A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNAiMet
2001; Springer Nature; Volume: 20; Issue: 11 Linguagem: Inglês
10.1093/emboj/20.11.2954
ISSN1460-2075
Autores Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle1 June 2001free access A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNAiMet Lon Phan Lon Phan Present address: National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD, 20894 USA Search for more papers by this author Lori W. Schoenfeld Lori W. Schoenfeld Present address: Massachusetts Institute of Technology, Department of Biology, Cambridge, MA, 02142 USA Search for more papers by this author Leoš Valášek Leoš Valášek Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20910 USA Search for more papers by this author Klaus H. Nielsen Klaus H. Nielsen Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20910 USA Search for more papers by this author Alan G. Hinnebusch Corresponding Author Alan G. Hinnebusch Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20910 USA Search for more papers by this author Lon Phan Lon Phan Present address: National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD, 20894 USA Search for more papers by this author Lori W. Schoenfeld Lori W. Schoenfeld Present address: Massachusetts Institute of Technology, Department of Biology, Cambridge, MA, 02142 USA Search for more papers by this author Leoš Valášek Leoš Valášek Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20910 USA Search for more papers by this author Klaus H. Nielsen Klaus H. Nielsen Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20910 USA Search for more papers by this author Alan G. Hinnebusch Corresponding Author Alan G. Hinnebusch Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20910 USA Search for more papers by this author Author Information Lon Phan2, Lori W. Schoenfeld3, Leoš Valášek1, Klaus H. Nielsen1 and Alan G. Hinnebusch 1 1Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, 20910 USA 2Present address: National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD, 20894 USA 3Present address: Massachusetts Institute of Technology, Department of Biology, Cambridge, MA, 02142 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:2954-2965https://doi.org/10.1093/emboj/20.11.2954 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1. We found that a stable subcomplex of His8-PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits. eIF5, eIF1 and HCR1 co-purified with this subcomplex, but not with distinct His8-PRT1–TIF34–TIF35 (P45) or His8-PRT1–TIF32 (P2) sub complexes. His8-PRT1 and NIP1 did not form a stable binary subcomplex. These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3. Heat-treated prt1-1 extracts are defective for Met-tRNAiMet binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself in these extracts. We could rescue 40S binding of Met- tRNAiMet and mRNA, and translation of luciferase mRNA, in a prt1-1 extract almost as well with purified PN2 subcomplex as with five-subunit eIF3, whereas the P45 subcomplex was nearly inactive. Thus, several key functions of eIF3 can be carried out by the PRT1–TIF32–NIP1 subcomplex. Introduction The initiation of protein synthesis in eukaryotic cells is dependent on multiple eukaryotic initiation factors (eIFs) that stimulate binding of mRNA and methionyl-initiator tRNA (Met-tRNAiMet) to the 40S ribosome. In mammals, eIF3 is the most complex of these factors, containing 11 different subunits, and participates in multiple steps of the initiation pathway. Mammalian eIF3 forms a complex with the 40S subunit and stimulates binding of the eIF2–GTP–Met-tRNAiMet ternary complex (TC) to produce the 43S pre-initiation complex. Binding of mRNA to the 43S complex, producing the 48S complex, is stimulated by the m7G cap-binding initiation factor eIF4F and by eIF3. The 48S complex locates the start codon in a process known as scanning, and AUG recognition triggers hydrolysis of the GTP bound to eIF2 in a reaction stimulated by eIF5. This leads to dissociation of eIF2–GDP and other eIFs from the 40S subunit, and subsequent joining of