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1 Department of Genetics, St. Petersburg State University, 199034, St. Petersburg, Russia
2 Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain
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Abstract |
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deletion cannot be cured from [ISP+] in the presence of GuHCl. Since Hal3p is a negative regulatory subunit of Ppz1 protein phosphatase, consequences of PPZ1 over-expression and deletion are opposite to those of HAL3. The observed effects are mediated by the catalytic function of Ppz1 and are probably related to the participation of Ppz1 in regulation of eEF1B
elongation factor activity. Importantly, [ISP+] status of yeast strains is determined by fluctuation in Hal3p level, since [ISP+] strains have less Hal3p than their [isp–] derivatives obtained by GuHCl treatment. A model considering epigenetic (possibly prion) regulation of Hal3p amount as a mechanism underlying [ISP+] status of yeast cell is suggested. |
Introduction |
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Read-through efficiency is determined not only by the status of termination machinery. Nonsense suppression was shown, in particular, for mutations affecting elongation factor eEF1A responsible for aminoacyl tRNA delivery and binding to ribosome A-site (Carr-Schmid et al. 1999a). In contrast, an increase of translation fidelity manifesting as anti-suppression was demonstrated for mutations affecting eEF1B, which is the nucleotide exchange factor regenerating eEF-1 A by GDP to GTP replacement (Carr-Schmid et al. 1999b).
We reported some time ago that the suppressor phenotype of strains bearing certain sup35 mutations is unstable. The non-suppressor phenotype acquired by these strains after several generations displayed properties of non-Mendelian inheritance. Moreover, treatment of these strains with 5 mM GuHCl, which is considered a universal agent curing yeast cells of prions, caused restoration of suppression. We have proposed, therefore, that loss of suppressor phenotype is related to the appearance of a prion-like anti-suppressor determinant called [ISP+] (Volkov et al. 2002).
To clarify the nature of [ISP+] phenotype, it is necessary to identify genes influencing its appearance, propagation and manifestation. Among other approaches, that will be described elsewhere, we carried out a screen using a YCp50-based gene library. This screen was aimed at (i) characterization of the genetic background of [ISP+] strains and detection of possible cryptic chromosomal mutations contributing to manifestation of [ISP+] phenotype, based on their complementation and consequent restoration of suppression in transformants; (ii) search of genes that influence stability or manifestation of [ISP+] phenotype through a modest increase of the amount of their products. One of conclusions derived from the screening performed was that [ISP+] strains contain not only sup35 suppressor mutations, but also specific sup45 mutations (Aksenova et al. 2006). These mutations do not have their own phenotypic expression, but are necessary for manifestation of anti-suppression, since in the presence of compensating plasmids we observe restoration of suppression despite of [ISP+] presence. Thus, the phenotype of [ISP+] strains is determined by a more complex mechanism than it was proposed initially (Volkov et al. 2002) and requires one more factor of genetic nature in addition to epigenetic determinant.
In this work, we present the results of the mentioned screen, which demonstrate a role played by HAL3 (SIS2) gene in [ISP+] propagation and manifestation. Hal3p (Sis2) protein is a negative regulatory subunit of the Ppz phosphatases (de Nadal et al. 1998) represented in yeast by two structurally and functionally related members, Ppz1 and Ppz2 (Posas et al. 1992, 1993; Lee et al. 1993). A link between Ppz phosphatases and regulation of translation accuracy was revealed several years ago (de Nadal et al. 2001). Our data emphasize the contribution of the Ppz phosphatases to translational fidelity control. We show also an involvement of the Ppz1 phosphatase and its regulatory subunit Hal3p to the [ISP+] manifestation and suggest that the amount of Hal3p determines the [ISP+] status of yeast cell.
