INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Eukaryotic cells respond to different conditions of starvation and stress by down-regulating the rates of protein biosynthesis. One aspect of this response in mammalian cells involves phosphorylation on serine 51 of the alpha subunit of translation initiation factor 2 (eIF2) (53). This phosphorylation event reduces the activity of eIF2B, the guanine nucleotide exchange factor for eIF2 that recycles eIF2-GDP to the active form eIF2-GTP (20, 40, 46, 59). Only eIF2-GTP can bind initiator methionyl-tRNA (tRNA_i^Met) to produce the eIF2-GTP-tRNA_i^Met ternary complex that delivers initiator tRNA to the 43S preinitiation complex; thus, phosphorylation of eIF2 inhibits general protein synthesis initiation. In the budding yeast Saccharomyces cerevisiae, eIF2 becomes phosphorylated when cells are deprived of an amino acid or purine, leading to depletion of ternary complex levels, just as occurs in mammalian cells (19, 31). Interestingly, it also specifically stimulates translation of the distinctive mRNA encoding Gcn4p, a transcriptional activator of more than 40 genes involved in amino acid biosynthesis (29), thereby alleviating the nutrient limitation conditions that trigger phosphorylation of eIF2 in yeast.

The molecular mechanism that couples GCN4 translation to amino acid availability has been explored in great detail over past several years (reviewed in references 31 and 32). Four short upstream open reading frames (uORFs) present in the GCN4 mRNA leader sequence prevent ribosomes from initiating translation at the GCN4 start codon under conditions of amino acid sufficiency. Amino acid starvation triggers activation of the protein kinase Gcn2p, which phosphorylates eIF2 and thereby inhibits eIF2-GTP-tRNA_i^Met ternary complex formation. This situation specifically favors GCN4 translation because, after translating uORF1, many ribosomes cannot acquire the ternary complex in time to recognize the start codons at uORF2, -3, and -4 and thus continue scanning downstream. The majority of these ribosomes rebind the ternary complex by the time they reach the GCN4 AUG codon and reinitiate there instead (19, 20).

Recessive mutations in any of the genes encoding the four essential subunits of eIF2B, GCD1, GCD2, GCD6, and GCD7, mimic the effects of eIF2 phosphorylation and derepress GCN4 mRNA translation in the absence of Gcn2p and amino acid starvation (29, 30). These gcd mutations also lead to unconditional slow growth or temperature sensitivity on nutrient-rich medium, phenotypes that result from defects in general translation initiation (9, 16, 22, 27). It is thought that gcd1, gcd2, gcd6, and gcd7 mutations impair the ability of eIF2B to recycle eIF2-GDP to eIF2-GTP and thereby decrease the rate of ternary complex formation in vivo (31, 46). This causes lower rates of translation initiation on most mRNAs but leads to increased translation of GCN4 mRNA, which is inversely coupled to the availability of ternary complexes in the cell (20).

Recessive mutations in GCD10 also lead to temperature-sensitive growth and constitutive derepression of GCN4 translation in the absence of Gcn2p (26, 42). GCD10 is an essential gene and was shown to encode a 62-kDa protein that copurified and coimmunoprecipitated with subunits of eIF3 (23). eIF3 stimulates several steps of the initiation pathway in mammalian cells (among others, binding of ternary complexes to 40S ribosomes [47]). Accordingly, we proposed that gcd10 mutations might derepress GCN4 translation by decreasing the ability of eIF3 to promote rebinding of ternary complexes to 40S subunits that have translated uORF1 and continued scanning downstream on the GCN4 mRNA leader. This would allow these subunits to skip uORF4 and reinitiate translation at the GCN4 AUG without any reduction in the level of ternary complex formation (23). More recently, we found that gcd10 mutations reduce the expression of mature tRNA_i^Met at a posttranscriptional step and that the phenotypes of gcd10 mutants are suppressed by multiple copies of the genes IMT1 to IMT4, encoding tRNA_i^Met (3). Interestingly, gcd10 mutants lack 1-methyladenosine (m¹A) at position 58 in tRNA_i^Met and in all other tRNAs (17 or more) containing this modified base. The absence of m¹A seems to impair specifically the expression of mature tRNA_i^Met through degradation of the unprocessed precursors containing 5' and 3' extensions (3). The reduction in mature tRNA_i^Met levels in gcd10 mutants should diminish ternary complex formation, decreasing the rate of general translation initiation and producing constitutive derepression of GCN4 translation (20). Thus, the impaired maturation of tRNA_i^Met can also explain the known phenotypes of gcd10 mutants (3).