the 60S subunit to form an 80S initiation complex (reviewed in Hershey and Merrick, 2000; Hinnebusch, 2000). Yeast eIF3 contains five stoichiometric core subunits (known as TIF32, NIP1, PRT1, TIF34 and TIF35) that are orthologs of human eIF3 subunits p170, p116, p110, p36 and p44, respectively. The yeast factor was affinity purified and shown to restore high-level binding of Met-tRNAiMet to 40S ribosomes in a heat-treated prt1-1 mutant extract, showing that it possesses a key activity of eIF3 (Phan et al., 1998). Interactions among the five core subunits have been studied by yeast two-hybrid analysis and in vitro binding assays (Asano et al., 1998; Valášek et al., 2001). These studies suggest that PRT1 is central to the complex, with the two smallest core subunits (TIF34/TIF35) binding to its extreme C-terminus (and also to one another), and the largest subunit (TIF32) binding to the RNA recognition motif (RRM) at the N-terminus of PRT1. NIP1 binds to TIF32 but not to PRT1 (Figure 1). Mutational analysis has shown that PRT1 and NIP1 are both required for eIF3-dependent TC binding to 40S subunits in yeast extracts (Feinberg et al., 1982; Phan et al., 1998); however, their molecular functions in this activity are not understood. Figure 1.Predicted interactions among yeast eIF3 subunits, eIF5, eIF1 and HCR1. Yeast eIF3 contains five core subunits (TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1. PRT1 and TIF35 contain RRMs, and TIF32 contains an HCR1-like domain (HLD). Interactions among these proteins detected by yeast two-hybrid or in vitro binding assays are depicted schematically as points of contact between the representative shapes. The CTD of eIF5 contains a conserved bipartite motif (AA-boxes) required for its interaction with eIF3–NIP1. NIP1 additionally interacts with eIF1, and eIF1 interacts with the eIF5-CTD in these binding assays. The N-terminal portion of eIF5 contains a zinc-finger motif depicted as a prong. (See text for references.) Download figure Download PowerPoint Yeast eIF3 also contains a protein related to human eIF3 subunit p35, known as HCR1. Although HCR1 was not detected in affinity-purified eIF3 preparations (Phan et al., 1998), it interacted genetically with TIF32 and PRT1, bound in vitro to both of these eIF3 subunits (Figure 1), and co-immunoprecipitated with eIF3 subunits from cell extracts. In vitro, HCR1 interacted with RRM in PRT1 and with TIF32 (Valášek et al., 2001). The absence of HCR1 from purified eIF3 preparations seems to result from its substoichiometric level and weak interaction with the core eIF3 complex (Valášek et al., 1999, 2001). Interestingly, affinity-purified yeast eIF3 contained nearly stoichiometric amounts of eIF5 (Phan et al., 1998). eIF1 (SUI1) is also physically associated with yeast eIF3 (Naranda et al., 1996), although this interaction is salt labile (Phan et al., 1998). Both eIF1 and eIF5 interacted with recombinant forms of the NIP1 subunit of yeast eIF3 (Figure 1) (Asano et al., 1998, 1999; Phan et al., 1998). The association of eIF5 with eIF3 has also been observed in mammals (Bandyopadhyay and Maitra, 1999), and both eIF5 and eIF1 interacted with the mammalian counterpart of yeast NIP1 (eIF3-p110) (Fletcher et al., 1999; Das and Maitra, 2000). eIF5 and eIF1 have been implicated in recognition of initiation codons during the scanning process (Pestova et al., 1998; Donahue, 2000), and the rate of eIF5-dependent GTP hydrolysis in the TC is an important determinant of the stringent selection of AUG as start codon (Huang et al., 1997). Although eIF3 stimulates binding of the TC to 40S ribosomes, no direct interaction has been observed between eIF3 and eIF2 that might underlie this function of eIF3. Interestingly, the C-terminal domain (CTD) of yeast eIF5 can interact simultaneously with the β-subunit of eIF2 and eIF3–NIP1, and thereby promotes formation of a multifactor complex (MFC) containing eIFs 1, 3, 5 and the TC. A mutation in the eIF5-CTD that destabilizes the MFC impaired translation initiation in yeast, suggesting that physical coupling between eIF3 and eIF2 