Functions of eIF3 downstream of 48S assembly impact AUG recognition and GCN4 translational control
2004; Springer Nature; Volume: 23; Issue: 5 Linguagem: Inglês
10.1038/sj.emboj.7600116
ISSN1460-2075
AutoresKlaus Nielsen, Béla Szamecz, Leoš Shivaya Valášek, Antonina Jivotovskaya, Byung‐Sik Shin, Alan G. Hinnebusch,
Tópico(s)RNA modifications and cancer
ResumoArticle19 February 2004free access Functions of eIF3 downstream of 48S assembly impact AUG recognition and GCN4 translational control Klaus H Nielsen Klaus H Nielsen Search for more papers by this author Béla Szamecz Béla Szamecz Search for more papers by this author Leoš Valášek Leoš Valášek Search for more papers by this author Antonina Jivotovskaya Antonina Jivotovskaya Search for more papers by this author Byung-Sik Shin Byung-Sik Shin Search for more papers by this author Alan G Hinnebusch Corresponding Author Alan G Hinnebusch Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Klaus H Nielsen Klaus H Nielsen Search for more papers by this author Béla Szamecz Béla Szamecz Search for more papers by this author Leoš Valášek Leoš Valášek Search for more papers by this author Antonina Jivotovskaya Antonina Jivotovskaya Search for more papers by this author Byung-Sik Shin Byung-Sik Shin Search for more papers by this author Alan G Hinnebusch Corresponding Author Alan G Hinnebusch Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Author Information Klaus H Nielsen, Béla Szamecz, Leoš Valášek, Antonina Jivotovskaya, Byung-Sik Shin and Alan G Hinnebusch 1 1Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, MD, USA *Corresponding author. Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Building 6A/Room B1A-13, Bethesda, MD 20892-2716, USA. Tel.: +1 301 496 4480; Fax: +1 301 496 6828; E-mail: [email protected] The EMBO Journal (2004)23:1166-1177https://doi.org/10.1038/sj.emboj.7600116 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The binding of eIF2–GTP–tRNAiMet ternary complex (TC) to 40S subunits is impaired in yeast prt1-1 (eIF3b) mutant extracts, but evidence is lacking that TC recruitment is a critical function of eIF3 in vivo. If TC binding was rate-limiting in prt1-1 cells, overexpressing TC should suppress the temperature-sensitive phenotype and GCN4 translation should be strongly derepressed in this mutant, but neither was observed. Rather, GCN4 translation is noninducible in prt1-1 cells, and genetic analysis indicates defective ribosomal scanning between the upstream open reading frames that mediate translational control. prt1-1 cells also show reduced utilization of a near-cognate start codon, implicating eIF3 in AUG selection. Using in vivo cross-linking, we observed accumulation of TC and mRNA/eIF4G on 40S subunits and a 48S 'halfmer' in prt1-1 cells. Genetic evidence suggests that 40S–60S subunit joining is not rate-limiting in the prt1-1 mutant. Thus, eIF3b functions between 48S assembly and subunit joining to influence AUG recognition and reinitiation on GCN4 mRNA. Other mutations that disrupt eIF2–eIF3 contacts in the multifactor complex (MFC) diminished 40S-bound TC, indicating that MFC formation enhances 43S assembly in vivo. Introduction Initiation of protein synthesis begins with the recruitment of initiator methionyl tRNA (Met-tRNAiMet) in a ternary complex (TC) with eIF2 and GTP to the 40S subunit, to form the 43S preinitiation complex. This is followed by recruitment of mRNA, prebound to the cap-binding complex eIF4F and the poly(A)-binding protein, to form the 48S preinitiation complex. The 43S complex scans the mRNA and AUG recognition triggers GTP hydrolysis in the TC, after which the 60S subunit joins and translation elongation begins. Following