We previously identified GCD14 and GCD15 as novel genes in S. cerevisiae that are required for translational repression of GCN4 mRNA under amino acid-replete conditions (17). Here we report the molecular cloning of GCD14 and show that it encodes an essential protein containing binding motifs for S-adenosylmethionine (S-AdoMet). We found that gcd14 mutants have reduced levels of mature tRNA_i^Met and accumulate tRNA_i^Met precursors containing 5' and 3' extensions. Similar to the case for gcd10 mutants, the presence of IMT1 to IMT4 in high copy number suppresses the phenotypes of gcd14-1 and gcd14-2 mutants, and overexpression of IMT4 overcomes the lethality of a gcd14::URA3 deletion. Gcd14p is physically associated with Gcd10p in cell extracts (3), and we observed additive effects of gcd14 and gcd10 mutations on cell growth and expression of mature tRNA_i^Met. Interestingly, we found that gcd14 mutations also lead to reduced expression of other tRNAs and the RNA components of RNases P (38) and MRP (54) when combined with a deletion of LHP1, encoding a yeast homologue of the human autoantigen La. These findings and others (3) suggest that Gcd10p and Gcd14p functionally cooperate with Lhp1p to promote the maturation of a subset of RNA polymerase III transcripts.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. Plasmids constructed in this work are represented in Fig. 1. Plasmid pRC5 was isolated from a yeast genomic library in the low-copy-number vector YCp50 (51) by selecting for complementation of the 3-amino-1,2,4-triazol resistance (3AT^r) and Slg phenotypes of strain Hm316 (gcd14-2). Plasmids pRC55 and pRC56 contain, respectively, the 2.3-kb XbaI and the 2.8-kb SpeI-ClaI fragments from pRC5 subcloned into the corresponding sites of pRS316 (56).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Analysis of the GCD14 coding region. A partial restriction map of the GCD14 region is shown in the center, indicating the positions of the two ORFs, GCD14 and SPT10 (44), identified in the plasmid isolated from the genomic library bearing GCD14 (pRC5). The direction of transcription is indicated by the arrows. The top line depicts the genomic DNA insert in pRC5, and below that are depicted the fragments present in subclones constructed from pRC5 in low-copy-number vector pRS316 (pRC55 and pRC56). The ability of each subclone to complement the phenotypes of gcd14-1 and gcd14-2 mutants is shown on the right. Below the map of the GCD14 region are shown enlargements of the 2.6-kb SpeI-ClaI region in plasmid pRC56 and of the 3.2-kb SpeI-ClaI region in pRC560 bearing the gcd14::URA3 allele, in which a 1.1-kb URA3 fragment replaces ~730 bp of the GCD14 coding sequence. Restriction enzyme sites: B, BamHI; Bg, BglII; C, ClaI; H, HindIII; Hp, HpaI; S, Sau3A; X, XbaI; Sp, SpeI.

Yeast strain constructions. The genotypes and origins of all yeast strains used in this study are summarized in Table 1. Strains Hm295 (gcd14-1), Hm296 (gcd14-2), and Hm316 (gcd14-2) were selected as 3AT^r and Slg ascospores from genetic crosses previously described (17). Isogenic Hm296G and Hm296g strains were obtained by transforming Hm296 to Ura⁺ with the integrating plasmid pRC57, containing an incomplete copy of GCD14. Depending on the location of the crossover, integration of pRC57 yielded a nontandem duplication of wild-type GCD14 and the truncated gcd14 allele, or the gcd14-2 and truncated gcd14 alleles, separated by plasmid sequences. The former (in Hm296G) led to a 3AT-sensitive phenotype, whereas the latter (in Hm296g) conferred 3AT resistance.

ura3-52 leu2-3,112 trp163

Media and genetic techniques. Sensitivity to 3AT (Sigma; catalog no. A8056) was tested as previously described (28). Transformation of yeast strains was carried out as described by Ito et al. (34). Standard genetic techniques and media were as previously described (55).

Chromosomal mapping of GCD14. GCD14 was physically mapped to chromosome X by using a Chromo-Blot (Clontech), in which yeast chromosomes were fractionated by clamped homogeneous electrical field electrophoresis and blotted to a nylon membrane. Two individual blot lanes were probed, hybridizing a radiolabeled 2.8-kb HpaI-BamHI fragment from pRC5 that contains most of the GCD14 coding sequence. The same probe and a radiolabeled 2.4-kb XbaI fragment from pRC55 were used to hybridize a set of filters containing an ordered lambda library of yeast genomic DNA (49). The first probe hybridized to clone 70692, and the second hybridized with the same clone and also with clone 70091, both bearing inserts corresponding to containing a region encompassing TIF2 and URA2 sequences on in the left arm of chromosome X. To map GCD14 genetically, we crossed H168 (gcn2-101 gcn3-101 gcd14-2 ino1) with H753 (gcn2::LEU2 GCD14 INO1) and analyzed cosegregation of the 3AT^r, Slg, and Ino phenotypes in 45 tetrads. We observed 32 parental ditype, 0 nonparental ditype, and 13 tetratype asci, thus locating GCD14 at 14.4 centimorgans from INO1.

DNA sequence analysis and cloning of gcd14 alleles. The nucleotide sequence of a 2.8-kb SpeI-ClaI fragment in pRC56 was obtained for both strands by using a Sequenase version 2.0 kit (U.S. Biochemical Corp). Reverse primer, M13-40 primer, and several oligonucleotides derived from the previously determined sequences were used as primers, and pRC56 was used as double-stranded DNA template. The DNA sequence was analyzed by using the DNAsis sequence analysis program (Hitachi software), and comparisons of the GCD14 sequence were made with BLAST programs (2).

