N-terminal region of Saccharomyces cerevisiae eRF3 is essential for the functioning of the eRF1/eRF3 complex beyond translation termination

Identification and characterization of mutants in which deletion of the eRF3 NM region is lethal

The SUP35-C strain YJV159-rS35C carrying the pHT-SUP35 (SUP35 ADE3) centromeric plasmid was mutagenized with UV irradiation and over 150,000 of grown colonies were screened using a yeast colony color sectoring assay. Among them, 23 were of Sect^- phenotype. Such clones could arise due to mutations in the SUP35-C gene, since it is essential [8], or due to mutations in other genes. To discriminate between these possibilities, the centromeric pRS315-SUP35C (SUP35-C LEU2) plasmid was introduced into the YJV159-rS35C strain carrying pHT-SUP35. In 22 cases this restored Sect⁺ phenotype indicating that mutations were within SUP35-C and corresponding mutants were discarded. One mutant, which segregated white sectors very poorly, preserved its leaky Sect^- phenotype in the presence of the pRS315-SUP35C plasmid. The mutant, designated as YJV-rS35C-SL23, could not grow at 37°C on YPD plate. This mutant carrying pHT-SUP35 was transformed with a genomic library based on the LEU2 centromeric plasmid p366, and two transformants able to grow at non-permissive temperature were selected. The obtained temperature resistant transformants were characterized by the frequent loss of pHT-SUP35 at permissive temperature, thus being Sect⁺. Plasmid from one transformant was isolated and partial sequencing of the cloned DNA fragment has shown that it contained the SUP45 gene. Subsequent delimitation of the functional region of the isolated DNA fragment indicated that SUP45 was responsible for both temperature resistance and Sect⁺ phenotype. Transformation of this mutant with various SUP45-carrying plasmids caused the same effects. Thus, mutation that made the NM region of eRF3 indispensable arose in the SUP45 gene. Transformation of the mutant YJV-rS35C-SL23 carrying pHT-SUP35 with the multicopy plasmid YEplac181-SUP35NM encoding the eRF3 NM region did not restore the Sect⁺ phenotype. This indicated that expression of the entire eRF3 protein is necessary for viability of this mutant.

The obtained sup45 mutation, designated as sup45-sl23 ^ts , caused growth inhibition in the strain YJV-rS35C-SL23, but was lethal in other sup45-sl23 ^ts strains with the SUP35-C allele. For example, the URA3 SUP35 plasmid pRS316-SUP35 of the SUP35-disrupted strain 33G-D373-rSL23-ΔS35 carrying sup45-sl23 ^ts could not be replaced with the LEU2 SUP35-C plasmids upon incubation on 5FOA medium (Fig. 1). It is also important that in this strain the plasmid pRS316-SUP35 also could not be replaced with the centromeric pRS315-SUP35MC plasmid, encoding eRF3 which lacks only the N domain (Fig. 1). Thus, just the absence of the eRF3 prion-forming domain was responsible for synthetic lethality.

Synthetic lethality was also reproduced in the S1-R-H8 strain. This strain, carrying SUP35-C, was disrupted for SUP45 and contained the URA3 SUP45 centromeric plasmid pRG415-SUP45, which could not be replaced with the plasmid pRS315-sup45-sl23 carrying the mutant sup45-sl23 ^ts allele. In contrast, in the control strain S1-H8 with chromosomal wild type SUP35, pRS315-sup45-sl23 could replace the resident pRG415-SUP45 plasmid. The lethal effect of combination with SUP35-C was also found for another sup45 mutation, sup45-36 ^ts [24]: the pRS315-sup45-36 plasmid did not replace pRG415-SUP45 in the S1-R-H8 strain, but replaced this plasmid in the SUP45 disruptant S1-H8 bearing the wild type SUP35 gene.

The possible reason of lethality manifested by the sup45 mutations in combination with SUP35-C is the decreased level of eRF1. However, the amount of eRF1 was not affected in the sup45-36 ^ts [25] and sup45-sl23 ^ts mutants (data not shown). S. cerevisiae eRF1 and eRF3 interact with each other through the binding sites located at their C-termini [26, 27], though the eRF3 N-terminal region also may be involved in this interaction [28]. Mutations in eRF1 breaking its interaction with eRF3 decrease cell viability [27]. The observed disparity in genetic interaction of the sup45-sl23 ^ts and sup45-36 ^ts mutations with SUP35 and SUP35-C suggests that these mutations influence binding of eRF1 to eRF3, and this effect is more pronounced when eRF3 lacks its N-terminal region. However, the study of the ability of Escherichia coli-expressed His₆-eRF1 and His₆-eRF1-sl23 immobilized on TALON beads to precipitate eRF3 and eRF3C from yeast cell lysates [27] did not show a noticeable effect of sup45-sl23 ^ts mutation on the interaction of eRF1 with both eRF3 and eRF3C (data not shown).