ejection of eIF2·GDP, the bound GDP is replaced with GTP by the guanine nucleotide exchange factor eIF2B in order to regenerate TC (Hershey and Merrick, 2000; Hinnebusch, 2000). Mammalian eIF3 binds to the 40S ribosome and stimulates the binding of both TC and mRNA to 40S subunits in vitro (Hershey and Merrick, 2000; Hinnebusch, 2000). Yeast eIF3, consisting of the five subunits eIF3a/TIF32, eIF3b/PRT1, eIF3c/NIP1, eIF3i/TIF34 and eIF3g/TIF35, can restore binding of Met-tRNAiMet (Danaie et al, 1995; Phan et al, 1998) and mRNA (Phan et al, 2001) to 40S ribosomes in heat-inactivated extracts of the prt1-1 (eIF3b) mutant. Thus, yeast eIF3 performs two key functions ascribed to the mammalian factor. The recruitment of TC to the 40S subunit is also stimulated by eIF1 and eIF1A in vitro (Hershey and Merrick, 2000; Algire et al, 2002; Majumdar et al, 2003); however, the relative importance of these factors and of eIF3 for 43S formation in vivo is unclear. In yeast, translational control of GCN4 mRNA is a sensitive in vivo indicator of the rate of TC binding to 40S ribosomes. GCN4 translation is activated by amino-acid starvation through a mechanism involving four upstream open reading frames (uORFs 1–4) in GCN4 mRNA. After translating uORF1, many 40S subunits remain attached to the mRNA and resume scanning; however, GCN4 translation is normally repressed because all of these 40S ribosomes rebind the TC before reaching uORF4, translate uORF4, and dissociate from the mRNA. Starvation leads to phosphorylation of eIF2α by GCN2, converting eIF2 from a substrate to a competitive inhibitor of eIF2B and reducing the concentration of TC (Hinnebusch, 1996). This allows ∼50% of the rescanning 40S ribosomes to rebind TC after bypassing uORF4 and reinitiate at GCN4 instead. gcn2Δ mutants fail to induce GCN4 in starved cells and have a Gcn− (general control nonderepressible) phenotype. Mutations in eIF2B that lower TC levels derepress GCN4 under nonstarvation conditions. This Gcd− (general control derepressed) phenotype does not require eIF2α phosphorylation and can be recognized in gcn2Δ cells (Hinnebusch, 1996). We recently described the first mutation in eIF1A with a Gcd− phenotype, a deletion of the C-terminal 45 residues of the protein (Olsen et al, 2003). Importantly, overexpression of TC from a high-copy (hc) plasmid containing the genes for eIF2α, -β and -γ and tRNAiMet (hc TC) suppressed the Gcd− phenotype of this mutation. This showed that eIF1A is required for optimal TC binding to 40S subunits in vivo and implicated eIF1A in the reinitiation process on GCN4 mRNA. Mutations in eIF3 that reduce TC binding to 40S subunits should also produce a Gcd− phenotype, but none has been isolated. In yeast, the TC is associated with eIF3, eIF1 and eIF5 in a multifactor complex (MFC) that can exist free of 40S ribosomes (Asano et al, 2000). eIF2 interacts indirectly with NIP1/eIF3c in a manner bridged by eIF5 and it also binds directly to the C-terminal domain (CTD) of TIF32/eIF3a (Asano et al, 2000, 2001; Valášek et al, 2002). The tif5-7A mutation in eIF5 disrupts the indirect contact between eIF2 and eIF3 and leads to temperature-sensitive (Ts−) growth in vivo and diminished TC recruitment in vitro (Asano et al, 1999, 2000). Reducing the direct contact between eIF2 and eIF3 by overexpressing a truncated TIF32 protein lacking the eIF2β-binding domain (hc-TIF32Δ6) confers a slow-growth (Slg−) phenotype that is partially suppressed by hc TC. Combining tif5-7A and hc-TIF32Δ6 produces a synthetic growth defect (Valášek et al, 2002), suggesting that the independent contacts between eIF2 and eIF3 in the MFC have additive effects on TC recruitment. However, the tif5-7A hc-TIF32Δ6 