ISSN 0026�8933, Molecular Biology, 2010, Vol. 44, No. 6, pp. 907–914. © Pleiades Publishing, Inc., 2010.Original Russian Text © M.S. Ivanov, E.A. Radchenko, L.N. Mironova, 2010, published in Molekulyarnaya Biologiya, 2010, Vol. 44, No. 6, pp. 1018–1026.

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Decoding of mRNA triplets by protein synthesismachinery according to the genetic code is an essen�tial property of the living cell, which ensures its stablefunctioning. Similarly to other template�directedprocesses, translation is characterized by a certainlevel of mistakes that result in misinterpretation ofsense codons and recognition of stop�codons as senseones. A convenient model for studying the geneticcontrol of translation accuracy is the suppression ofnonsense mutations that result in the prematureappearance of stop�codons in the open readingframe. Suppression of nonsense mutations might bedetected at phenotypic level, since the reading ofstop�codons restores the synthesis of a full�lengthprotein, aborted by nonsense mutation.

The increase or reduction of nonsense suppres�sion efficiency in Saccharomyces yeasts can becaused by genetic and epigenetic factors. Mostmutations that affect the efficiency of nonsensesuppression are localized in genes encoding tRNAs,rRNAs, ribosomal proteins, elongation, and termi�nation factors of translation [1]. A well�studiedexample of epigenetic factor that enhances non�sense suppression is yeast prion [PSI +]—a productof prion transformation of eRF3 (Sup35p) transla�tion termination factor [2–4]. The increase of non�sense suppression efficiency in the presence of this

factor results from the reduction of functionalmonomeric Sup35p in cells [PSI +]. Another exam�ple is an yeast prion�like factor [ISP +], whichreduces the efficiency of codon decoding [5].[ISP +] is assumed to be a prion form of transcrip�tion factor Sfp1p [6]. However, molecular mecha�nisms relating prionization of this protein to thesuppression efficiency still remain unknown.

Many proteins directly or indirectly involved intranslation are phosphorylated, and their functionalactivity is regulated by specific kinases and phos�phatases [7–9]. Therefore, the modulation of kinaseand phosphatase level might affect the efficiency oftranslation. Translation in yeasts is regulated amongothers by homological phosphatases Ppz1p andPpq1p. Deletion of PPZ1 and PPQ1 genes results inallosuppression, while overexpression of these genesentails antisuppression of various nonsense suppres�sion factors [10–12]. The level of nonsense suppres�sion was shown to be regulated not only by PPZ1 phos�phatase but also by its regulatory subunit Hal3p [13].Therefore, Hal3p/Ppz1p protein complex is involvedin the regulation of nonsense suppression efficiency inyeasts but the mechanism relating this complex to theprocess of translation still remains undiscovered. Thiswork is aimed to study this regulatory mechanism.

Protein Complex Ppz1p/Hal3p and the Efficiency of Nonsense Suppression in Yeasts Saccharomyces cerevisiae

M. S. Ivanov, E. A. Radchenko, and L. N. Mironova Department of Genetics and Breeding, St. Petersburg State University, St. Petersburg, 199034 Russia;

e�mail: lnmiron@gmail.com Received March 10, 2010

Accepted for publication April 2, 2010

Abstract—It is known that the efficiency of nonsense suppression in yeasts is controlled both genetically andepigenetically. Since many components of translation machinery are represented by phosphoproteins, theefficiency depends, in particular, on the activity of kinases and phosphatases that include the Ppz1p/Hal3pcomplex. It contains Ppz1p phosphatase, which is a catalytic subunit, and Hal3p that negatively regulates itsfunction. The aim of this work was to study the mechanisms which relate the activity of Ppz1p/Hal3p com�plex to nonsense suppression efficiency. In this study, we used a genetic approach implicating the analysis ofnonsense suppression phenotype of the strains overexpressing HAL3 or PPZ1 genes and also bearing deletionsor mutant alleles of genes, which presumably could participate in the manifestation of these overexpressions.We have shown that Hal3p inhibits not only Ppz1p but also the homologous phosphatase Ppz2p. Our dataindicate that Ppz2p is also involved in the control of nonsense suppression efficiency. In the course of searchfor Ppz1p target protein, it was shown that Ppz1p dephosphorylates at least two proteins involved in transla�tion. Moreover, Ppz1p affects the efficiency of nonsense suppression not only due to its phosphatase activitybut also due to another mechanism triggered by its interaction with Hsp70 chaperones.