Production of Gcd14p-specific antiserum. The TrpE-Gcd14p fusion protein encoded in plasmid pTRPC14 was overexpressed in E. coli HB101 and partially purified from the insoluble fraction of whole-cell extracts by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as already described (21). Gel slices containing ~200 µg of the fusion protein were used to inoculate rabbits. Immunization of the rabbits and collection of the immune antisera were conducted by Hazelton Laboratories (Vienna, Va.).

RNA isolation and Northern blot analysis. Total RNA was isolated as previously described (36). Samples were dried, resuspended in loading buffer (90% formamide, 1 mM EDTA [pH 8.0], 0.1% bromophenol blue, 0.1% xylene cyanol), and heated to >90°C for 10 min before separation on a 6 or 8% polyacrylamide-bisacrylamide (19:1)-8.3 M urea gels by electrophoresis at 250 V for approximately 3.5 h. Gels were run at 250 V for 20 min prior to separation of RNAs. Following electrophoresis, gels were soaked in 0.5× Tris-borate-EDTA for 20 min, and RNA was transferred to positively charged nylon membranes (Boehringer Mannheim) in the same buffer by electrotransfer (Trans-Blot Cell; Bio-Rad) for 3.5 h at 19 V. RNA was immobilized on membranes by UV cross-linking with a UV-Stratalinker 2400 (Stratagene) according to the manufacturer's instructions. tRNAs on the membranes were detected by hybridization with radiolabeled oligonucleotides in hybridization buffer (0.25 M Na₂HPO₄ [pH 7.5], 7% SDS, 1% bovine serum albumin, 1 mM EDTA) at 50 to 52°C for 12 to 20 h. Oligonucleotides were radiolabeled at the 5' ends with [-³²P]ATP (6,000 Ci/mmol) and T4 polynucleotide kinase (Pharmacia). Following hybridization, membranes were washed once with 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS for 30 min at room temperature and once in 1× SSC-0.1% SDS at 60°C for 20 min prior to detection by autoradiography. Direct quantitation of all hybridization signals was conducted by phosphorimager analysis with a BAS-1500 PhosphorImager and MacBAS Ver.2.x software (Fuji Film). For reprobing, membranes were stripped of radioactive probes by incubation at 100°C in 1.0% SDS and cooled to room temperature.

Nucleotide sequence accession number. The EMBL accession number for the GCD14 nucleotide sequence is Z54149.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Isolation and characterization of the wild-type GCD14 gene and of the gcd14-1 and gcd14-2 mutant alleles. Mutations in GCD14 were isolated as suppressors of the 3AT phenotype of a gcn2-101 gcn3-101 double mutant (17). All gcn mutations confer sensitivity to 3AT because they impair derepression of GCN4, and of histidine biosynthetic genes regulated by Gcn4p, in response to histidine starvation imposed by 3AT (28). gcd14 mutations restore derepression of GCN4 translation and thus confer 3AT resistance in a gcn2 gcn3 background. In addition, recessive gcd14 mutations lead to a slow-growth phenotype (Slg) on rich media at 28°C which is exacerbated at both lower and higher temperatures (18 and 37°C) (Ts phenotype) (data not shown). We cloned the wild-type allele of GCD14 from a yeast genomic library on plasmid pRC5 (Fig. 1) by complementing the 3AT^r and Slg phenotypes conferred by gcd14-2 in the gcn2-101 gcd14-2 strain Hm316. To prove that we had cloned authentic GCD14, we mapped the chromosomal location of the genomic insert in pRC5 to chromosome X of S. cerevisiae. A radiolabeled 2.8-kb HpaI-BamHI fragment obtained from pRC5 hybridized exclusively to chromosome X in a yeast Chromo-Blot (see Materials and Methods). The cloned sequence was mapped to the left arm of chromosome X, in a region close to SPT10/PBS2/TRK1, by hybridizing the same HpaI-BamHI fragment to filters containing an ordered lambda library of yeast genomic clones (see Materials and Methods). From tetrad analysis we estimated that gcd14-2 is about 14.4 centimorgans from INO1 (see Materials and Methods). The fact that INO1 is located 50 kb from the cloned sequence on the left arm of chromosome X verifies that the insert in pRC5 contains the wild-type allele of GCD14.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Deduced amino acid sequence of Gcd14p. The predicted amino acid sequence of S. cerevisiae Gcd14p is given in single-letter code. Two regions of sequence similarity observed in several S-AdoMet-dependent methyltransferases are boxed and indicated as motif I and motif II (35). The conserved G residue at position 5 of motif I is G118 in Gcd14p. A glutamate residue commonly located 17 to 19 residues C terminal to motif I is found 17 residues C terminal to motif I in Gcd14p, at position 139 (*), and a cluster of hydrophobic residues, hhXh (D/E), at positions 135 to 138 (underlined, LFSF) precedes the E element. The central invariant aspartate in motif II is conserved at position 209 in Gcd14p. Motifs I and II are separated by 83 residues in Gcd14p. A putative nuclear localization signal (7) (shaded box) is located between residues K282 R294 in the Gcd14p sequence.