double mutant does not have a Gcd− phenotype, neither does it show reduced binding of TC to 40S subunits in cell extracts (Valášek et al, 2002). Thus, it appears that a reduction in TC recruitment is not the rate-limiting defect conferred by disruption of the MFC in this strain. In an effort to implicate eIF3 directly in TC recruitment in vivo, we examined the effect of prt1-1 on the level of 43S complexes in mutant cells at the restrictive temperature. This mutation replaces Ser-518 with Phe, and does not affect eIF3 integrity (Phan et al, 2001). Surprisingly, we saw an accumulation of eIF2 on 40S subunits in extracts of heat-treated prt1-1 cells, and confirmed this result using a new technique for cross-linking preinitiation complexes in living cells. Consistent with the idea that 43S assembly is not rate-limiting in the prt1-1 mutant, its Ts− phenotype was not suppressed by hc TC and we observed a strong Gcn− phenotype that likely results from a delay in scanning by reinitiating ribosomes on GCN4 mRNA. Interestingly, prt1-1 also increases the probability of rejecting a non-AUG start codon, implicating eIF3 in AUG recognition. These findings fit well with the fact that an eIF3 ortholog is lacking in prokaryotes, where scanning does not occur and AUG recognition relies heavily on base-pairing between rRNA in the 30S subunit and mRNA. Results TC and mRNA binding to 40S subunits is not diminished in prt1-1 cells at the restrictive temperature We asked whether incubating prt1-1 cells at the restrictive temperature would reduce the amount of eIF2 associated with 40S subunits, as predicted from a defect in 40S binding by the TC. Incubating prt1-1 cells for 20 min at 37°C produced a run-off of polysomes and accumulation of 80S monosomes (Figure 1A, right panel), indicating a severe reduction in translation initiation (Hartwell and McLaughlin, 1968). Substantial amounts of eIF3, eIF2, eIF1 and eIF5 (components of the MFC), and eIF1A were found in the fractions containing free 40S subunits in the wild-type (WT) extracts (Figure 1B, left panel). Unexpectedly, there was an even greater proportion of eIF2 subunits in the 40S fraction from prt1-1 cells (Figure 1B, right panel), at odds with the previous finding that prt1-1 impairs TC binding to 40S subunits in heat-treated extracts (Danaie et al, 1995; Phan et al, 1998). Figure 1.eIF2 remains bound to 40S subunits in extracts of prt1-1 cells incubated at the nonpermissive temperature. (A) Isogenic PRT1 (H2879) and prt1-1 (H1676) cells were grown in YPD medium at 25°C and treated for 20 min at 37°C. Cyclohexamide was added to 50 μg/ml just prior to harvesting and WCEs prepared with heparin (200 μg/ml) in the breaking buffer were separated on a 4.5–45% sucrose gradient by centrifugation at 39 000 r.p.m. for 2.5 h. The gradients were collected and scanned at 254 nm to visualize the ribosomal species. (B) WCEs described in (A) were separated on a 7.5–30% sucrose gradient by centrifugation at 41 000 r.p.m. for 5 h. Proteins were subjected to Western analysis using antibodies against the proteins listed between the blots. An aliquot of each WCE was analyzed in parallel (In, input). Download figure Download PowerPoint Owing to this discrepancy, we considered the possibility that the results in Figure 1 were an artifact of extract preparation. The binding of MFC components to 40S subunits during sedimentation through sucrose gradients requires heparin in the extraction buffer (Asano et al, 2000), and it was possible that heparin suppressed a defect in eIF2 binding in the prt1-1 extract. To address this possibility, we developed a protocol for cross-linking 43S–48S complexes in vivo by formaldehyde (HCHO) treatment