bridged by the eIF5-CTD is required for efficient TC binding or AUG recognition (Asano et al., 2000). The function of eIF3 in stimulating mRNA binding to the 40S ribosome is poorly understood at the molecular level. Interactions between eIF3 and the eIF4G subunit of eIF4F are thought to be instrumental in this activity; however, a strong eIF3–eIF4G interaction has not been reported in yeast. Because binding of the TC is a prerequisite for mRNA binding to 40S ribosomes in vitro (Hinnebusch, 2000), eIF3 may also stimulate mRNA binding indirectly by promoting recruitment of the TC. In this study, we investigated the contributions of different yeast eIF3 subunits to its interactions with other initiation factors and its activities in recruiting TC and mRNA to 40S ribosomes. We overexpressed different combinations of eIF3 subunits, including a polyhistidine-tagged version of PRT1 (His8-PRT1), and affinity purified the subcomplexes by nickel chelation chromatography. Analysis of these subcomplexes confirmed our protein linkage map for eIF3 subunits (Asano et al., 1998) and demonstrated a requirement for NIP1 in the association of eIFs 1, 5 and HCR1 with native eIF3 in vivo. When the subcomplexes were tested for the ability to rescue TC and mRNA binding in a yeast prt1-1 mutant extract, the results showed that a PRT1–TIF32–NIP1 subcomplex restored both activities almost as well as the five-subunit eIF3 complex did, while a PRT1–TIF34–TIF35 subcomplex was nearly inactive. Thus, the key biochemical activities of eIF3, and its ability to interact with eIFs 1, 5 and HCR1, reside in the PRT1–TIF32–NIP1 subcomplex. Results The prt1-1 mutation impairs mRNA binding to 40S subunits in vitro The prt1-1 mutation in eIF3 subunit PRT1 leads to temperature-sensitive growth and a severe reduction in translation initiation in vivo (Hartwell and McLaughlin, 1969). In vitro analysis of heat-inactivated prt1-1 extracts revealed a defect in binding of the TC to 40S ribosomal subunits that could be complemented with purified eIF3 complexes (Danaie et al., 1995; Phan et al., 1998). As mammalian eIF3 stimulates binding of mRNA and TC to 40S subunits (Hinnebusch, 2000), we investigated whether mRNA binding was temperature sensitive in the prt1-1 extract. Aliquots of PRT1 and prt1-1 extracts were heat treated at 37°C for 5 min and incubated with 32P-labeled MFA2 mRNA at 26°C in a reaction containing all components required for in vitro protein synthesis. The non-hydrolyzable GTP analogue guanylyl-(β,γ-imido)diphosphonate (GMPPNP) was included to promote accumulation of 48S complexes by preventing release of factors from 48S complexes and joining of 60S subunits. Reactions were resolved by sedimentation through sucrose gradients and the labeled mRNA in each fraction was determined. Whereas the wild-type extract showed substantial binding of [32P]MFA2 mRNA to 40S ribosomes (Figure 2A), the mutant extract was almost completely defective for binding (Figure 2B). Importantly, mRNA binding was rescued in the prt1-1 extract with purified eIF3 (Figure 2B), while addition of eIF3 did not stimulate mRNA binding in the wild-type extract (Figure 2A). Thus, yeast eIF3 is required for efficient mRNA binding to 40S ribosomes in cell extracts. Figure 2.Rescue of [32P]mRNA binding to the 40S ribosome in heat-inactivated prt1-1 extract by purified eIF3. Twenty microliters (∼300 μg) of extract prepared from PRT1 strain LPY200 (A) or prt1-1 strain H1676 (B) were heat treated at 37°C for 5 min and incubated in a 40 μl reaction containing ∼2 pmol of [32P]MFA2 mRNA (∼200 000 c.p.m.), 1× translation buffer, 1.2 mM GMPPNP, and either 1.5 pmol purified eIF3 (+) or buffer alone (−), at 26°C for 20 min. The reactions were stopped by adding formaldehyde to 0.3% and chilled on ice for 10 min before loading on a 7.5–30% sucrose gradient and centrifuging for 5 h at 41 000 r.p.m. in an SW41 rotor at 4°C. Fractions of 0.6 ml were collected using an ISCO gradient fraction collector and assayed for [32P]mRNA by mixing 0.2 ml of each fraction with 1 ml of water and 10 ml of scintillation fluid, and