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Results |
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HAL3 expression from low copy plasmid induces high frequency appearance of Lys+ secondary clones in [ISP+] strain
As we have shown earlier, the [ISP+] phenotype is mitotically stable; the rate of its spontaneous change to [isp–] phenotype is about 1.2–3.5 x 10–7 per cell per generation (Volkov et al. 2002). Among 80 000 transformants of [ISP+] strain 4 V-P4482 with YCp50-based gene library we isolated several plasmids masking anti-suppressor effect of [ISP+] (Aksenova et al. 2006) and one plasmid, designated as YCp50-165, which significantly increased the rate of secondary Lys+ clones (Fig. 1A). This plasmid contained the region of the right arm of chromosome XI flanked by YKR070W and YKR078W ORFs, as was shown by sequencing of insertion ends (Fig. 1B).
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Over-expression of HAL3 causes allosuppression in [ISP+] strain, while PPZ1 over-expression causes anti-suppression in [isp–] strain
In the next step, the effect of HAL3 over-expression in [ISP+] strain was examined. To this aim, the multicopy plasmid YEplac195-HAL3 was used for transformation of [ISP+] strain 4 V-P4482. All 32 transformants examined displayed suppressor phenotype, that is, complete loss of [ISP+] manifestation instead of Lys+ papillation phenotype observed when low copy plasmids were used (Fig. 2A, top line).
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Nonsense suppression displayed by transformants over-expressing HAL3 may result from specific suppressor effect of HAL3 over-expression, independent of sup35-25 effect, or from increase of sup35-25 suppressor efficiency (i.e., allosuppression) caused by Hal3p overproduction. To distinguish between these possibilities we transformed the strain 2 V-P3982 containing wild-type SUP35 with YEplac195-HAL3. Notably, transformants obtained did not differ phenotypically from the initial strain (not shown). This fact indicates that masking of [ISP+] phenotype is governed by allosuppressor effect of over-expressed HAL3 toward sup35-25.
As was stated in the Introduction, Hal3p is a negative regulatory subunit of Ppz1 protein phosphatase (de Nadal et al. 1998). It can be suggested that enhancement of nonsense suppression efficiency in cells overproducing Hal3 is related to inhibition of Ppz1 activity. Consequently, the effect of PPZ1 over-expression should be opposite to that of HAL3 over-expression. To examine this idea, we transformed [isp–] and [ISP+] strains with the multicopy plasmid YEplac195-PPZ1 and observed that transformants of [isp–] strain manifested a strong anti-suppressor phenotype; that is, manifestation of sup35-25 in these transformants was completely neutralized (Fig. 2A, top line). The anti-suppressor effect of PPZ1 over-expression was visible even in transformants of [ISP+] strain, as growth of these transformants was inhibited yet more profoundly than growth of the control [ISP+] strain; in particular, PPZ1 over-expression neutralized suppression of ade1-14 (Fig. 2A, medium line). Thus, anti-suppression caused by over-expressed PPZ1 is stronger than the anti-suppressor effect of [ISP+], which has no evident effect on ade1-14 manifestation.
Remarkably, over-expression of Ppz1-R451L, a catalytically inactive version of Ppz1 (Clotet et al. 1996) did not demonstrate an anti-suppressor effect (Fig. 2B). Thus, anti-suppression caused by PPZ1 over-expression is related to the catalytic function of this phosphatase.
Mutations of Hal3 that affect its interaction with Ppz1 do not influence [ISP+] manifestation
Additional confirmation of the idea that effects of HAL3 high-copy number expression on termination efficiency are mediated by the regulatory role of Hal3p on Ppz1 phosphatase were obtained expressing specific hal3 mutated versions. As it was described earlier (Munoz et al. 2004), mutations of some residues near the C-terminal region of Hal3p affect moderately (Y313, N466I and I480) or strongly (V390, I446 and W452) its binding to Ppz1. Change of other residues retained normal binding but abolished moderately (V462 and N478) or in full (E460) the ability of Hal3p to inhibit Ppz1 activity.
When the [ISP+] strain 4 V-P4482 was transformed with a series of YEplac181-based plasmids carrying mutant hal3 alleles listed above, we observed that I446K, W452G, E460G, V462A, N466I and N478D replacements failed to cause suppression, while transformants containing Y313D, V390G and I480F alleles did not differ phenotypically from transformants expressing wild-type HAL3 and showing the suppressor phenotype (Fig. 3A). This result was confirmed when hal3 strain (dh3-a16-4 V-P4482) was used as a recipient for transformation with the same plasmids (not shown).