of living cells that eliminated the need for heparin. Cyclohexamide is also omitted because the cross-linking prevents polysome run-off in the extracts (data not shown). Substantial proportions of eIF2, eIF3 and eIF1A cosedimented with the free 40S subunits in the whole-cell extracts (WCEs) from cross-linked WT cells (Figure 2B, left panel), although smaller proportions of 40S-bound eIF5 and eIF1 were observed compared to the conventional protocol (Figure 1B). A significant proportion of eIF4G also cosedimented with 40S subunits (Figure 2B, left panel), whereas this factor is undetectable in the 40S fraction of WCEs prepared with heparin (data not shown). Total RNA was extracted from each fraction and probed for Met-tRNAiMet and RPL41A mRNA by Northern analysis. The short length of this mRNA, 340 nt, ensures that free mRNP complexes sediment more slowly than 40S subunits. As expected, both tRNAiMet and RPL41A mRNA peaked in the 40S fraction of cross-linked WT cells (Figure 2C, left panel). Figure 2.TC and mRNA remain bound to 40S subunits in HCHO-treated prt1-1 cells at the nonpermissive temperature. PRT1 (H2879) and prt1-1 (H1676) cells were grown in YPD at 25°C, heat-treated for 20 min at 37°C, and cross-linked with HCHO for 15 min (A) or 1 h (B, C). (A) WCEs were separated and analyzed as in Figure 1A. (B, C) WCEs were separated and treated as in Figure 1B, except that each fraction was divided and analyzed by Western and Northern blotting. Download figure Download PowerPoint Comparison of equivalent A260 units of WCEs from HCHO-treated WT and prt1-1 cells revealed the expected reduction in polysomes and accumulation of 80S monosomes in the mutant cells incubated at 37°C (Figure 2A). Interestingly, the 40S fraction from the prt1-1 cells had similar levels of eIF3, eIF5 and eIF1, and relatively greater amounts of eIF2, eIF4G, eIF1A, tRNAiMet and mRNA compared to that seen in WT (Figure 2B and C). Quantification of the results from several experiments revealed that the amounts of 40S-bound eIF2γ, tRNAiMet and mRNA were increased in the prt1-1 cells by 166±58, 180±10 and 250±60%, respectively. The amount of free 40S subunits was generally greater in the mutant cells due to polysome run-off. If we normalize the amounts of 40S-bound eIF2, tRNAiMet and mRNA for the RPS22 signals, then the levels of these 40S-bound factors are nearly identical between prt1-1 and WT cells. However, because it is unknown whether 40S subunits are limiting for 43S/48S formation, we chose to compare the absolute levels of 40S-bound factors determined in replicate experiments. Regardless of how the data are quantified, the results indicate that 40S binding of TC, mRNA and eIF4G is not diminished in prt1-1 cells at 37°C. We also found that similar amounts of eIF2 and eIF4G were present in the 80S and polysome fractions of WT and prt1-1 mutant cells at 37°C, representing ∼50% of the total pools of these factors, despite the low polysome content of the mutant at 37°C (data not shown). Thus, high levels of eIF2 and eIF4G are associated with both free and polysome-associated 48S complexes in the mutant. We conclude that the rate-limiting defect occurs at a step following assembly of 48S complexes in prt1-1 cells at 37°C. To validate the cross-linking technique, we wished to show that mutations in eIF2 would reduce the 40S binding of tRNAiMet in cross-linked cells. Hence, we constructed a strain harboring a temperature-sensitive degron allele, sui3-td, encoding a fusion protein containing ubiquitin and a thermolabile dihydrofolate reductase moiety attached to the N-terminus of eIF2β, and expressed from a copper-dependent promoter. The strain also expresses the ubiquitin