counting in a scintillation counter. The arrow in each panel marks the migration position of 40S ribosomes. Download figure Download PowerPoint The prt1-1 mutation reduced binding of eIF3 and initiation factors 1, 2 and 5 to the 40S ribosome in cell extracts It was possible that prt1-1 impaired binding of TC and mRNA to 40S ribosomes because the mutant eIF3 complexes dissociated following heat treatment. The levels of all five eIF3 subunits were indistinguishable between the prt1-1 and PRT1 extracts following heat treatment (see Figure 4, 5% input lane), providing no indication of accelerated degradation in the mutant extract. To examine directly the stability of the mutant complex, we inserted a polyhistidine tag at the C-terminus of the prt1-1 allele (producing prt1-1-His) to allow affinity purification of the mutant complexes. Extracts were prepared from isogenic strains LPY202 and LPY201 containing prt1-1-His or the corresponding tagged wild-type allele PRT1-His (Phan et al., 1998), respectively. Aliquots were incubated at 37°C for 5 min, or maintained at 25°C, and complexes containing His8-prt1-1 or His8- PRT1 were isolated on Ni2+-nitrilotriacetic acid (NTA)–silica resin. Western analysis revealed similar amounts of the mutant or wild-type PRT1 proteins and other eIF3 subunits (TIF32, NIP1, TIF34 and TIF35) in equivalent proportions of all four purified preparations. Additionally, similar amounts of eIF5 and eIF1 were associated with the mutant and wild-type eIF3 complexes with or without prior heat treatment of the extracts (Figure 3). We conclude that heat treatment of prt1-1 extracts did not lead to dissociation of eIF3 or disruption of its interactions with eIF5 and eIF1. Figure 3.Ni2+ affinity purification of intact eIF3 complexes containing eIF1 and eIF5 from heat-treated extract containing His8-prt1-1. Translation extracts were prepared from strains LPY201 (PRT1-His) and LPY202 (prt1-1-His) grown in YPD medium (Sherman et al., 1974) at 26°C to an OD600 of 1.0. The extracts were supplemented with imidazole to a final concentration of 20 mM, incubated at 25 or 37°C for 5 min, as indicated, and subjected to Ni2+ chelation chromatography using buffer A (see Materials and methods). Equivalent aliquots (2.5, 5 and 10 μl) of the Ni2+-NTA–silica eluates were separated by SDS–PAGE using 4–20% gels and subjected to immunoblot analysis using rabbit polyclonal antibodies against the proteins shown on the left, at the following dilutions: PRT1, 1:3000; TIF32, 1:3000; NIP1, 1:1000; TIF34, 1:500; TIF35, 1:5000; eIF5, 1:10000; SUI1/eIF1, 1:1000. Immune complexes were detected by chemiluminescence (ECL™, Amersham Pharmacia Biotech) using horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech). Download figure Download PowerPoint Figure 4.Binding of eIF3, eIF2, eIF5 and eIF1 to 40S ribosomes is defective in the heat-treated prt1-1 extract. (A) Twenty microliters (∼300 μg) of translation extracts prepared from PRT1 strain LPY200 or prt1-1 strain H1676 were heat treated at 37°C for 5 min and incubated in a 40 μl reaction containing 1× translation buffer and 1.2 mM GMPPNP at 26°C for 20 min. The reactions were stopped by adding formaldehyde to 0.3% and incubating on ice for 10 min. A portion of each reaction (5%) was removed (input samples) and the remainder was separated on a 7.5–30% sucrose gradient as described in Figure 2. Fractions (0.6 ml) were collected and precipitated with 1.0 ml of ethanol at −20°C. The precipitates were washed once with ethanol, dried and resuspended in 50 μl of loading buffer, and separated by SDS–PAGE using 4–20% gradient gels. The separated proteins were subjected to immunoblot analysis using antibodies against the proteins indicated on the left. Antibodies were used at the same dilutions described in Figure 3, with the addition of antibodies against HCR1 (1:500) (Valášek et al., 2001) and GCD11 (1:10000). The position of 40S ribosomes in the gradients is indicated over fractions 10–12. (B) Heat-treated PRT1 and prt1-1 extracts were analyzed as in (A) except that 4.5 pmol of highly purified eIF3 (+) or buffer