GCD14 is an essential gene. The slow-growth phenotype of the gcd14-1 and gcd14-2 mutations suggests that Gcd14p has an essential function beyond its role in GCN4-specific translational control. To test this possibility, we constructed a deletion-insertion null allele, gcd14::URA3, in plasmid pRC560 (Fig. 1) and used it to replace one allele of GCD14 in ura3-52/ura3-52 diploid strains YRC1 (gcn2/gcn2 GCD14/gcd14-2 ura3-52/ura3-52) and YRC2 (GCN2/GCN2 GCD14/GCD14 ura3-52/ura3-52) (see Materials and Methods). We verified by Southern blot analysis that the gcd14::URA3 allele replaced one of the two copies of GCD14 in the Ura⁺ transformants of YRC1 and YRC2 that were obtained (data not shown). Tetrad analysis of these transformants revealed that in 24 asci from each diploid, only two of the four spores formed colonies on rich medium after 4 days at 28°C (data not shown). All viable spores were Ura (ura3-52) (and thus contain GCD14), and those from the YRC1 transformant were also 3AT^s (ura3-52 gcn2 GCD14), indicating that the gcd14-2 allele had been replaced by gcd14::URA3. These results demonstrate that GCD14 is essential for growth.

gcd14-2 cells exhibit a modest defect in general translation initiation. The essential nature of the GCD14 gene together with its role as a repressor of GCN4 mRNA translation (17) suggested to us that Gcd14p could be required for general initiation of translation. To investigate that possibility, we analyzed total polysome profiles from wild-type and mutant gcd14-2 strains by fractionating whole-cell extracts on sucrose gradients by velocity sedimentation. The gcd14-2 mutants do not form colonies at 37°C; however, they exhibited little or no growth defect in liquid medium after several hours at 37°C (data not shown). Quantitation of total polysome profiles for gcd14-2 and GCD14 strains grown at 23°C to mid-logarithmic phase and then incubated for 4 h at 37°C revealed no significant differences in the polysome/monosome (P/M) ratio. Accordingly, we next examined polysome profiles for the isogenic strains Hm296g (gcd14-2) and Hm296G (GCD14) grown at 39°C, where the gcd14-2 mutant exhibits a more detectable growth defect in liquid medium. As shown in Fig. 3, the P/M ratio was considerably lower in the gcd14-2 mutant (P/M ratio, 2.4) than in its isogenic GCD14 parent strain (P/M ratio, 5.7) after 1.5 h at 39°C. There was also a small reduction in the average size of the polysomes in the gcd14-2 mutant at 39°C (see Fig. 3 legend). Although these differences in polysome size and content are not dramatic, they were consistently observed in three independent experiments (see Fig. 3 legend). These data suggest that the gcd14-2 mutants exhibit a modest defect in general translation initiation. The magnitude of this defect is less than that observed in gcd1 or gcd2 mutants, consistent with the lesser derepression of GCN4 translation associated with gcd14 versus gcd1 or gcd2 mutations (17, 26).

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Polysome profiles of isogenic gcd14-2 and GCD14 strains. Isogenic strains Hm296g and Hm296G were cultured overnight in yeast extract-peptone-dextrose at 23°C and used to inoculate two flasks containing 300 ml of yeast extract-peptone-dextrose to an optical density at 600 nm of ~0.1. Cultures were incubated at room temperature in a rotary shaker at 250 rpm. At an optical density at 600 nm of ~4.0, cells were collected and resuspended in the appropriate volume of yeast extract-peptone-dextrose prewarmed to 39°C and grown to an optical density at 600 nm of ~2.0. From both flasks, 150 ml was collected immediately for the 39°C, t = 0 samples, and 150 ml of fresh prewarmed yeast extract-peptone-dextrose was added back to each flask. Samples of 150 ml were collected from both flasks after 1.5 h and processed for polysome analysis by velocity sedimentation of whole-cell extracts on sucrose gradients (22). Gradients were fractionated while being scanned at 254 nm, and the resulting absorbance profiles are shown, with the tops of the gradients on the left. The positions of 40S, 60S, and 80S ribosomal species are indicated by arrows. For each strain, the top two gradients correspond to samples incubated at 39°C for 0 h (t = 0) and the two bottom gradients contain samples obtained after 1.5 h of incubation at 39°C. Areas delimited inside the profiles correspond to the fractions of the total absorbance profiles represented by 2-mer and 3-mer polysomes. In the gcd14-2 strain, the 2-mers and 3-mers represented 26% of the polysome mass at the permissive temperature but comprised 32% of the polysomes after 1.5 h at 39°C. In contrast, 2-mers and 3-mers represented 24 and 25% of the polysome mass at 23°C and after 1.5 h at 39°C, respectively, in the wild-type strain Hm296G. The P/M ratios were calculated at t = 0 and t = 1.5 h for three independent experiments, and the following mean values and standard errors (in parentheses) were obtained: at t = 0, P/M = 4.2 (0.83) for GCD14 and 2.8 (0.44) for gcd14-2; at t = 1.5 h, P/M = 5.2 (0.35) for GCD14 and 3.5 (0.72) for gcd14-2.