The sup45-sl23 ^ts and sup45-36 ^ts mutations altered the eRF1 N domain (Ser-30 to Phe and Leu-34 to Ser substitutions, respectively), increased nonsense codon readthrough and caused temperature sensitivity [25]. At elevated temperature corresponding mutations did not cause any noticeable increase in the levels of nonsense codon readthrough (Fig. 2 and data not shown), but affected cell morphology. The sup45-36 ^ts mutation caused defect of cytokinesis and resulted in appearance of multinucleated cells with elongated buds. The defect of cytokinesis in this mutant is related to mislocalization of the myosin light chain Mlc1p and its temperature sensitivity could be suppressed by Mlc1p overproduction [25]. In contrast, temperature sensitivity of the sup45-sl23 ^ts mutant could not be alleviated by excess of Mlc1p. Cells of the sup45-sl23 ^ts mutant were increased in size (Fig. 3) and contained single nuclei (data not shown). Temperature sensitivity of the sup45-sl23 ^ts mutant can not be explained by a defect of translation termination, since increase of the number of sup45-sl23 ^ts copies allowed mutant to grow at 37°C, but did not decrease the levels of nonsense codon readthrough either at permissive or non-permissive temperature (Fig. 2). It is also noteworthy that extra copies of the sup45-sl23 ^ts allele did not restore viability of the strain YJV-rS35C-SL23 carrying chromosomal sup45-sl23 ^ts and SUP35-C, indicating that temperature sensitivity and synthetic lethality were caused by the impairment of different cellular processes.

Mutations in SUP35-C abolishing its synthetic lethality with sup45-sl23^ts or sup45-36^ts

Mutations in SUP35-C which make it compatible with sup45-sl23 ^ts were obtained by passing the corresponding plasmid in the E. coli "mutator" strain. Five independent pools of the mutagenized plasmid were obtained and used to transform the strain YJV-rS35C-SL23 which carried the chromosomal sup45-sl23 ^ts and SUP35-C alleles and the centromeric SUP35 ADE3 pHT-SUP35 plasmid. Over 100,000 of transformed cells were incubated on leucine omission medium at 37°C and 116 Leu⁺ temperature resistant clones were selected. Streaking of these transformants on leucine omission medium showed that 75 of them produced colonies with Sect⁺ phenotype. The plasmid DNA from 8 Sect⁺ transformants was isolated and used for repeated transformation of the strain YJV-rS35C-SL23 carrying pHT-SUP35. All Leu⁺ transformants bearing the isolated plasmids were temperature resistant and able to lose the resident pHT-SUP35 (SUP35 ADE3 URA3) plasmid on medium supplemented with uracil, suggesting that they carried mutant SUP35-C. It is noteworthy that, since these mutations were dominant, they restored the normal growth rate and temperature resistance of the sup45-sl23 ^ts mutant YJV-rS35C-SL23 carrying the chromosomal SUP35-C allele. These SUP35-C mutant alleles supported viability of the sup45-sl23 ^ts mutant 33G-D373-rSL23-ΔS35 disrupted for SUP35 and allowed its growth at non-permissive temperature, indicating that suppression of temperature sensitivity did not depend on genetic background.

Sequencing of the mutant SUP35-C alleles revealed that they had nucleotide substitutions that caused either Glu-539 to Lys (five cases, isolated from four mutagenized plasmid pools), Ala-596 to Val (two cases, both derived from the same plasmid pool) or Ala-597 to Val (one case) replacements. It is noteworthy that these amino acid substitutions occurred in the positions located downstream to the eRF3 regions implicated in GTP hydrolysis [3, 8]. The corresponding alleles were respectively designated as SUP35-C14, SUP35-C11 and SUP35-C42.

