptides in fractions 7-9 (Fig. 1B, lanes3-6). They are most likely associated in a protein complex (see below). Two less abundant polypeptides (arrowhead and arrow in Fig. 1B) are probably not true subunits of the complex; the 50-kDa polypeptide (arrow) does not comigrate with the other polypeptides under the high salt concentration condition of the gradient but appears to migrate slower. The 116-kDa subunit (arrowhead) appears as a minor band after sucrose gradient centrifugation in this preparation (and not at all in another preparation, result not shown) but becomes more prominent upon further purification (compare Fig. 2B, lanes 2-4). This indicates that it may be a proteolytic digestion product of the largest polypeptide. The main polypeptides have apparent molecular masses of about 130, 80, 75, 40, and 32 kDa. The 75-kDa polypeptide is the Prt1 protein as judged from its reaction with the polyclonal rat anti-Prt1 antibody (Fig. 1C). The vast majority of Prt1 protein comigrates with the complex, and we estimate the protein complex to be at least 70% pure after sucrose density gradient centrifugation (Fig. 1B).Figure 1:SDS-PAGE and Western blot analysis of sucrose density gradient fractions. A, optical density profile of sucrose gradient loaded with approximately 1.5 mg of protein of 300 mM KCl eluate fraction from phosphocellulose column (see "Materials and Methods"). Sedimentation was from right to left. B, Coomassie Brilliant Blue-stained SDS-polyacrylamide gel. Lane1, marker proteins (prestained SDS-PAGE standards from Bio-Rad): phosphorylase b (106 kDa), bovine serum albumin (80 kDa), ovalbumin (49 kDa), carbonic anhydrase (32 kDa), soybean trypsin inhibitor (27 kDa), and lysozyme (17 kDa); lane2, 12 μg of 300 mM KCl eluate fraction from the phosphocellulose column; lanes 3-6, 20 μl of sucrose gradient fractions 6-9. C, peroxidase-stained Western blot. Samples were the same as in panelB.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2:Non-denaturing gel electrophoresis. A, lane1, marker proteins (see legend to Fig. 1); lane2, about 15 μg of sucrose density gradient-purified protein was fractionated on a polyacrylamide gel as described for SDS-PAGE, except that the gel, running buffer, and sample buffer did not contain SDS (non-denaturing condition). The gel was stained with Coomassie Blue. B, lane1, marker proteins; lane2, about 1.5 μg of protein complex after sucrose density gradient centrifugation; lane3, the slowest migrating band in panelA (arrow) was cut out, the gel piece loaded onto a SDS-polyacrylamide gel, and proteins fractionated by SDS-PAGE and stained with Coomassie Brilliant Blue; lane4, as lane3, but the gel was stained with silver.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further substantiate that the Prt1 protein is associated with additional polypeptides in a stable protein complex we subjected the sucrose gradient-purified protein to non-denaturing gel electrophoresis (Fig. 2). Two main bands (arrow and arrowhead) and several minor bands were observed under non-denaturing conditions (Fig. 2A, lane2). We cut the main bands individually out of the gel and analyzed them by SDS-PAGE and staining with Coomassie Brilliant Blue (Fig. 2B, lane3) and with silver (Fig. 2B, lane4). Both produced the same band pattern; we obtained the five main bands discussed in Fig. 1B. Protein blotting verified that the 75-kDa band in the complex after non-denaturing gel electrophoresis is Prt1 protein (not shown). These findings support our observation that the 130-, 80-, 40-, and 32-kDa polypeptides form a stable protein complex with Prt1 protein.The Prt1-containing Protein Complex Is a Translation FactorCell-free translation systems derived from prt1-1 strains (strain P501-1 in this study) are defective in translation initiation when the cells are incubated at 37°C (13Feinberg B. McLaughlin C.S. Moldave K. J. Biol. Chem. 1982; 257: 10846-10851Abstract Full Text PDF PubMed Google Scholar) or when the extract from cells grown at a lower temperature is preincubated at 37°C(17Mandel T. Trachsel H. Biochim. Biophys. Acta. 1989; 1007: 80-83Crossref PubMed Scopus (2) Google Scholar). We tested our Prt1-containing protein complex for biological activity in a cell-free system derived from strain P501-1 (Fig. 3). Preincubation of this extract at 37°C for 5 min results in very low methionine incorporation in the absence of mRNA or with total yeast RNA as mRNA (Fig. 3B). Addition of sucrose gradient-purified Prt1-containing protein complex (100 (0.28 pmol) or 200 ng (0.56 pmol)) stimulates translation 8-12-fold (Fig. 3B), whereas the same protein fraction stimulates translation in an untreated extract (kept at low