ligase UBR1 from a galactose-inducible promoter. Shifting the sui3-td cells from medium containing copper and raffinose at 25°C to medium containing galactose and lacking copper at 36°C represses new synthesis of the degron-tagged protein (eIF2βtd), and the pre-existing eIF2βtd is rapidly eliminated by proteosomal degradation (Dohmen et al, 1994; Labib et al, 2000). After incubating under the nonpermissive condition for 16 h to deplete eIF2βtd, the sui3-td cells were treated with HCHO to cross-link the 43–48S complexes. As expected, we observed polysome run-off in the cross-linked sui3-td cells under the nonpermissive condition (Figure 3A). Furthermore, eIF2βtd was undetectable and little or no eIF2γ and eIF2α cosedimented with free 40S subunits (Figure 3B, right panel). Importantly, the amount of tRNAiMet in the 40S fraction (Figure 3C, right panel) declined to 14±4% of that seen under permissive conditions. There was little reduction in 40S binding of eIF3 subunits, and a small increase in 40S-bound eIF1A under the nonpermissive condition (Figure 3B), indicating a specific loss of TC from preinitiation complexes. We also examined the effects of a nonconditional mutation in eIF2γ, gcd11-506, which reduces the affinity of eIF2 for Met-tRNAiMet in vitro (Erickson and Hannig, 1996). Consistently, we observed a threefold reduction in the amount of eIF2 on 40S subunits in cross-linked gcd11-506 cells compared to WT (data not shown). Figure 3.sui3-td and cdc33-1 mutations reduce the binding of TC and mRNA to 40S subunits in vivo. (A–C) sui3-td (YAJ18-3) cells were grown in SC-raffinose in the presence of 0.1 M copper sulfate at 25°C and shifted to SC-raffinose+galactose in the absence of copper and grown overnight at 37°C. Cells were cross-linked with HCHO and analyzed as described in Figure 2A–C, except that eIF2βtd was detected in (B) using HA antibody. (D) Strain F324 was transformed with YEplac195-CDC33-URA3 (CDC33) or empty vector (cdc33-1) and the resulting transformants were grown in SC-Ura medium at 25°C, heat treated for 2 h at 37°C, and cross-linked with HCHO. WCEs were prepared and analyzed as (B–C). Download figure Download PowerPoint We next examined the cdc33-1 mutant to examine the consequences of depleting the cap-binding protein, eIF4E, on mRNA recruitment. Incubating these cells at 37°C for 2 h leads to the disappearance of eIF4E, presumably due to proteolytic degradation (Figure 3D). The amount of 40S-bound mRNA also declined in the cdc33-1 cells at 37°C to 47±8% of the level observed in WT cells, while the level of tRNAiMet was reduced to only 86±10% of WT (Figure 3E). The ability of eIF4G to interact with both poly(A)-binding protein and eIF4E (Sachs, 2000) likely accounts for the residual mRNA binding to 40S ribosomes in cdc33-1 cells at 37°C. As expected, we saw little reduction in the amounts of eIF3 or eIF2 subunits bound to free 40S subunits in the cdc33-1 mutant (Figures 3D). Thus, it appears that HCHO cross-linking provides a faithful depiction of the composition of preinitiation complexes in living cells. If the rate-limiting defect in prt1-1 cells is downstream from 48S complex assembly, we would expect to observe a halfmer shoulder on the 80S monosome formed by mRNAs containing an elongating 80S ribosome plus a 48S complex in the mRNA leader. As most monosomes in this mutant are 80S couples lacking mRNA (data not shown), the concentration of such 1½-mers should be small. Nevertheless, we consistently detected a halfmer on the 80S peak in cross-linked prt1-1 cells incubated for 20 min or 1 h at 37°C (Figure 4B and data not shown). In some experiments, the 1½-mer was less prominent (Figure 2A, right panel), possibly