The isolated compensatory SUP35-C mutations were studied for the ability to alleviate temperature sensitivity caused by the sup45-36 ^ts mutant allele as well as to suppress its synthetic lethality with SUP35-C. Introduction of either one of the plasmids with the SUP35-C14, SUP35-C11 or SUP35-C42 alleles into the sup45-36 ^ts mutant 33G-D373-r36-ΔS35, which was disrupted for SUP35 and carried the pRG415-SUP35 (URA3 SUP35) plasmid, allowed the loss of the resident plasmid on 5-FOA-containing medium at permissive temperature (Fig. 4), but did not cause resistance to 37°C. Thus, the compensatory mutations suppressed lethality caused by the combination of either sup45-sl23 ^ts or sup45-36 ^ts with SUP35-C, but did not suppress temperature sensitivity of sup45-36 ^ts .

Synthetic lethality between sup45-sl23^ts or sup45-36^ts and SUP35-C is not caused by the increased levels of nonsense codon readthrough

We also tested, whether the SUP35-C compensatory mutations influenced the nonsense codon readthrough caused by the sup45-sl23 ^ts and sup45-36 ^ts mutations. For this, the strain 33G-D373-rSL23 carrying the chromosomal sup45-sl23 ^ts mutation and wild type SUP35, was transformed with centromeric plasmids containing either the SUP35-C allele (control) or alleles with the SUP35-C14, SUP35-C11 or SUP35-C42 compensatory mutations along with either one of the pUKC815, 817, 818, 819 plasmids. Quantitative assay demonstrated that the nonsense codon readthrough was noticeably decreased only in the presence of SUP35-C11. This compensatory mutation also decreased the levels of nonsense readthrough in the sup45-36 ^ts mutant 33G-D373-r36 carrying wild type SUP35 but to a lesser extent than in the isogenic sup45-sl23 ^ts mutant 33G-D373-rSL23 (Fig. 5). Thus, though the effect of SUP35-C compensatory mutations on the levels of nonsense readthrough was not significant in most cases, they all abolished synthetic lethality, making unlikely that the increased levels of readthrough was the reason of lethal interaction between the sup45 ^ts mutations and SUP35-C allele.

To confirm this we have constructed the strains 33G-D373-rSL23-r35C and 33G-D373-r36-r35C, which carried, respectively, the sup45-sl23 ^ts and sup45-36 ^ts mutations and the SUP35-C allele, as well as the centromeric pCM183-SUP45 plasmid with the TRP1 selectable marker and SUP45 under the control of regulatable tetO₂ promoter. The synthetic lethality of sup45 ^ts and SUP35-C combination in these strains was manifested only upon the repression of wild type SUP45 by doxycycline. As a control we used the isogenic strains 33G-D373-rSL23 and 33G-D373-r36, which had the wild type SUP35 gene instead of the SUP35-C allele. To monitor the levels of nonsense codon readthrough upon the repression of wild type SUP45, all strains were transformed with either one of the pUKC815, 817, 818, 819 plasmids.

Since chromosomally-encoded mutant eRF1 and its wild type counterpart encoded by the plasmid were electrophoretically indistinguishable, we could not estimate a decline in the amount of eRF1 upon the repression of the plasmid SUP45 in the studied strains. However, in another strain with the same tetO₂-SUP45 plasmid, 17 h repression decreased the amount of eRF1 to approximately 5% of its initial levels. Importantly, this drop in wild type eRF1 amount was accompanied with enrichment of cultures with dead cells up to 20% for the 33G-D373-rSL23-r35C and 33G-D373-r36-r35C strains, while the number of such cells in the 33G-D373-rSL23 and 33G-D373-r36 cultures was less than 10%. The in vivo quantification of UAA, UAG, and UGA nonsense readthrough has shown that its levels in the strains with synthetic lethal mutations was not higher than in the control strains (Fig. 6). This disagrees with the assumption that the cause of synthetic lethality between sup45 mutations and SUP35-C allele is too high levels of nonsense codon readthrough. Incubation of the strain 33G-D373-rSL23-r35C carrying the tetO₂-SUP45 plasmid in medium containing doxycycline did not cause any change in the polyribosome content or distribution (data not shown), indicating absence of the sharp effect on translation.

Media and strains

Yeast strains were grown on standard organic (YPD) and synthetic (SD) media [34]. The modified YPD media (YPDm) [2% peptone, 0.5% yeast extract (Oxoid), 3% glucose and 2% agar] was used in colony color sectoring assay experiments, because it favored accumulation of red pigment in the ade2 mutants. The 5-fluoroorotic acid (5FOA) medium was prepared as described [35]. The expression of the tetO_2-controlled SUP45 was repressed by incubation of corresponding strains on medium which contained 10 μg/ml doxycycline and was selective for the pCM183-SUP45 plasmid. LB and 2 × YT media were used for bacteria [36]. Appropriate amounts of antibiotics, amino acids, and bases were added when necessary. Yeast cells were grown at 30°C, if not indicated otherwise, and bacteria at 37°C. DNA transformation of lithium acetate-treated yeast cells was performed as described previously [37]. E. coli cells were transformed by the method described in [38].