Genes encoding initiator tRNA^Met or Lhp1p are dosage suppressors of the Gcd and Slg phenotypes of gcd14 mutants. gcd10 and gcd14 mutations lead to constitutive derepression of GCN4 translation in the absence of Gcn2p (Gcd phenotype), conferring resistance to 3AT in a gcn2 strain background (17, 26). We found recently (3) that the 3AT^r and Ts phenotypes conferred by gcd10 mutations in a gcn2 background can be fully suppressed by the presence in high copy number of any of the four genes, IMT1 to IMT4, encoding tRNA_i^Met (11, 14) and can be partially suppressed by high-copy-number LHP1 (39, 60). In view of the physical interaction found between Gcd10p and Gcd14p (3), we asked whether the dosage suppressors of gcd10 mutations could also suppress the 3AT^r and Slg phenotypes conferred by gcd14 mutations. Each of the four IMT genes on high-copy-number plasmids (hcIMT) suppressed the 3AT^r (data not shown) and Slg (Fig. 4) phenotypes in gcd14-1 gcn2-101 and gcd14-2 gcn2-101 mutants to about the same extent as did a low-copy-number plasmid containing GCD14, whereas high-copy-number LHP1 (hcLHP1) only partially suppressed the mutant Slg phenotype (Fig. 4 and data not shown). hcGCD10 had no suppressor activity in the gcd14 mutants (Fig. 4), and neither single-copy GCD14 nor hcGCD14 suppressed the phenotypes of mutants containing different gcd10 alleles (data not shown). Furthermore, suppression by hcIMT and hcLHP1 seems to be specific for gcd10 and gcd14, as temperature-sensitive mutations in genes encoding subunits of translation initiation factors eIF2 and eIF3 (sui2-1 and prt1-1, respectively) were not suppressed by these dosage suppressors (3).

View larger version (84K): [in this window] [in a new window]   FIG. 4.   Phenotypes of gcd14 dosage suppressors. Eight independent transformants of strains Hm295 (gcd14-1) and Hm296 (gcd14-2) carrying empty vector pRS426 (gcd14), a low-copy-number plasmid bearing GCD14 (pRC56), or high-copy-number plasmids bearing IMT1 (p2632), IMT2 (p2633), IMT3 (p2634), IMT4 (p2635), GCD10 (pE107), or LHP1 (p2636) were streaked for single colonies on minimally supplemented SD plates and incubated at 28°C (top plates) or 37°C (bottom plates) for 2 days. The locations of the transformants on plates are indicated inside the central schematic.

gcd14 mutants are defective in processing the primary precursors of the initiator tRNA^Met. High-copy-number IMT1, -2, -3, and -4 and LHP1 suppress the phenotypes of gcd10 mutants because these mutants contain reduced amounts of mature tRNA_i^Met, and the dosage suppressors compensate for this defect by increasing mature tRNA_i^Met to nearly wild-type (hcLHP1) or higher-than-wild-type (hcIMT) levels (3). Accordingly, we used Northern analysis to investigate whether steady-state levels of mature tRNA_i^Met were also reduced in gcd14 mutants H160 (gcd14-1) and H168 (gcd14-2) compared to that in their parental wild-type strain (H117). The probe for tRNA_i^Met, containing sequences complementary to nucleotides 33 to 64 in tRNA_i^Met, hybridizes to both mature tRNA_i^Met and different precursor tRNA_i^Met species bearing unique 5'-3' extensions encoded by the different IMT genes (3) (see Materials and Methods). The Northern data in Fig. 5 were quantified with a phosphorimager, and the results are given in Table 2. Relative to the level of elongator tRNA^Met (tRNA_e^Met), we calculated that the amounts of mature tRNA_i^Met in the mutant strains were ca. half of that in the wild-type strain both at 28°C and after 1.5 h at 37°C (Fig. 5A; Table 2). In addition, the levels of tRNA_i^Met precursors were 2.3- and 3.2-fold higher at 28 and 37°C, respectively, leading to a fivefold increase in the ratio of precursor to mature tRNA_i^Met in the gcd14 mutants versus the wild type (Fig. 5A; Table 2). These data strongly suggest that Gcd14p is required for efficient processing of the nascent tRNA_i^Met transcripts. It is noteworthy that tRNA_i^Met species migrating faster than the wild-type mature form were detected in both gcd14 mutants under all conditions (species f in Fig. 5A), suggesting that fully 5'-3'-processed tRNA_i^Met is either unstable or incorrectly processed in these mutants. Similar results were obtained for a gcd14-2 mutant by Anderson et al. (3), although the accumulation of pre-tRNA_i^Met at 37°C was less extensive than that observed here.