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As previously described (Munoz et al. 2004), the H378 residue in Hal3 is not involved in its regulatory function over Ppz1, although it is relevant for additional, Ppz1 unrelated functions of the Hal3 protein. As expected, the H378A mutation still allows suppression in transformants of [ISP+] strain. However, H378A is the only allele that manifests when expressed in [isp–] derivative of 4 V-P4482 (Fig. 3B).
Different phenotypes of [ISP+] and [isp–] strains are related to different cellular amounts of Hal3p
Since HAL3 over-expression caused suppression in the [ISP+] strain, it may be proposed that HAL3 inactivation should cause anti-suppression in the [isp–] strain. To check this, it was necessary to avoid spontaneous appearance of [ISP+] clones (which also have anti-suppressor phenotype) in the mitotic progeny of hal3 derivatives of [isp–] strain. Thus the hal3 allele was obtained in [ISP+] strain a16-4 V-P4482 [ISP+] (see Experimental procedures). Strains containing hal3 not only retained the anti-suppressor phenotype of the initial strain, but also did not generate Lys+ clones, which is typical for [ISP+] strains (Fig. 4A). Transformation of the hal3 strains with the YCp111-HAL3 plasmid restores all the features of initial HAL3 [ISP+] strain. Therefore, one of hal3 derivatives transformed with compensating plasmid YCp111-HAL3 had been chosen for further analysis. Eight independent transformants of this derivative with YCp111-HAL3 plasmid (carrying the LEU2 marker) were treated with GuHCl. For each transformant, one Lys+ clone appeared after GuHCl treatment was selected and grown on YPD to allow loss of the compensating plasmid YCp111-HAL3. After that, several Leu– clones (from 9 to 19) were selected in the progeny of each clone. Notably, all Leu– clones examined changed their phenotype from Lys+ to Lys– (Fig. 4A). We can conclude that HAL3 deletion causes anti-suppression of sup35-25 mutation. Notably the anti-suppressor phenotype of hal3 derivatives is not curable with GuHCl (Fig. 4B). Retaining of [ISP+] by these derivatives after GuHCl treatment was confirmed by their cross with [isp–] strain (Fig. 4C). This fact, together with observation that Hal3p overproduction in [ISP+] strain causes suppression (i.e., mimics the [isp–] phenotype), suggests that [ISP+] status of the strain depends on Hal3p presence and that [ISP+] and [isp–] phenotypes may correspond to different amounts of Hal3p.
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Deletion of PPZ1 in [ISP+] strain displays a weak suppressor effect
If Hal3p effects are mediated by Ppz1 phosphatase, then consequences of inhibition of this phosphatase should be also opposite to the consequences of HAL3 inactivation and similar to effects of HAL3 over-expression; that is [ISP+] strains containing inactivated PPZ1 should change their phenotype from anti-suppressor to suppressor one. In fact we observed that [ISP+] strain 4 V-P4482 ppz1 displayed weaker suppression of lys2-87 than [isp–] derivative of 4 V-P4482 (Fig. 5A). This may indicate that Ppz1 is not the only target regulated by Hal3p. This possibility was confirmed in another experiment where HAL3 was over-expressed in a [ISP+]
ppz1 strain (dz1-4 V-P4482). In spite of the absence of Ppz1, this strain displayed suppression, albeit weak, but expressed more profoundly than in the same strain transformed with the control (empty) vector (not shown).