The E. coli mutator strain XL1-Red [endA 1 gyrA 96 thi- 1 hsdR 17 supE 44 relA 1 lac mutD 5 mutS mutT Tn10 (Tet^r)] (Stratagene) was used for obtaining mutations in the plasmid SUP35-C gene. For this, the plasmid pRS315-SUP35C was introduced into this E. coli strain. After incubation for 48 h, over 500 transformant colonies were suspended in 10 ml of 2 × YT supplemented with ampicillin (100 μg ml^-1). Cell culture was additionally incubated for 8 h with shaking, plasmid DNA was isolated and used to transform yeast. The E. coli strain DH5α [supE 44 Dlac U169 (f80 lacZ DM15) hsdR 17 recA 1 endA 1 gyrA 96 thi-1 relA 1] [36] was used in cloning experiments.

Plasmids

The SUP45 gene was cloned using a yeast genomic library based on the plasmid p366, which was identical to YCp50 [41], but had LEU2 instead of URA3 (gift of P. Hieter). Isolation and sequencing of the sup45-sl23 ^ts allele was described earlier [25]. The plasmid YEplac181-sup45-sl23 was obtained by insertion of the Xba I-Xho I DNA fragment of pRS315-sup45-sl23 containing the mutant sup45-sl23 ^ts allele into the Xba I and Sal I sites of YEplac181. The Xho I-Sac I DNA fragments carrying the sup45 mutant alleles of the plasmids pRS315-sup45-36 and pRS315-sup45-sl23 were cloned between the Sal I and Sac I sites of the integrative URA3 vector pJJ244 [40] to obtain the plasmids pJJ244-sup45-36 and pJJ244-sup45-sl23, respectively. The pCM183-SUP45 plasmid bearing the SUP45 gene under the control of regulatable tetO₂ promoter was constructed as follows. Using the plasmid pRS315-SUP45 as template, PCR was applied to place the Nco I site (CCATGG) at the SUP45 translation initiation codon. The obtained PCR fragment was digested with Nco I and Xho I and cloned into the same sites of the E. coli plasmid pET-30a(+) (Novagen) to generate the plasmid pL51-3. The Kpn I-Xho I fragment of pL51-3, containing the coding sequence of SUP45, was inserted into the Kpn I and Sal I sites of YEplac112 resulting in the YEplac112-atgSUP45 plasmid. Then, the Eco ICRI-Pst I fragment of YEplac112-atgSUP45 was cloned into the Hpa I and Pst I sites of pCM183 [42].

β-galactosidase activity in cell lysates was quantified as described [43]. The levels of UAA, UAG and UGA readthrough were determined using the pUKC815/817/818/819 series vectors, as described previously [44].

Colony color sectoring assay

The assay is based on the facts that ade2 yeast cells accumulate a red pigment and form red colonies but ade2 ade3 cells do not accumulate this pigment and, consequently, form white colonies because the ade3 mutation blocks the pathway at a point prior to the accumulation of the pigment [45]. Thus, a strain that is ade2 ade3, but carries ADE3 on an autonomously replicating plasmid, forms on medium non-selective for this plasmid red colonies (cells with the plasmid) that contain white sectors (cells that have lost the plasmid). We refer this phenotype as Sect⁺ (ability to lose the ADE3 plasmid), and, consequently, the phenotype of red clones without white sectors or with a negligible number of such sectors is Sect^- (inability to lose the ADE3 plasmid). Multiple suspensions of the SUP35-C ade2 ade3 strain YJV159-rS35C carrying the centromeric plasmid pHT-SUP35 (SUP35 ADE3) were prepared in liquid uracil omission medium; each suspension deriving from a separate colony grown on uracil omission medium was diluted, and plated on ten YPDm plates at approximately 5,000 cells per plate. These cells were mutagenized with UV irradiation to approximately 10% survival and then incubated for 4 or 5 days. As expected, most clones were Sect⁺. Cells from Sect^- colonies were streaked on YPDm and after 2 days, individual colonies were restreaked on YPDm. Only strains that continued to give red colonies or red colonies with rare white sectors were studied further.