temperature) only 1.5-3-fold (Fig. 3A). Apparently, the endogenous temperature-sensitive Prt1 protein can be inactivated by preincubation at 37°C, and Prt1 activity can be restored by addition of Prt1-containing protein complex. The stimulation of untreated extract by Prt1-containing protein complex varied somewhat between no stimulation and 2-3-fold stimulation.Figure 3:In vitro translation. Extract of strain P501-1 was treated with micrococcal nuclease and either kept at 0°C (A) or incubated at 37°C for 5 min (B) before the addition of [35S]methionine (4 μCi/reaction). Where indicated, 5 μg of total yeast RNA was added as mRNA. Incubation mixtures (12 μl of total volume) were incubated at 25°C, and 4-μl aliquots were assayed for methionine incorporation into protein. A, lysate kept at 0°C. □, minus mRNA; ◇, plus mRNA; ○, plus mRNA plus 100 ng (0.28 pmol) of protein complex; ▵, plus mRNA plus 200 ng (0.56 pmol) of protein complex. B, lysate preincubated at 37°C. □, minus mRNA; ◇, plus mRNA; ○, plus mRNA plus 100 ng (0.28 pmol) of protein complex; ▵, plus mRNA plus 200 ng (0.56 pmol) of protein complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since it was shown earlier that the yeast mutant strain prt1-1 is deficient in ternary complex eIF-2•GTP•Met-tRNAi binding to the 40 S ribosomal subunit(13Feinberg B. McLaughlin C.S. Moldave K. J. Biol. Chem. 1982; 257: 10846-10851Abstract Full Text PDF PubMed Google Scholar), it was pertinent to test whether Prt1-containing protein complex is able to stimulate this reaction. We incubated preheated P501-1 extracts under translation conditions for 5 min and fractionated them on sucrose density gradients. A dose-dependent stimulation of methionine binding to ribosomes was obtained (Fig. 4). Since the extract was analyzed under translation conditions the subunit-joining reaction converted most of the 40 S•Met-tRNAi complexes into 80 S initiation complexes.Figure 4:Methionine binding to ribosomes. Extract of strain P501-1 was treated as described for Fig. 3, heated for 5 min at 37°C, and supplied with [35S]methionine (5 μCi/reaction). Incubation mixtures (12 μl, about 5 pmol of ribosomes) were incubated at 25°C for 5 min, diluted with 38 μl of dilution buffer (20 mM Tris-HCl, 100 mM KCl, 2 mM MgCl2), and layered onto 4 ml of 5-30% sucrose density gradients in dilution buffer. Gradients were centrifuged at 370,000 × g for 90 min at 4°C, and fractions (170 μl) were collected and counted in a scintillation counter. □, no factor added; ◇, plus 100 ng (0.28 pmol) of protein complex; ○, plus 200 ng (0.56 pmol) of protein complex. Sedimentation was from right to left. The positions of the 40 and 80 S ribosomes in the gradient are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)These results show that Prt1 protein in the complex is active as a translation factor in vitro.The N Terminus of Prt1Two in-frame AUG codons separated by 39 codons are potential start codons for the Prt1 amino acid sequence (AUG codons +373 and +490 in the Prt1 sequence of Hanic-Joyce et al.(16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar). Mapping of the 5′ end of PRT1 mRNA by S1 nuclease digestion experiments (Keierleber et al.(15Keierleber C. Wittekind M. Qin S. McLaughlin C.S. Mol. Cell. Biol. 1986; 6: 4419-4424Crossref PubMed Scopus (17) Google Scholar) indicated that the major transcription product might begin at position +481 (numbering of Hanic-Joyce et al.(16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar), only 9 nucleotides upstream of the second AUG codon. This would result in the synthesis of a polypeptide chain of 82.7 kDa molecular mass. This is closer to the molecular mass of the Prt1 band we identify by Western blotting in our protein complex than the molecular mass (88.1 kDa) originally proposed(16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar). To find out whether Prt1 protein starting with the methionine encoded by the second AUG (position +490) is functional in vivo we expressed this protein under the control of the GAL1/10 promoter in the temperature-sensitive strain T92A (see "Materials and Methods"). Cells carrying the construct encoding Prt1 protein but not cells transformed with the vector alone were able to grow at 37°C (Fig. 5). This shows that Prt1 protein starting with methionine encoded by the second AUG codon (position +490 in the sequence of Hanic-Joyce et al.(16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar) is active in vivo.Figure 5:Growth of yeast transformants at 37°C. Strain T92A was transformed with either the vector K10 (clone K10) or K10 containing the PRT1 open reading frame starting at position +490 in the sequence of Hanic-Joyce et al.