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ppz2 deletion in [ISP+] strain. But contrary to data obtained previously and indicating normal growth of strains containing double ppz1
ppz2 deletion at permissive temperature (Posas et al. 1995; de Nadal et al. 2001; Yenush et al. 2002), strains used in this work grew very slowly even at 26 °C on YPD (Fig. 5B). This fact impeded the correct comparison of suppression efficiency in ppz1
ppz2 strains to the control strains. TEF5 expression from high copy number plasmid has anti-suppressor effect in [isp–] strain
As was stated above (see Introduction), a known target of Ppz1 phosphatase is the translation elongation factor eEF1B encoded by the TEF5 gene. We supposed that the effects observed in strains with variable levels of HAL3 and PPZ1 expression are due to fluctuations in the level of different (phosphorylated vs. dephosphorylated) forms of eEF1B. To check this proposal, we over-expressed a tef5 allele, causing S86A replacement in eEF1B, in [ISP+] and [isp–] derivatives of 4 V-P4482. This replacement generates an eEF1B form that cannot be phosphorylated (de Nadal et al. 2001). Consequences of overproduction of this form manifested as anti-suppression in [isp–] strain; a similar effect was demonstrated for the over-expressed wild-type TEF5 allele (Fig. 6).
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Discussion |
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Search of genes involved in the control of [ISP+] maintenance and manifestation revealed, in particular, the HAL3 (SIS2) gene which being expressed from low copy plasmids increases the rate of clones with suppressor phenotype in the mitotic progeny of transformants. However, HAL3 expression from multicopy plasmid changes the phenotype of [ISP+] strain to [isp–] one not due to [ISP+] elimination but due to allosuppressor effect of HAL3 over-expression toward sup35-25 mutation. Thus, HAL3 displays a dosage dependent manifestation in [ISP+] strain. This dependence could be explained assuming that HAL3 extra-copy has slight effect on translation termination that allows growing subpopulation of cells, which carry translational modifiers not manifesting when only one copy of HAL3 presents in the cell. At the same time high-copy number expression of HAL3 has strong allosuppressor effect itself. Therefore, increasing HAL3 gene dosage does not affect [ISP+] propagation but abolishes [ISP+] manifestation elevating stop codon read-through level.
Contrary to over-expression effects, HAL3 deletion has drastic consequences not only for nonsense-suppression but also for [ISP+] propagation. Thus, it prevents [ISP+] curing with GuHCl. As to independent phenotypic manifestation of HAL3 deletion, it causes strong anti-suppression in [isp–] strain, that is, complete inhibition of sup35-25 manifestation, and enhances anti-suppression in [ISP+] strain.
Dependence of nonsense suppression efficiency from the level of HAL3 expression can be understood if it is thought that Hal3p represents a negative regulatory subunit of Ppz protein phosphatases (de Nadal et al. 1998). Yeast Z-type Ser/Thr protein phosphatases are represented in yeast by two proteins, Ppz1 and Ppz2, which are involved in a variety of cell processes, including regulation of salt tolerance, maintenance of cell wall integrity and regulation of cell cycle at the G1/S transition (for review, Arino 2002). As mentioned in the Introduction Ppz phosphatases contribute also to regulation of translation accuracy, since Ppz-deficient strains display from three to fourfold elevated level of nonsense codons read-through (de Nadal et al. 2001).
Since Hal3p negatively regulates Ppz phosphatases, its over-expression in [ISP+] strain should cause an increased efficiency of stop codons read-through, or suppression. Contrary to this, Hal3p inactivation should decrease the level of stop codons read-through and cause anti-suppression in [isp–] strain. Both effects were observed in our experiments. The opposite consequences have been shown for the high-copy number expression and deletion of the PPZ1 gene.
Additional facts supporting the proposal that Hal3 effects in our strains are mediated by its interaction with Ppz phosphatases were obtained using hal3 mutations decreasing Hal3p ability to interact with Ppz1 phosphatase or inhibit its activity. The majority of these alleles, contrary to the wild-type HAL3, do not show suppression when used for transformation of [ISP+] strain in multicopy state. The only apparent inconsistency would be V390G, which precludes Hal3p binding to Ppz1p, but results in suppression. However, it should be noted that this allele in a multicopy state also manifests as wild-type for other Ppz-dependent phenotypes such as salt tolerance (Munoz et al. 2004).
Finally, we have shown that the anti-suppressor effect of PPZ1 over-expression is related to the catalytic function of Ppz1 phosphatase since this effect was abolished when the catalytically inactive Ppz1 (R451L) mutant was used.