View larger version (47K): [in this window] [in a new window]   FIG. 5.   gcd14 mutants are defective in processing the primary precursors of initiator tRNAMet and tRNAUAUIle. Northern blot analysis of total RNA (10 µg) isolated from strains H117 (GCD14), H160 (gcd14-1), and H168 (gcd14-2) grown in yeast extract-peptone-dextrose medium at 28°C to mid-exponential phase (0 h at 37°C) and shifted to 37°C for 1.5 h is shown. The blot was probed with a radiolabeled oligonucleotide that specifically hybridized to tRNAiMet (A) and then was stripped and reprobed with radiolabeled oligonucleotides specific for tRNAeMet (B) or tRNAUAUIle (C) (see Materials and Methods). The positions of pre-tRNAiMet species, mature tRNAiMet, tRNAeMet, and primary (upper band) and 5'- and 3'-end-processed intron-containing (lower band) pre-tRNAUAUIle and mature tRNAUAUIle are indicated on the left. Indicated on the right are the positions of pre-tRNAiMet containing 5' and 3' extensions encoded by IMT2 and IMT3 (b), pre-tRNAiMet containing 5' and 3' extensions encoded by IMT1 and IMT4 (c), mature tRNAiMet (e), and aberrantly processed tRNAiMet species (f) (see text for details).

Additive effects of gcd10-504 and gcd14-2 mutations on processing and accumulation of tRNA_i^Met. In view of the similar phenotypes of gcd14 and gcd10 mutations, we investigated possible genetic interactions between them. The gcd10-505 gcd14-2 double mutant Hm397 was transformed with low-copy-number plasmids containing GCD10, GCD14, or empty vectors to generate four isogenic transformants with the following genotypes: (i) gcd10-505 gcd14-2, (ii) GCD10 gcd14-2, (iii) gcd10-505 GCD14, and (iv) GCD10 GCD14. As shown in Fig. 6A, the gcd10-505 gcd14-2 double mutant grew more slowly at 28 and 34°C than did either single mutant, indicating additivity of the growth defects conferred by these mutations. Northern analysis of the transformants grown at 37°C showed that the gcd14-2 single mutant contained high levels of tRNA_i^Met precursors and a modest reduction in mature tRNA_i^Met levels relative to the wild-type strain (Fig. 6B; Table 3), similar to the results described above. Compared to the gcd14-2 mutant, the gcd10-505 single mutant at 37°C showed a similar accumulation of the precursors but a somewhat greater reduction in mature tRNA_i^Met expression at 37°C (Fig. 6B, lanes 6 to 8). Both mutants showed 7- to 10-fold increases in the precursor tRNA/mature tRNA ratios for tRNA_i^Met (Table 3). These last findings are interesting because they suggest that Gcd10p is also required for efficient end processing of tRNA_i^Met. The gcd10-505 gcd14-2 double mutant at 37°C showed a greater reduction in mature tRNA_i^Met levels than did either single mutant, exhibiting levels only 30% of that seen in the wild-type transformant (Fig. 6B, lanes 5 to 8; Table 3). The additivity of these defects in mature tRNA_i^Met expression suggests that Gcd10p and Gcd14p participate in the same process involved in pre-tRNA_i^Met processing.

View larger version (36K): [in this window] [in a new window]   FIG. 6.   Exacerbation of gcd14-2 phenotypes by a gcd10-505 mutation. (A) The double mutant Hm423 (gcd10-505 gcd14-2) and isogenic GCD10 gcd14-2 (Hm420), gcd10-505 GCD14 (Hm421), and GCD10 GCD14 (Hm422) strains were streaked for single colonies on minimally supplemented SD plates and incubated at 28, 34, or 37°C for 2 days. The genotypes of the transformants are indicated adjacent to the appropriate sectors of the 28°C plate. (B) Northern blot analysis of total RNA (10 µg) isolated from the same strains analyzed in panel A, conducted as described for Fig. 5 except that cells were grown in minimally supplemented SD at 28°C (t = 0) (lanes 1 to 4) and then shifted to 37°C for 4 h (lanes 5 to 8). The blot was probed for 5S rRNA, tRNAiMet, and tRNAUAUIle as described for Fig. 5.

GCD14 is dispensable in yeast strains overexpressing initiator tRNA^Met. Having shown that GCD14 is essential and that gcd14 mutations impair the maturation of pre-tRNA_i^Met, we asked whether overexpression of an IMT gene would allow cells to survive in the absence of Gcd14p. To test this possibility, we replaced one of the two GCD14 alleles in the wild-type diploid strain YNG1 with the gcd14::URA3 deletion-insertion allele and verified the gene replacement by Southern blot analysis (data not shown). After sporulation of the resulting GCD14/gcd14::URA3 diploid, tetrad analysis revealed the expected 2⁺:2 segregation for cell viability, and all viable spores were Ura (bearing GCD14). When the GCD14/gcd14::URA3 diploid, which is homozygous for leu2, was first transformed with a high-copy-number LEU2 plasmid p1775 bearing IMT4 (20) and then subjected to tetrad analysis, 20 of 32 tetrads showed a 4⁺:0 segregation for cell viability and 2⁺:2 segregation for the Ura phenotype. Importantly, all of the Ura⁺ spores (bearing gcd14::URA3) were Leu⁺ (bearing hcIMT4 on p1775) (data not shown). These results showed that p1775 (hcIMT4) overcame the lethality of the gcd14::URA3 deletion. The gcd14::URA3 hcIMT4 and GCD14 hcIMT4 strains derived from one tetrad grew similarly at 28 and 37°C (Fig. 7A, spores 7A to 7D). In a second tetrad, however, the two gcd14::URA3 hcIMT4 strains grew more slowly than did the GCD14 hcIMT4 strains, particularly at 37°C (Fig. 7A, spores 5A to 5D). Thus, at least in certain genetic backgrounds (e.g., spores 7C and 7D), overexpression of tRNA_i^Met from IMT4 makes Gcd14p dispensable for growth at 28 and 37°C.