(16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar) (clones 1-3). Transformants were plated on a YPGal plate (1% yeast extract, 2% peptone, 2% galactose), and the plate was incubated for 2 days at 37°C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe polypeptide Prt1 is most likely expressed from the AUG at position +490 in the sequence published by Hanic-Joyce et al.(16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar) and has therefore a molecular mass of 82.7 kDa. This is supported by (a) the S1 nuclease digestion experiments of Keierleber et al.(15Keierleber C. Wittekind M. Qin S. McLaughlin C.S. Mol. Cell. Biol. 1986; 6: 4419-4424Crossref PubMed Scopus (17) Google Scholar), (b) the finding that the polypeptide starting with methionine encoded by the AUG at position +490 is active in vivo (Fig. 5), and (c) the apparent molecular weight of Prt1 protein on SDS gels (Figure 1:, Figure 2:). Prt1 is a subunit of a protein complex, which is stable under conditions of high salt concentration on sucrose density gradients (Fig. 1) and during non-denaturing acrylamide gel electrophoresis (Fig. 2). The abundance of the Prt1-containing protein complex in yeast cells was estimated to be about 0.2 copies/ribosome. This estimation is based on our recovery of protein complex during purification and the determination of the ribosome concentration by absorption at 260 nm after the high salt washing step. Since Prt1 protein is stably integrated in the protein complex and active in the restoration of translation in an extract in which endogenous Prt1 has been inactivated, we assume that Prt1 protein is active as a subunit of the protein complex.Our data support (but do not prove) the earlier claim (14Moldave K. McLaughlin C.S. NATO ASI Ser. 1988; 14: 271-281Google Scholar) that Prt1 is a subunit of the yeast homologue of mammalian eIF-3: (a) the purification scheme for Prt1 described in this work is very similar to the one used earlier by Trachsel et al.(24Trachsel H. Erni B. Schreier M.H. Staehelin T. J. Mol. Biol. 1977; 116: 755-767Crossref PubMed Scopus (205) Google Scholar) to purify the mammalian factor; (b) the protein complex is a translation factor as shown previously (12Hartwell L.H. J. Bacteriol. 1967; 93: 1662-1670Crossref PubMed Google Scholar, 13Feinberg B. McLaughlin C.S. Moldave K. J. Biol. Chem. 1982; 257: 10846-10851Abstract Full Text PDF PubMed Google Scholar, 14Moldave K. McLaughlin C.S. NATO ASI Ser. 1988; 14: 271-281Google Scholar) and in Figure 3:, Figure 4: of this work; and (c) like eIF-3, the Prt1-containing protein complex has RNA binding activity. The latter was shown by incubating the Prt1-containing protein complex with capped and uncapped (in vitro transcribed) RNA of 227 nucleotides in length containing an AUG translation initiation codon and measuring RNA-protein complex formation by filtration through a nitrocellulose filter. In this assay the Prt1-containing protein complex retains RNA on the filter (results not shown).The Prt1-containing protein complex has fewer subunits and a lower molecular weight than mammalian eIF-3. The estimated molecular mass (340 kDa) is in agreement with data from Cigan et al.(25Cigan M.A. Foiani M. Hannig M.E. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3217-3228Crossref PubMed Scopus (93) Google Scholar) showing that Prt1 is in a complex, which has a similar sedimentation behavior in glycerol gradients like yeast eIF-2B whose molecular mass is estimated to be 300 kDa(26Cigan M.A. Bushman J.L. Boal T.R. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5350-5354Crossref PubMed Scopus (69) Google Scholar). We cannot exclude the possibility that the Prt1-containing protein complex contains additional subunits and that we lose these proteins during purification. Such proteins could reassociate with our purified complex in the in vitro translation system and restore active factor.Based on our findings we believe that the Prt1-containing protein complex very likely represents the yeast homologue of mammalian eIF-3. The isolation of eIF-3 from the yeast S. cerevisiae is an important step toward the elucidation of the functions of this complex factor in translation initiation in eukaryotes. INTRODUCTIONTranslation initiation is a multistep pathway, which positions an 80 S ribosome with bound initiator Met-tRNAi at the initiator AUG codon of an open reading frame on mRNA(1Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar, 2Merrick W.C. Microbiol. Rev. 1992; 56: 291-315Crossref PubMed Google Scholar). In eukaryotes, the main initiation pathway is cap-dependent; ribosomes bind near the cap structure m7GpppN at the 5′ end of mRNA and then scan the mRNA in the 5′ to 3′ direction until they encounter an initiator AUG codon(3Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2794) Google Scholar). These reactions are catalyzed by a large number of polypeptides, the eukaryotic initiation factors (eIFs) 1The abbreviations used are: eIFeukaryotic initiation factorPAGEpolyacrylamide gel electrophoresis. (1Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar, 2Merrick W.C. Microbiol. Rev. 1992; 56: 291-315Crossref PubMed Google Scholar).Experiments with the yeast Saccharomyces cerevisiae revealed that translation initiation in this lower eukaryote strongly resembles cap-dependent initiation in mammals(4Müller P.P. Trachsel H. Eur. J. Biochem. 