DOI: 10.1134/S0026893310060075

Keywords: yeast, Saccharomyces cerevisiae, translation, nonsense suppression, phosphatase, genes HAL3,PPZ1, TEF5, SUP45, SLA1, SSB1, SSB2

UDC 577.21

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MATERIALS AND METHODS

5V�H19 strain of Saccharomyces cerevisiae (MATaade2�1 can1�100 ura3�52 leu2�3,112 SUQ5 [PSI+] and[psi–]) [14] was a kind gift of Dr. V.V. Kushnirov (Car�diology Research Complex MH RF, Moscow, Russia).

Plasmids pRS315�TEF5 and pRS315�TEF5�S86Awere obtained as a result of cloning of PCR�productscorresponding to either wild�type allele TEF5 ormutant allele tef5�S86A into pRS315 vector at BamHIrestriction site. Centromeric plasmids pDB800,pDB902, and pDB858 carrying wild�type allele SUP45or mutant alleles sup45�S421/432A and sup45�S421/432D, respectively [15], were generously giftedby Dr. D. Bedwell (Alabama University, Birmingham,United States). The effects of HAL3 and PPZ1 overex�pression were studied using multicopy plasmidYEplac195 [16] and its derivates YEplac195�HAL3,YEplac195�PPZ1, and YEplac195�PPZ1�R451L[17], kindly gifted by Dr. J. Ariño (Auonomous Uni�versity of Barselona, Spain). Gene deletions wereobtained using pFA6 [18] and pAG32[19] plasmids.

Conventional methods of yeast genetics [20, 21]and standard media were used: YAPD, synthetic com�plete medium SMM, and selective media lacking indi�vidual components of SMM. YAPD�based media,containing hygromycin B (HygB) (Sigma) or geneti�cin (G418) (GibcoBRL), and SMM supplementedwith 5�fluoroorotic acid (Angus) were also used.

The efficiency of ade2�1 nonsense mutation sup�pression was estimated visually by the growth rate of

strains on SMM�Ade medium. Yeast suspension wasplated onto corresponding selective medium in a seriesof threefold dilutions [22]. The method of threefolddilutions ensures the detection of even slight differ�ences in the efficiency of nonsense suppression. Allexperiments were performed in three repeats withindependently isolated transformants.

Polymerase chain reaction (PCR), restrictionanalysis, isolation of genomic and plasmid DNA,transformation of yeasts and Escherichia coli, and DNAgel�electrophoresis were performed according to stan�dard protocols [23–26]. Mutant tef5�T622C allele wasobtained by Overlap Extension PCR [27]. The dele�tions of TEF5, SUP45, SLA1, SSB1, SSB2, HAL3, PPZ1,and PPZ2 genes were obtained using site�specificmutagenesis with oligonucleotides, whose 3'�endswere complimentary to the regions of pFA6 or pAG32plasmids [18, 19] that flanked the gene of selectivemarker, and whose 5'�ends were complimentary to theregions of chromosomal gene to be deleted.

RESULTS

Interaction of Hal3p and Ppz2p Phosphatase

We have previously demonstrated that phenotypicmanifestation of nonsense suppression mutations inSUP35 and SUP45 genes, and [PSI+] and [ISP+] priongenes depends on the level of PPZ1 expression. Over�expression of PPZ1 gene reduces the efficiency of sup�pression in the presence of these factors, while thedeletion enhances it [12, 13]. Hal3p protein is knownas a regulatory subunit of Ppzlp phosphatase [28];therefore, alteration of HAL3 expression should causean opposite effect. To verify this, we deleted HAL3 genefrom the cells of 5V�H19[PSI+] strain, and trans�formed this strain with a multicopy YEplac195�HAL3plasmid. The phenotype analysis of the obtainedstrains revealed that HAL3 deletion indeed resulted inantisuppression, while overexpression of HAL3enhanced the efficiency of suppression, i.e., causingallosuppression of [PSI+] factor (Fig. 1).