A known target of Ppz1 phosphatase is translation elongation factor eEF1B (de Nadal et al. 2001). eEF1B, encoded by the essential TEF5 gene, accelerates the rate of GDP hydrolysis on eEF1A, maintaining a pool of active eEF1A-GTP (Hiraga et al. 1993; Kinzy et al. 1994). It was found that mutation in the TEF5 gene reducing total translation speed results in anti-suppression (Carr-Schmid et al. 1999b). It was proposed that Ppz1 phosphatase might regulate the efficiency of stop codons read-through by eEF1B dephosphorylation (de Nadal et al. 2001).
Our data also may be interpreted as indicating the functional importance of eEF1B phosphorylation for translation fidelity. In our experiment, high-copy number expression of mutant TEF5, encoding the eEF1B version, in which phosphorylatable Ser-86 is changed to Ala (tef5S86A), manifests the anti-suppressor effect toward sup35-25 mutation, which can be explained by a decrease of active eEF1A-GTP amount.
At the same time the exact interpretation of these data is complicated by contradictions regarding the role played by eEF1B phosphorylation status in regulation of translation fidelity. A popular model connects decreased translation speed with enhancement of its fidelity (Dong & Kurland 1995). However, other data show that there is no correlation between general slowing of translation rate and nonsense suppression (Carr-Schmid et al. 1999a; Andersen et al. 2000). It was suggested that eEF1B could participate in the control of translational fidelity not only through regulation of GDP to GTP exchange on eEF1A (Valente & Kinzy 2003). Probably, the similarity of effects observed for over-expressed tef5S86A allele and the TEF5 wild-type allele could be explained by the existence of unknown regulatory effects of eEF1B toward eEF1A. The other hypothetical possibility is that eEF1B provides functioning not only eEF1A but also eRF3. eRF3 is a GTPase whose C domain is structurally similar to eEF1A and contains potential GTP-binding sites (see Inge-Vechtomov et al. 2003 for review). However, no recycling factor is known for GDP to GTP replacement on eRF3. If eEF1B were this factor, it might influence not only elongation but also termination efficiency.
Although Hal3p effects observed in our work are mediated by Ppz1 phosphatase, some facts indicate that not all of them depend on Hal3p–Ppz1p interaction only. We have shown that PPZ1 deletion does not cause a strong suppressor effect, similar to the effect of HAL3 over-expression. We cannot attribute this effect to substitution of Ppz1-functions by Ppz2 phosphatase, as ppz2 mutants obtained in [ISP+] strain did not demonstrate a nonsense-suppressor phenotype. These facts may indicate the existence of non-Ppz-related targets of Hal3p participating in regulation of stop-codons read-through.
Anyway, in the light of the results obtained, we can conclude that there is a pathway of translation fidelity regulation by HAL3 gene based on Hal3p–Ppz1p interaction (Fig. 7). We propose that activity of Ppz1 phosphatase influences the relative amount of eEF1B Ser-86 phosphorylated vs. unphosphorylated form and that excess of dephosphorylated form of eEF1B enhances the translation fidelity, manifesting in our system as anti-suppression of sup35 mutation.
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strains also indicates that [isp–] phenotype may be related to Hal3p presence. Thus, some mechanism affecting the Hal3p amount may underlie the [ISP+] status of yeast strains. This mechanism may be provided, for example, by a protein regulating the Hal3p amount. If [ISP+] phenotype is settled on by prion-like determinant, as was proposed in consequence of [ISP+] properties (Volkov et al. 2002), this protein may be prionogenic and its inactivation due to prionization might decrease the Hal3p amount, providing the anti-suppressor phenotype of [ISP+] strains. Here, it should be referred to that effects observed are unlikely caused by prionization of Hal3p itself. Hal3p does not have structural properties typical for known yeast prion proteins and is not included in the lists of potential prions (Michelitsch & Weissman 2000; Harrison & Gerstein 2003), albeit this fact does not rule out the possibility of its prionization. In addition, we have found that [ISP+] and [isp–] strains differ only by total amount of Hal3p, but not by redistribution of the protein between fractions. It should be mentioned also that HAL3 over-expression does not induce the [ISP+] appearance, but causes the opposite phenotype. On the other hand, it can be proposed that HAL3 expression is influenced in some way by GuHCl treatment. It should be emphasized once more that we obtain [isp–] strains by GuHCl treatment of [ISP+] strains and, as we have demonstrated earlier, [isp–] strains are less sensitive to GuHCl than [ISP+] strains (Volkov et al. 2002). Thus the elevated level of Hal3p in [isp–] strains correlates with their GuHCl-resistance. However, regardless of the fact that HAL3 is one of the main determinants providing salt tolerance in yeast and that Hal3p overproduction confers halotolerance, it was shown that high salt conditions do not influence HAL3 expression (Ferrando et al. 1995). So, if the elevated level of Hal3p in [isp–] strains is caused by GuHCl treatment, this effect should be specific just for this salt.