alone (−) were added to each reaction, as indicated on the right, prior to incubation at 26°C for 20 min. The reactions were stopped and analyzed as described in (A). Download figure Download PowerPoint To determine whether prt1-1 impaired binding of eIF3 to 40S subunits, aliquots of PRT1 and prt1-1 extracts were heated at 37°C for 5 min and added to translation reaction mixtures containing GMPPNP, as described above. After incubating at 26°C, the reactions were separated on sucrose gradients and the fractions were analyzed by western blotting. As expected, large proportions of all five core eIF3 subunits in the wild-type extract co-sedimented with small ribosomal subunits in the 40–48S region of the gradient (Figure 4A, WT, fractions 10–12 versus 1–4). In the prt1-1 extract, co-sedimention of eIF3 core subunits with 40S subunits was greatly reduced. As we did not see a commensurate increase in the amounts of unbound eIF3 subunits in fractions 1–4 for the mutant extract, it appears that a large proportion of the mutant complexes were degraded during centrifugation. Nevertheless, prt1-1 clearly reduced the absolute amounts and proportion of eIF3 subunits that co-sedimented with 40S ribosomes following heat treatment of the extract. Substantial fractions of eIF1, eIF5 and eIF2γ also co-sedimented with 40S ribosomes in the PRT1 extract, and this behavior was impaired by heat treatment of the prt1-1 extract (Figure 4A). These data suggest that eIF3 promotes binding of all these factors to 40S subunits, dependent on PRT1. The fact that eIF2γ binding to 40S subunits was reduced by prt1-1 is in keeping with previous observations that binding of initiator tRNAMet is impaired in heat-treated prt1-1 extracts (Danaie et al., 1995; Phan et al., 1998). As noted above, eIF1 and eIF5 co-purified with eIF3, and both factors interacted with the NIP1 subunit of eIF3. The results in Figure 4A confirm that physical association of eIF1 and eIF5 with eIF3 stimulates incorporation of these factors into 43–48S initiation complexes. Thus, the prt1-1 mutation impairs 40S binding by all components of the MFC (Asano et al., 2000) in heat-treated extracts. The effect of prt1-1 on 40S binding by HCR1 was distinctive. In the wild-type extract, HCR1 consistently peaked in the 40S region one fraction earlier than the other factors (Figure 4A). In the mutant extract, the most rapidly sedimenting form of HCR1 in fraction 11 was diminished, but a substantial proportion of HCR1 was retained in fraction 10. Recently, we found that HCR1 has a dual function in translation initiation, being required for normal levels of 40S ribosomes and for high levels of the MFC (Valášek et al., 2001). We suggest that HCR1 binds to 40S subunits with the MFC, but remains bound to the ribosome following dissociation of other factors on heat treatment of the prt1-1 extract. This eIF3-independent binding of HCR1 to 40S subunits may be related to its function in ribosome biogenesis. To confirm the involvement of eIF3 in recruitment of eIF5 to the 40S ribosome, we attempted to rescue 40S binding of eIF5 in the heat-treated prt1-1 mutant extract with purified eIF3. As shown in Figure 4B, addition of eIF3 to the mutant extract significantly increased the proportions of eIF5, eIF2γ and eIF3–TIF32 sedimenting in the 40–48S region (fractions 10–12) at the expense of the unbound forms of these factors (fractions 1–4). These results provide strong evidence that eIF3 promotes stable incorporation of eIF5 into 43–48S initiation complexes. Physical association between overexpressed eIF3 subcomplexes and other initiation factors In an effort to determine which subunits of eIF3 are required for its ability to stimulate binding of TC, mRNA and other initiation factors to 40S ribosomes, we overexpressed and purified various eIF3 subcomplexes containing His8-PRT1, and tested them for the presence of co-purifying initiation factors and the ability to rescue Met-tRNAiMet and mRNA binding in heat-treated prt1-1 extracts. Our previous analysis of protein linkages among eIF3 subunits revealed that PRT1, TIF34 and