due to the shorter HCHO treatment in those cases; however, we never detected a 1½-mer peak in cdc33-1 (Figure 4D) or sui3-td cells (Figure 3A, right panel). (We attribute the absence of the halfmer peak in Figure 1A to the presence of heparin, which could compete with mRNA for 40S binding.) Figure 4.The prt1-1 mutant displays a halfmer phenotype at the nonpermissive temperature. Polysome profiles of (A) PRT1 (H2879) and (B) prt1-1 (H1676) cells after 20 min at 37°C. The halfmer shoulder on the 80S peak is indicated along with an explanatory schematic of a 1½-mer (see text). Polysome profiles of the (C) CDC33 and (D) cdc33-1 transformants described in Figure 3D after 2 h at 37°C. Download figure Download PowerPoint Genetic evidence that TC binding to 40S subunits is not rate-limiting in prt1-1 cells If binding of TC to 40S ribosomes was the principal deficiency in the prt1-1 mutant, then its growth defect should be reduced by overexpressing TC from an hc plasmid. As expected, hc TC suppressed the growth defect of a gcd1-502 mutant containing a defective subunit of eIF2B (Figure 5A, right panels) (Dever et al, 1995). By contrast, the growth defect of prt1-1 cells at a semipermissive temperature of 33°C was not suppressed by hc TC (Figure 5A, left panels). Figure 5.The prt1-1 strain has a strong Gcn− phenotype and its Ts− phenotype is not suppressed by TC overexpression. (A) prt1-1 (H1676) and gcd1-502 (H70) cells were transformed with hc plasmid p3000 encoding hc TC, or empty vector, streaked on SC-Ura plates and incubated for 4 (left panels) or 2 days (right panels) at 33°C. (B) prt1-1 (YKHN60) cells were transformed with p3000 (hc TC) or empty vector, grown overnight in SC-Ura, and 10-fold serial dilutions were spotted in rows 1 and 2 on SC-Ura plates or SC-Ura-His plates containing 40 mM leucine with 30 mM 3-AT and incubated for 7 and 10 days, respectively, at 33°C. PRT1 GCN2 (H2879) and PRT1 gcn2Δ (H2881) cells transformed with empty vector were analyzed in parallel in rows 3 and 4. (C) PRT1 (H2879) and prt1-1 (H1676) cells were transformed with p3000 (hc TC) or empty vector and analyzed essentially as in (B). (D) ssu2-1 (F708) cells were transformed with empty vector or p3342 encoding SSU2 (TIF5), and analyzed in rows 1 and 2 as in (B), except using SC-Ura and SC-Ura-Ile-Val+1 μg/μl SM plates and incubating for 2 or 3 days (row 1) and 1 or 2 days (rows 2–4) at 30°C. SSU2 gcn2Δ (H2881) and SSU2 GCN2 (H2879) cells, transformed with empty vector, were analyzed in parallel in rows 3 and 4. (E) prt1-1 (H1676) cells were transformed with p3993 encoding SUI5-R31G or p3992 encoding SUI5 (TIF5) and analyzed essentially as in (B), except using SC-Leu plates or SC-Leu-His plates containing 10 mM 3-AT and incubating for 5 (row 1) or 3 days (row 2) at 33°C. PRT1 gcn2Δ (H2881) and PRT1 GCN2 (H2879) cells, transformed with empty vector, were analyzed in parallel in rows 3 and 4, incubating for 2 days. (F) tif5-7A GCN2 (YKHN206) cells were transformed with p3927 encoding TIF32Δ6–His or empty vector or p3342 encoding TIF5 and analyzed essentially as in (B), except that plates were incubated at 30°C for 2 or 3 days (rows 2–5) and 3 or 4 days (row 1) due to the synthetic growth defect of the tif5-7A TIF32Δ6–His strain. TIF5 GCN2 (YKHN205) and TIF5 gcn2Δ (H2898), transformed with empty vector, were analyzed in parallel in rows 4 and 5 and incubated for 2 or 3 days. Download figure Download PowerPoint If prt1-1 lowers the rate of TC binding to 40S subunits, then it should confer a Gcd− phenotype. Gcd− mutations suppress the sensitivity of gcn2Δ cells to 3-aminotriazole (3-AT), an inhibitor of the HIS3 product, because they derepress GCN4 translation, with