In conclusion, it is necessary to stress that albeit direct arguments in favor of regulation of Hal3p amount by a prion mechanism are still absent, we can assert that efficiency of stop-codons read-through in yeast depends on the Hal3p-Ppz1p activity and the level of this activity is regulated epigenetically.
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Experimental procedures |
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Media and cultivation procedures
All media were prepared as described (Kaiser et al. 1994) and included supplemented minimal medium (SMM), SMM lacking one or more specific supplements (e.g., SMM-lysine), YPD as a rich medium and 5-FOA medium. The growth temperature for yeast cultures was 28 °C. Yeast transformation was performed according to Gietz protocol (<http://www.umanitoba.ca/faculties/medicine/biochem/gietz/method.html>). Selection of strains transformed with PCR cassettes from pFA6-KanMX4 and pAG32 plasmids was performed on YPD medium supplemented with G418 (GibcoBRL) or Hygromycin B (Sigma), respectively, at concentrations of 200 mg/L. Fluctuation assay for estimation of the rate of suppressor clones in the mitotic progeny of [ISP+] strain was performed as described earlier (Volkov et al. 2002).
Plasmids and libraries
YCp50 based yeast genome library described in (Rose et al. 1987) was obtained from ATCC (<http://www.atcc.org>). Plasmids used in this study are listed in Table 2. To generate YCp33-PPZ1, a 2.8 kbp fragment was excised from PYC1Z1 plasmid (Clotet et al. 1996) using BamHI and HindIII restriction endonucleases and cloned into the same sites of the YCplac33 (low copy) or YEplac195 (high copy) plasmids. This fragment contains the entire PPZ1 open reading frame downstream of a 456-bp region that displays full promoter activity. For further details see Clotet et al. (1996). The YEplac195-Ppz1(R451L) construct contains a version of the Ppz1 phosphatase that has been shown to be catalytically inactive (Clotet et al. 1996).
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Plasmids p426/TEF5 and p426/TEF5(S86A) have been previously described (de Nadal et al. 2001). Briefly, they contain the wild-type allele or a Ser-86 to Ala allele of the TEF5 gene cloned in the plasmid p426TEG2. This vector is based on the high copy yeast plasmid pRS426 (Christianson et al. 1992) and allows expression of GST fusion proteins under the control of the TEF1 promoter.
Sequencing of the insert's flanks in the YCp50-165 plasmid and of the HAL3 gene
Insertion of genomic DNA in the YCp50-165 plasmid was identified by sequencing using PSB and ycp50r2 primers (see Table S1). The HAL3 gene isolated from YCp50-165 plasmid was sequenced using primers SIS2sense, SIS2anti, SISSEQ2, SISSEQ3, sis2_343f50, sis2_624f50, sis2_1221f50, sis2_1550f50, sis2_407r50, sis2_732r50, sis2_1028r50, sis2_1337r50, sis2_1647r50, sis2_1932r50 (see Table S1).