View larger version (24K): [in this window] [in a new window]   FIG. 7.   GCD14 is not essential in the presence of hcIMT4. (A) Ascospore clones from two four-spored tetrads (designated 5 and 7) were obtained from a heterozygous GCD14/gcd14::URA3 diploid (YNG1) containing high-copy-number plasmid p1775 (hcIMT4) (20). The four strains from each tetrad (5A to 5D and 7A to 7D), all containing hcIMT4, were streaked for single colonies on minimally supplemented SD medium and incubated at 28 or 37°C for 3 days. The position of each spore clone and its GCD14 genotype is indicated adjacent to the appropriate plate sector. +, wild-type GCD14; , gcd14::URA3. (B) Northern blot analysis of total RNA (10 µg) isolated from the spore clones of tetrad 5 (strains 5A to 5D) bearing hcIMT4 (described for panel A). The strains were grown to mid-logarithmic phase in minimally supplemented SD medium at 28°C (t = 0 at 37°C) and shifted to 37°C for 4 h. The blot was probed for tRNAiMet, tRNAeMet, or tRNAUAUIle as described for Fig. 5.

Deletion of LHP1 exacerbates the phenotypes of gcd14 mutations. To investigate the role of Lhp1p in processing of tRNA_i^Met precursors in gcd14 mutants, we disrupted LHP1 with the LEU2 gene in wild-type GCD14 and gcd14 mutant strains (see Materials and Methods). The lhp1::LEU2 mutation did not affect the growth rate of the wild-type strain H117, in agreement with previous results (60); however, it greatly exacerbated the Slg phenotypes of the gcd14-1 and gcd14-2 mutants (Fig. 8A). In addition, we observed additive effects of the lhp1::LEU2 and gcd14 mutations on the levels of mature tRNA_i^Met. Whereas the lhp1::LEU2 allele reduced mature tRNA_i^Met by only ca. 27% in GCD14 cells, it caused reductions of ca. 60% in gcd14-1 and gcd14-2 cells (Fig. 8B; Table 4). The levels of tRNA_i^Met precursors also were greatly diminished by the lhp1::LEU2 mutation in all three backgrounds, with reductions of 77, 86, and 68% in the GCD14, gcd14-1, and gcd14-2 strains, respectively (Fig. 8B; Table 4). Lhp1p has been implicated in stabilizing precursors and promoting correct 3' end processing of certain tRNAs (61). Thus, to account for the reduced amounts of mature tRNA_i^Met seen in the double mutants, we suggest that deletion of LHP1 leads to degradation of a substantial fraction of the tRNA_i^Met precursors and that this degradation is more extensive in the gcd14 mutants than in the GCD14 strain.

View larger version (141K): [in this window] [in a new window]   FIG. 8.   Synthetic interactions of gcd14 and lhp1::LEU2 mutations. (A) Strains YNG174 (GCD14), YNG175 (gcd14-1), and YNG176 (gcd14-2) and the isogenic lhp1::LEU2-containing derivatives Hm406 (GCD14 lhp1::LEU2), Hm407 (gcd14-1 lhp1::LEU2), and Hm408 (gcd14-2 lhp1::LEU2) were streaked for single colonies on minimally supplemented SD plates and incubated at 28 or 37°C for 2 days. The locations of the strains are indicated by their relevant genotypes in the schematic at the top. (B) Northern blot analysis of total RNA (20 µg) isolated from the strains described for panel A and separated by electrophoresis in a 6% polyacrylamide-8.3 M urea gel. Strains were grown in minimally supplemented SD at 28°C. The blot was probed for tRNAiMet, tRNAUAUIle, tRNAeMet, and tRNACGASer by using the appropriate oligonucleotides (see Materials and Methods). Indicated on the right of the top panel are the positions of various precursor and processed forms of tRNAiMet, as described in the legend to Fig. 5. The positions of precursor and mature tRNACGASer species are indicated on the right of the bottom panel: a primary precursor containing 5' and 3' extensions (g), a processing intermediate containing only the 3' extension (h), a 5'- and 3'-end-processed intron-containing precursor (i), and mature tRNACGASer (j). (C) The Northern blot in panel B was probed for RPR1 RNA, NME1 RNA, 5S rRNA, and U6 RNA by using the appropriate oligonucleotides (see Materials and Methods). The positions of precursor and mature RPR1 species are indicated on the left.