attendant derepression of HIS3, independently of eIF2α phosphorylation by GCN2. We found that prt1-1 gcn2Δ cells displayed a weak Gcd− phenotype, allowing slightly better growth on 3-AT plates at 33°C compared to PRT1 gcn2Δ cells (Figure 5B, rows 2 and 3). This weak Gcd− phenotype was not suppressed by hc TC, however (Figure 5B, rows 1 and 2), suggesting that it does not result from impaired TC recruitment. Unexpectedly, prt1-1 cells containing GCN2 are sensitive to 3-AT (3-ATs) at 33°C (Figure 5C, rows 2 and 4), the phenotype characteristic of Gcn− mutants. Consistently, derepression of a GCN4–lacZ reporter containing all four uORFs was conditionally defective in the prt1-1 GCN2 cells, showing nearly WT induction by 3-AT at 25°C, but no induction at 33°C (Figure 6A, rows 2–4, 28 versus 30 U). Compared to the PRT1 gcn2Δ strain, GCN4–lacZ was partially derepressed under noninducing conditions at 33°C in the prt1-1 GCN2 mutant (weak Gcd− phenotype) but did not increase further upon 3-AT induction. At 34°C, we observed only the strong Gcn− phenotype in the prt1-1 GCN2 strain (Figure 6A, row 4, 7 versus 6 U), suggesting that it reflects the most severe defect produced by prt1-1. Figure 6.The prt1-1 mutation impairs GCN4 translational control, leads to hyperaccurate start codon selection, and does not show a synthetic growth defect with depletion of 60S subunit protein RPL11A. (A) prt1-1 (H1676), PRT1 (H2879), YEF3 (F1006) or yef3 (F650S) (F1007) cells were transformed with p180 containing the GCN4–lacZ fusion with all four uORFs and grown in SC-Ura in the presence or absence of 10 mM 3-AT, or 0.06 μg/μl SM, as shown, at the indicated temperatures. In row 1, the prt1-1 strain was also transformed with hc LEU2 plasmid (p832) containing GCN2 (hcGCN2) or empty vector, and the PRT1 strain also harbored an empty vector. β-Galactosidase activities were measured in WCEs and expressed in units of nmol of o-nitrophenyl-β-D-galactopyranoside hydrolyzed per min per mg of protein. The mean activities and standard errors obtained from independent transformants are indicated. (B) The prt1-1 (H1676) and PRT1 (H2879) strains were transformed with plasmids p180, pM199, pM226 or p226 (rows 1–4), respectively, and contained the GCN4–lacZ constructs shown schematically to the right and analyzed as in (A). Row 1 contains the same data as in row 4 of (A) shown for comparison. (C) prt1-1 (H1676), PRT1 (H2879), YEF3 (F1006) or yef3 ts (F1007) cells were transformed with p367 or p391 containing a HIS4–lacZ reporter harboring AUG or UUG start codons, respectively. The PRT1 and prt1-1 transformants were grown at 34°C, while the YEF3 and yef3 ts transformants were grown at 35°C, in SC-Ura medium, and β-galactosidase was assayed in WCEs. The graph shows the mean ratios of expression from the UUG to the AUG reporter measured for four independent transformants of each strain (for each reporter), with standard errors indicated as error bars. (D) Growth of prt1-1 (H1676) and prt1-1 rpl11aΔ (H2925) strains in YPD medium for 3 days at 25°C (upper panel) or 4 days at 33°C (lower panel). Download figure Download PowerPoint The Gcn− phenotype of prt1-1 cells could arise from a defect in eIF2α phosphorylation by GCN2. Western analysis using antibodies specific for eIF2α phosphorylated on Ser-51 (eIF2α-P) revealed an ∼40% reduction in the level of eIF2α-P in the prt1-1 mutant grown at 34°C with 10 mM 3-AT. Introducing GCN2 on an hc plasmid into the prt1-1 strain restored eIF2α-P to levels greater than or equal to those seen in the PRT1 strain (data not shown), but derepression of GCN4–lacZ expression remained impaired (Figure 6A, row 1). Hence, the partial reduction in eIF2α phosphorylation