Protein extraction and Western blot analysis
Strains were grown in SMM medium until they reached an approximate A600 of 1.5 and were recovered by centrifugation. Cells were disrupted by vortexing in the presence of glass beads (Sigma) in the extraction buffer containing 50 mM Tris–HCl (pH 7.6); 0.2 mM EGTA; 150 mM NaCl; 0.1% Triton X-100; 10% Glycerol plus 2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol and a protease inhibitor mixture. Lysates were clarified by centrifugation at 750 g during 10 min at 4 °C. Protein concentration was determined for every sample using a colorimetric (Bradford) assay. Forty micrograms of total protein were analyzed in each case by SDS-PAGE, and Hal3p was inmunodetected using anti-Hal3 polyclonal antibodies (kindly supplied by R. Serrano). Anti-actin antibodies (Sigma) were used to monitor the total amount of protein loaded in each lane.
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Acknowledgements |
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Footnotes |
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Present address:
aDepartment of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden;
bMedical Research Council Protein Phosphorylation Unit, Welcome Trust Biocentre/Medical Sciences Institute Complex, Dow Street, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
* Correspondence: E-mail: lmiron{at}mail.ru |
References |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Andersen, G.R., Pedersen, L., Valente, L., Chatterjee, I., Kinzy, T.G., Kjeldgaard, M. & Nyborg, J. (2000) Structural basis for nucleotide exchange and competition with tRNA in the yeast elongation factor complex eEF1A: eEF1B. Mol. Cell. 6, 1261–1266.[CrossRef][Medline]
Arino, J. (2002) Novel protein phosphatases in yeast. Eur. J. Biochem. 269, 1072–1077.[Medline]
Carr-Schmid, A., Durko, N., Cavallius, J., Merrick, W.C. & Kinzy, T.G. (1999a) Mutations in a GTP-binding motif of eukaryotic elongation factor 1A reduce both translational fidelity and the requirement for nucleotide exchange. J. Biol. Chem. 274, 30297–30302.
Carr-Schmid, A., Valente, L., Loik, V.I., Williams, T., Starita, L.M. & Kinzy, T.G. (1999b) Mutations in elongation factor 1ß, a guanine nucleotide exchange factor, enhance translational fidelity. Mol. Cell. Biol. 19, 5257–5266.
Christianson, T.W., Sikorski, R.S., Dante, M., Shero, J.H. & Hieter, P. (1992) Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119–122.[CrossRef][Medline]
Clotet, J., Gari, E., Aldea, M. & Arino, J. (1999) The yeast ser/thr phosphatases Sit4 and Ppzl play opposite roles in regulation of the cell cycle. Mol. Cell. Biol. 19, 2408–2415.
Clotet, J., Posas, F., de Nadal, E. & Arino, J. (1996) The NH2-terminal extension of protein phosphatase PPZ1 has an essential functional role. J. Biol. Chem. 271, 26349–26355.
Dong, H. & Kurland, C.G. (1995) Ribosome mutants with altered accuracy translate with reduced processivity. J. Mol. Biol. 248, 551–561.[CrossRef][Medline]
Ferrando, A., Kron, S.J., Rios, G., Fink, G.R. & Serrano, R. (1995) Regulation of cation transport in Saccharomyces cerevisiae by the salt tolerance gene HAL3. Mol. Cell. Biol. 15, 5470–5481.
Gietz, R.D. & Sugino, A. (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527–534.[CrossRef][Medline]
Goldstein, A.L. & McCusker, J.H. (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553.[CrossRef][Medline]
Harrison, P.M. & Gerstein, M. (2003) A method to assess compositional bias in biological sequences and its application to prion-like glutamine/asparagine-rich domains in eukaryotic proteomes. Genome Biol. 4, R40.[CrossRef][Medline]
Hiraga, K., Suzuki, K., Tsuchiya, E. & Miyakawa, T. (1993) Cloning and characterization of the elongation factor EF-1ß homologue of Saccharomyces cerevisiae. EF-1ß is essential for growth. FEBS Lett. 316, 165–169.[CrossRef][Medline]
Inge-Vechtomov, S., Zhouravleva, G. & Philippe, M. (2003) Eukaryotic release factors (eRFs) history. Biol. Cell 95, 195–209.[CrossRef][Medline]
Kaiser, C., Michaelis, S. & Mitchell, A. (1994) Methods in Yeast Genetics. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Kinzy, T.G., Ripmaster, T.L. & Woolford, J.L. Jr (1994) Multiple genes encode the translation elongation factor EF-1
in Saccharomyces cerevisiae. Nucleic Acids Res. 22, 2703–2707.