TIF35 interacted with one another, that PRT1 interacted with TIF32, but that NIP1 interacted only with TIF32 (Figure 1) (Asano et al., 1998). Based on these findings, we predicted that two different trimeric subcomplexes could be overproduced, one containing His8-PRT1, TIF34 and TIF35, and the other containing His8-PRT1, TIF32 and NIP1. We also expected to observe a stable His8-PRT1–TIF32 binary complex, but since PRT1 did not interact directly with NIP1 (Asano et al., 1998), a His8-PRT1–NIP1 binary complex should not exist. Similarly, a four-subunit complex containing His8-PRT1, TIF34, TIF35 and TIF32 was predicted, whereas one containing NIP1 in place of TIF32 should not occur. To test these predictions, we constructed yeast strains containing different subsets of eIF3 subunit genes under their native promoters on high-copy plasmids. All of the strains overexpressed His8-PRT1, and the overexpressed TIF34 and TIF35 subunits contained hemagglutinin (HA)- and FLAG-epitope tags, respectively. Western analysis of whole-cell extracts (WCEs) confirmed that eIF3 subunits were overexpressed in the appropriate strains between 10- and 20-fold above the endogenous levels, except for NIP1, which was only ∼5-fold overexpressed (data not shown). We affinity purified His8-PRT1 and associated proteins on Ni2+-NTA–silica from ribosomal high-salt washes (RSWs) and the eluates were subjected to western analysis using PRT1 antibodies. Aliquots containing equivalent amounts of His8-PRT1 were then examined for levels of other eIF3 subunits (Figure 5A). A mock purification using RSW from a strain containing untagged chromosomal PRT1 was conducted to ensure that purification of eIF3 subunits was dependent on binding of His-tagged PRT1 to the Ni2+-NTA resin. Indeed, none of the eIF3 subunits was detectable in this control preparation, henceforth designated 'Vector' (Figure 5A, lanes 1–3). As expected, the sample purified from the strain overexpressing only His8-PRT1 (designated sample P) contained detectable amounts of the other four eIF3 subunits (Figure 5A, lanes 4–6), which we attribute to incorporation of His8-PRT1 into endogenous eIF3 in place of untagged PRT1 expressed from the chromosome. This background level of five-subunit eIF3 should also occur in the preparations containing His8-PRT1 and other overexpressed eIF3 subunits. Figure 5.eIF5 and eIF1 co-purify with eIF3 subcomplexes containing His8-PRT1, NIP1 and TIF32. (A) His8-PRT1 and associated proteins were purified from the PRS by nickel chelation chromatography in buffer containing 350 mM KCl (buffer B; see Materials and methods) from the following strains overexpressing different combinations of eIF3 subunits: LPY60 (empty vectors), LPY65 (P), LPY66 (PN), LPY67 (PN2), LPY68 (P2), LPY85 (P45), LPY86 (P45N) and LPY87(P45N2). The letters and numbers in parentheses designate overexpression of His8-PRT1 (P), HA-TIF34 (4), FLAG-TIF35 (5), NIP1(N) and TIF32 (2). Three serial dilutions of the Ni2+-NTA–silica eluates for each preparation were resolved by 4–20% SDS–PAGE and subjected to immunoblot analysis as described in Figures 3 and 4. Samples of the control extract loaded in lanes 1–3 (Vector) contained 0.5, 1 and 2 μg of total protein, respectively. Samples of the P preparation containing overexpressed His8-PRT1 alone in lanes 4–6 contained 0.38, 0.75 and 1.5 μg total protein, respectively. For the remaining preparations, the amounts loaded were predetermined to contain the same quantities of His8-PRT1 as in lanes 4–6. (B) Yeast strains LPY60, LPY65, LPY67, LPY68, LPY85 and LPY87, described in (A), were transformed with low-copy-number plasmid YCpLVHM-T encoding c-myc-tagged HCR1 to create strains LPY142 (Vector), LPY134 (P), LPY136 (PN2), LPY137 (P2), LPY138 (P45) and LPY140 (P45N2), respectively. In addition, strain LPY191 (P*) was constructed containing YCpLVHM-T and a low-copy plasmid bearing PRT1-His. His8-PRT1 and associated proteins were purified from PRS by nickel chelation chromatography in a buffer containing 100 mM KCl (buffer A; see Materials and methods). Two dilutions of the Ni2+-NTA–silica