1990; 191: 257-261Crossref PubMed Scopus (14) Google Scholar, 5Linder P. Antonie Leeuwenhoek. 1992; 62: 47-62Crossref PubMed Scopus (14) Google Scholar, 6Feng L. Donahue T.F. Ilan J. Translational Regulation of Gene Expression 2. Plenum Press, New York1993: 69-86Crossref Google Scholar). This is perhaps most convincingly demonstrated by the finding that some mammalian initiation factors can substitute for yeast factors in vivo(7Altmann M. Müller P.P. Pelletier J. Sonenberg N. Trachsel H. J. Biol. Chem. 1989; 264: 12145-12147Abstract Full Text PDF PubMed Google Scholar, 8Schwelberger H.G. Kang H.A. Hershey J.W.B. J. Biol. Chem. 1993; 268: 14018-14025Abstract Full Text PDF PubMed Google Scholar). The availability of yeast cell-free translation systems (9Gasior E. Herrera F. Sadnik I. McLaughlin C.S. Moldave K. J. Biol. Chem. 1979; 254: 3965-3969Abstract Full Text PDF PubMed Google Scholar, 10Tuite M.F. Plesset J. Yeast. 1986; 2: 35-52Crossref PubMed Scopus (31) Google Scholar) and powerful genetic approaches make this system an attractive model system to study the mechanism and regulation of translation in eukaryotes.Most of the translation initiation factors identified earlier in the mammalian system have also been isolated and their genes cloned from S. cerevisiae(4Müller P.P. Trachsel H. Eur. J. Biochem. 1990; 191: 257-261Crossref PubMed Scopus (14) Google Scholar, 5Linder P. Antonie Leeuwenhoek. 1992; 62: 47-62Crossref PubMed Scopus (14) Google Scholar, 6Feng L. Donahue T.F. Ilan J. Translational Regulation of Gene Expression 2. Plenum Press, New York1993: 69-86Crossref Google Scholar). In addition, yeast initiation factors were identified and their genes cloned whose mammalian homologues are not yet known(6Feng L. Donahue T.F. Ilan J. Translational Regulation of Gene Expression 2. Plenum Press, New York1993: 69-86Crossref Google Scholar, 11Hinnebusch A.G. Wek R.C. Dever T.E. Cigan A.M. Feng L. Donahue T.F. Ilan J. Translational Regulation of Gene Expression 2. Plenum Press, New York1993: 87-115Crossref Google Scholar).Among the few initiation factors that have not yet been isolated from yeast is the factor eIF-3. Mammalian eIF-3 is composed of eight polypeptide chains with a total mass of about 550 kDa(1Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar, 2Merrick W.C. Microbiol. Rev. 1992; 56: 291-315Crossref PubMed Google Scholar). Reconstitution experiments showed that this factor is involved in several consecutive steps in the initiation pathway including ribosome dissociation, Met-tRNAi binding to 40 S ribosomes, and mRNA binding to 40 S•Met-tRNAi complexes(1Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar).The analysis of the yeast mutant strain prt1-1 originally isolated by Hartwell (12Hartwell L.H. J. Bacteriol. 1967; 93: 1662-1670Crossref PubMed Google Scholar) showed that Prt1 protein stimulates the interaction of the ternary complex eIF-2•GTP•Met-tRNAi with the 40 S ribosomal subunit(13Feinberg B. McLaughlin C.S. Moldave K. J. Biol. Chem. 1982; 257: 10846-10851Abstract Full Text PDF PubMed Google Scholar). Based on these findings and unpublished data, 2K. Moldave and C. S. McLaughlin, unpublished data. Moldave and McLaughlin (14Moldave K. McLaughlin C.S. NATO ASI Ser. 1988; 14: 271-281Google Scholar) suggested that the PRT1 gene may encode a subunit of eIF-3. The cloning of the PRT1 gene (15Keierleber C. Wittekind M. Qin S. McLaughlin C.S. Mol. Cell. Biol. 1986; 6: 4419-4424Crossref PubMed Scopus (17) Google Scholar, 16Hanic-Joyce P.J. Singer R.A. Johnston G.C. J. Biol. Chem. 1987; 262: 2845-2851Abstract Full Text PDF PubMed Google Scholar) and the development of a cell-free system in which Prt1 activity can be measured (17Mandel T. Trachsel H. Biochim. Biophys. Acta. 1989; 1007: 80-83Crossref PubMed Scopus (2) Google Scholar) allowed attempts to purify Prt1. Here, we show that Prt1 is a subunit of a protein complex that may be the yeast homologue of mammalian eIF-3.
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