in the prt1-1 strain cannot account for its strong Gcn− phenotype. It was possible that the reduced rate of translation initiation in prt1-1 cells at 34°C would diminish the consumption of TC and restore TC to high levels even when eIF2α is phosphorylated. To test this, we asked whether a Ts− mutation in elongation factor eEF3 (encoded by YEF3) would produce a Gcn− phenotype at a semipermissive temperature (35°C) where the doubling time of the yef3 mutant (∼20 h) is similar to that of prt1-1 cells at 34°C. The yef3 mutant showed no Gcn− phenotype at 35°C (Figure 6A, row 5), making it improbable that the Gcn− phenotype of prt1-1 cells results from a reduced rate of translation. A third possibility to explain the Gcn− phenotype of prt1-1 cells would be if 40S subunits cannot resume scanning following translation of uORF1, as only rescanning subunits can bypass uORFs 2–4 and reinitiate at GCN4 (Mueller and Hinnebusch, 1986). For example, 40S subunits might dissociate from the mRNA at the uORF1 stop codon or while scanning downstream from uORF1. To address this possibility, we examined a GCN4–lacZ construct containing only uORF1 and lacking the segment containing uORFs 2–4, (pM199). Expression of this construct is constitutively high in WT cells because a large proportion of ribosomes that translate uORF1 can resume scanning and reinitiate at GCN4 regardless of TC levels (Grant et al, 1994). As shown in Figure 6B (row 2), there was no significant difference in the expression of this construct between prt1-1 and PRT1 cells in the presence of 3-AT. Hence, the proportion of ribosomes able to resume scanning and reach GCN4 following uORF1 translation was unaltered by prt1-1. The same result was obtained for a construct containing solitary uORF1 at its natural distance from GCN4 (data not shown). The fourth mechanism we addressed was the possibility that ribosomes leaky scan uORF1 in prt1-1 cells, making it impossible for them to bypass the remaining uORFs and reinitiate at GCN4. To test this possibility, we analyzed a GCN4–lacZ construct in which uORF1 is elongated and overlaps the beginning of GCN4. This elongated version of uORF1 blocks 98–99% of all scanning ribosomes from reaching the GCN4 start site, indicating that only 1–2% of ribosomes leaky scan uORF1 in WT cells (Grant et al, 1994). We observed little or no increase in GCN4–lacZ expression from this construct under 3-AT-inducing conditions in prt1-1 cells (Figure 6B, row 3). We also observed no increase in leaky scanning past uORF4 by assaying a construct containing solitary uORF4 at its normal location (Figure 6B, row 4). The results in rows 2–4 additionally eliminate the possibility that induction of GCN4–lacZ is reduced in prt1-1 cells by a reduction in reporter mRNA level, as this effect should apply equally to the mutant constructs containing solitary uORFs 1 or 4, yet expression of these constructs was unaffected by prt1-1. The fact that eliminating uORFs 2–4 completely suppresses the inability of prt1-1 cells to induce GCN4 expression (Figure 6B) provides strong genetic evidence that the Gcn− phenotype results from the inability of rescanning ribosomes to bypass uORFs 2–4 under starvation conditions when eIF2 is highly phosphorylated (Mueller and Hinnebusch, 1986). We envision two ways in which this could occur: (i) a reduction in the rate of scanning or (ii) a delay in GTP hydrolysis by 48S complexes at the start codons of uORFs 2–4, creating a barrier to movement of all 40S subunits through the leader to GCN4. Either defect would increase the time required for 40S subunits lacking TC to scan past uORFs 2–4 and thereby compensate f
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