Kisselev, L., Ehrenberg, M. & Frolova, L. (2003) Termination of translation: interplay of mRNA, rRNAs and release factors? EMBO J. 22, 175–182.[CrossRef][Medline]
Lee, K.S., Hines, L.K. & Levin, D.E. (1993) A pair of functionally redundant yeast genes (PPZ1 and PPZ2) encoding type 1-related protein phosphatases function within the PKC1-mediated pathway. Mol. Cell. Biol. 13, 5843–5853.
Liebman, S.W. & Derkatch, I.L. (1999) The yeast [PSI+] prion: making sense of nonsense. J. Biol. Chem. 274, 1181–1184.
Michelitsch, M.D. & Weissman, J.S. (2000) A census of glutamine/asparagine-rich regions: implications for their conserved function and the prediction of novel prions. Proc. Natl. Acad. Sci. USA 97, 11910–11915.
Munoz, I., Ruiz, A., Marquina, M., Barcelo, A., Albert, A. & Arino, J. (2004) Functional characterization of the yeast Ppz1 phosphatase inhibitory subunit Hal3: a mutagenesis study. J. Biol. Chem. 279, 42619–42627.
de Nadal, E., Clotet, J., Posas, F., Serrano, R., Gomez, N. & Arino, J. (1998) The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase. Proc. Natl. Acad. Sci. USA 95, 7357–7362.
de Nadal, E., Fadden, R.P., Ruiz, A., Haystead, T. & Arino, J. (2001) A role for the PpZ Ser/Thr protein phosphatases in the regulation of translation elongation factor 1B. J. Biol. Chem. 276, 14829–14834.
Posas, F., Camps, M. & Arino, J. (1995) The PPZ protein phosphatases are important determinants of salt tolerance in yeast cells. J. Biol. Chem. 270, 13036–13041.
Posas, F., Casamayor, A. & Arino, J. (1993) The PPZ protein phosphatases are involved in the maintenance of osmotic stability of yeast cells. FEBS Lett. 318, 282–286.[CrossRef][Medline]
Posas, F., Casamayor, A., Morral, N. & Arino, J. (1992) Molecular cloning and analysis of a yeast protein phosphatase with an unusual amino-terminal region. J. Biol. Chem. 267, 11734–11740.
Rose, M.D., Novick, P., Thomas, J.H., Botstein, D. & Fink, G.R. (1987) A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60, 237–243.[CrossRef][Medline]
Serio, T.R. & Lindquist, S.L. (2001) The yeast prion [PSI+]: molecular insights and functional consequences. Adv. Protein Chem. 59, 391–412.[Medline]
Storici, F., Lewis, L.K. & Resnick, M.A. (2001) In vivo site-directed mutagenesis using oligonucleotides. Nat. Biotechnol. 19, 773–776.[CrossRef][Medline]
Valente, L. & Kinzy, T.G. (2003) Yeast as a sensor of factors affecting the accuracy of protein synthesis. Cell. Mol. Life Sci. 60, 2115–2130.[CrossRef][Medline]
Volkov, K.V., Aksenova, A.Y., Soom, M.J., Osipov, K.V., Svitin, A.V., Kurischko, C., Shkundina, I.S., Ter-Avanesyan, M.D., Inge-Vechtomov, S.G. & Mironova, L.N. (2002) Novel non-Mendelian determinant involved in the control of translation accuracy in Saccharomyces cerevisiae. Genetics 160, 25–36.
Wach, A., Brachat, A., Pohlmann, R. & Philippsen, P. (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808.[CrossRef][Medline]
Yenush, L., Mulet, J.M., Arino, J. & Serrano, R. (2002) The PpZ protein phosphatases are key regulators of K+ and pH homeostasis: implications for salt tolerance, cell wall integrity and cell cycle progression. EMBO J. 21, 920–929.[CrossRef][Medline]
Received: 10 September 2006
Accepted: 17 December 2006
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