View larger version (26K): [in this window] [in a new window]   FIG. 9.   Gcd14p, but not Lhp1p, is tightly associated with Gcd10p in cell extracts. (A) Whole-cell extracts were prepared as described previously (47) from strains H1515 (LHP1) and YJA113 (lhp1) and from transformants of YJA142 (GCD10HA) or YJA143 (GCD10) bearing high-copy-number plasmid p2626 containing LHP1 or empty vector YEp24. Aliquots containing 200 µg of total cell protein were mixed with 2 to 4 µg of anti-HA monoclonal antibody HA.11 (Babco), which was prebound to protein A-Sepharose for 2 h on ice, and the mixture was incubated for 1 h at 4°C. After three washes with lysis buffer (20 mM Tris-HCl [pH 7.4], 100 mM KCl, 1.0 mM Mg acetate, 0.1% [vol/vol] Triton X-100) containing one complete protease inhibitor tablet (Boehringer Mannheim) per 25 ml, immunoprecipitates were collected by centrifugation and proteins were eluted by boiling in Laemmli buffer (37). The total proteins immunoprecipitated from 200 µg (pellet [P]; lanes 4, 6, and 8) or 50 µg of the starting whole-cell extracts (input [I]; lanes 3, 5, and 7) were separated by SDS-polyacrylamide gel electrophoresis (36) and transferred to a nitrocellulose membrane (Millipore) in 25 mM Tris-192 mM glycine-0.1% SDS containing 20% (vol/vol) methanol. Lanes 1 and 2 contain 50 µg of the starting whole-cell extracts from strains H1515 and YJA113, respectively, which were included to establish the identity of Lhp1p. The membrane was blocked overnight at 4°C in BLOTTO (5% [wt/vol] nonfat dry milk, 10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.05% [vol/vol] Tween 20). A single immunoblot was probed with 3 µg of rabbit anti-HA antibody HA.11 (Babco) per ml, stripped, reprobed with a 1:1,000 dilution of anti-Gcd14p antibodies (see Materials and Methods), and reprobed with a 1:1,000 dilution of anti-Lhp1p antibodies (60). Immune complexes were detected with horseradish peroxidase-conjugated sheep antimouse (Amersham) or donkey antirabbit (Amersham) secondary antibodies and an enhanced chemiluminescence kit (ECL; Amersham). (B) A longer exposure of lanes 5 and 6 of the blot in panel A probed with anti-Lhp1p antibodies, included to detect small amounts of Lhp1p in the immunoprecipitates.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Evidence that Gcd14p is required for efficient processing of tRNA_i^Met. Recessive gcd14 mutations confer constitutive derepression of GCN4 mRNA translation in the absence of eIF2 phosphorylation (17), a phenotype generally indicating reduced formation of the eIF2-GTP-tRNA_i^Met ternary complex (20). gcd14 mutants also have a Slg phenotype, and we found that general translation initiation is slightly impaired in these mutants, at least at elevated temperatures (Fig. 3). In addition, deletion of GCD14 is lethal. These findings suggest that Gcd14p has an essential function required for ternary complex formation in vivo. In agreement with this conclusion, we found that gcd14 mutants contain approximately 50% lower steady-state levels of mature tRNA_i^Met than are present in isogenic wild-type strains (Tables 2 to 4). They also contain processed tRNA_i^Met molecules that appear to be smaller than wild-type mature tRNA_i^Met (Fig. 5 to 7, species f) and thus may be defective in aminoacylation or ternary complex formation. The Gcd and Slg phenotypes of the gcd14 mutants were fully suppressed by introducing multiple copies of the IMT genes encoding tRNA_i^Met, which restored expression of mature tRNA_i^Met to greater-than-wild-type levels. Moreover, the presence of hcIMT4 overcame the lethality of a gcd14 deletion. The phenotypes of nonlethal gcd14 mutations also were partially suppressed by hcLHP1, which increased the level of mature tRNA_i^Met to just below wild-type levels. Together, these findings indicate that the essential function of Gcd14p is required for efficient and accurate production of mature tRNA_i^Met.

Evidence that Gcd14p and Gcd10p function together to promote maturation of tRNA_i^Met in vivo. Nonlethal gcd10 mutations lead to reduced expression of mature tRNA_i^Met and can be suppressed by hcIMT and hcLHP1 (3), just as shown here for gcd14 mutations (Fig. 4). Moreover, the lethal effect of deleting GCD10 also could be suppressed by overexpression of tRNA_i^Met from high-copy-number IMT4 (3). Thus, the essential functions of both proteins, at least at low growth temperatures, are required only for expression of mature tRNA_i^Met. In addition to these genetic similarities, we found that Gcd10p and Gcd14p can be coimmunoprecipitated (Fig. 9) and copurified (3) from whole-cell extracts. The physical association between these two proteins has been confirmed by using a polyhistidine-tagged form of Gcd10p to isolate a Gcd10p-Gcd14p complex from whole-cell extracts by affinity chromatography (3) and by yeast two-hybrid analysis (4). In addition, epitope-tagged forms of both proteins Gcd10p and Gcd14p were shown by immunofluorescence to exhibit prominent nuclear localization in yeast cells (3). These findings strongly suggest that Gcd10p and Gcd14p reside in a nuclear complex that functions directly in the maturation of tRNA_i^Met.