eluates for each preparation were resolved by 4–20% SDS–PAGE and subjected to immunoblot analysis as described in Figures 3 and 4, except that anti-c-Myc monoclonal antibodies (Boehringer-Mannheim; 1:2000) were used to probe for c-Myc-tagged HCR1. Samples of the control preparation in lanes 1 and 2 (Vector) contained 0.25 (1×) and 0.5 μg (2×) of total protein, respectively, as did the P* preparation in lanes 3 and 4. Samples of the P preparation in lanes 5 and 6 contained 0.125 (1×) and 0.25 μg (2×) of total protein. For the remaining preparations, the samples were predetermined to contain approximately the same amounts of His8-PRT1 as in lanes 5 and 6. Download figure Download PowerPoint As expected, higher levels of the other four eIF3 subunits co-purified with His8-PRT1 from the strain overexpressing all five subunits (sample P45N2) (Figure 5A, lanes 22–24 versus 4–6). Interestingly, the sample purified from the extract containing overexpressed HA-TIF34, FLAG-TIF35 and His8-PRT1 (P45) contained high levels of HA-TIF34 and FLAG-TIF35 compared with their levels in the P sample (Figure 5A, lanes 16–18 versus 4–6). These data support our prediction that HA-TIF34 and FLAG-TIF35 can form a stable subcomplex with His8-PRT1 in the absence of TIF32 and NIP1. Analogous results were obtained for the PN2 sample purified from the extract containing overexpressed His8-PRT1, NIP1 and TIF32, showing that TIF32 and NIP1 can form a stable subcomplex with PRT1 in the absence of TIF34 and TIF35 (Figure 5A, lanes 10–12). As predicted, TIF32 and PRT1 formed a stable binary complex, whereas NIP1 and PRT1 did not (Figure 5A, lanes 13–15 and 7–9, respectively). Similarly, the overexpressed NIP1 did not co-purify with the His8-PRT1–HA-TIF34–FLAG-TIF35 subcomplex in the P45N preparation, whereas a fraction of the overexpressed TIF32 was recovered with His8-PRT1–HA-TIF34–FLAG-TIF35 from the P452 preparation (Figure 5A, lanes 19–21 and data not shown). Thus, in accordance with our subunit interaction map for eIF3, TIF32 is required to bridge interaction between NIP1 and His8-PRT1 in vivo. The P45N2 sample contained a much higher amount of eIF5 compared with that present in the P sample, consistent with physical association of eIF5 with the eIF3 complex (Phan et al., 1998). (A low level of eIF5 was present in the P sample, but was not visible at the exposure chosen for Figure 5A.) Additionally, the increased yield of eIF5 co-purifying with His8-PRT1 was dependent on the presence of excess NIP1, occurring only for the PN2 and P45N2 complexes (Figure 5A, lanes 10–12 and 22–24). In the experiments of Figure 5A, we detected only a small amount of eIF1 associated with the P45N2 complex (data not shown). This can be attributed to the fact that association of eIF1 with eIF3 is salt labile (Phan et al., 1998) and the complex was purified from high-salt extracts of ribosomes. In contrast, high levels of eIF1 were recovered with the P45N2 and PN2 complexes when they were purified from WCEs at a lower salt concentration (Figure 5B). Under these conditions, increased amounts of eIF1 co-purified with His8-PRT1 only for the PN2 and P45N2 complexes, which contained NIP1 (Figure 5B, lanes 9–10 and 13–14 versus 5–6). These results provide in vivo evidence that NIP1 is the principal binding partner for eIFs 1 and 5 in the eIF3 complex (Figure 1). For the experiment in Figure 5B, we employed a strain overexpressing Myc-tagged HCR1 (Myc-HCR1) in addition to untagged HCR1 produced from the chromosomal allele. Using Myc antibodies to probe the eIF3 subcomplexes, we found that Myc-HCR1 specifically co-purified with the PN2 trimeric complex and with five-subunit eIF3 (Figure 5B, lanes 9–10 and 13–14 versus 5–6). Thus, HCR1 interacts with the same trimeric subcomplex that binds eIFs 1 and 5. Recently, we found that recombinant TIF32 and PRT1 contain independent binding domains for HCR1 (Valášek et al., 2001); however, here we observed only weak association of Myc-HCR1 with the P2 binary complex (Figure 5B, lanes 7–8). Thus, it a
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