We assume that the observed effects result from thealteration of phosphatase activity of Ppz1p. Therefore,the allosuppression effect of HAL3 overexpression isexpected to be hindered in [PSI+] strain, carryingPPZ1 deletion. To verify this hypothesis, we obtained5V�H19 [PSI+] strain with the deletion of PPZ1 gene.Deletion of PPZ1 enhanced the suppression effect ofin [PSI+] (Fig. 2a). Then 5V�H19�ppz1Δ[PSI+] strainwas transformed with a multicopy YEplac195�HAL3plasmid. Contrary to our assumption, the effect ofallosuppression caused by HAL3 overexpression wasobserved also in the presence of PPZ1 deletion(Fig. 2b).

Therefore, Ppz1p is not the only phosphatasewhose activity is regulated by Hal3p protein. Mostprobably, one of such phosphatases might be Ppz2p,an isoform of Ppz1p. Moreover, using PCA (protein–

SMM SMM�Ade

wt

hal3Δ

Ø

HAL3

(a)

(b)

Fig. 1. The influence of HAL3 gene expression on the man�ifestation of [PSI+]. (a) HAL3 deletion results in antisup�pression in [PSI+] strain. (b) overexpression of HAL3 generesults in allosuppression in [PSI+] strain. wt refers to theoriginal 5V�H19 [PSI+] strain; Ø is the strain transformedwith YEplac195 vector; HAL3 is the strain carrying aYEplac195�HAL3 plasmid; hal3Δ is the strain with HAL3deletion.

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PROTEIN COMPLEX Ppz1p/Hal3p 909

fragment complementation assay), Hal3p and Ppz1pwere shown to interact with each other [29]. Thus,Hal3p might work as a regulatory subunit for bothPpz1p and Ppz2p. If so, the sequences of HAL3 over�expression should not be observed in strains with dele�tion of both PPZ genes.

We obtained a deletion of PPZ2 gene and a doubledeletion of PPZ1 and PPZ2 in the 5V�H19[PSI+]strain. Deletion of PPZ2 and PPZ1 resulted in allosup�pression of [PSI+] (see Fig. 2a). Allosuppression effectof PPZ2 deletion was less pronounced than that ofPPZ1. Moreover, allosuppression effects of the dele�tions of these genes were additive, since, in the case ofdouble deletion of PPZ1 and PPZ2, it was more signif�icant than in the case of each single deletion (see Fig.2a). Evidently, Ppz2p phosphatase was involved in thecontrol of nonsense suppression efficiency. Deletion�containing strains were then transformed withYEplac195�HAL3 plasmid. The strain with PPZ2deletion exhibited allosuppression effect of HAL3overexpression, while the phenotype of the strain withdouble deletion remained unaltered (Fig. 2b). There�fore, Hal3p protein indeed functioned as a regulatorysubunit of Ppz1p and Ppz2p phosphatases.

The Role of EF1Bα Elongation Factor in the Effect of Ppz1p Overexpression

As we have shown previously, the overexpression ofPPZ1 results in antisuppression of [PSI+]. Moreover,overexpression of mutant allele of this gene encodingcatalytically inactive phosphatase did not cause suchan effect [12]. Therefore, antisuppression effect ofPpz1p overexpression resulted from its phosphataseactivity.

Based on the available information about phos�phoproteins directly or indirectly involved in transla�tion, Tef5p, Sup45p, Sla1p and Ssb1/2p proteins canbe considered as potential targets of Ppz1p. Are theseproteins indeed the targets of Ppz1p dephosphoryla�tion that results in antisuppression? We verified thishypothesis using a genetic approach that enabled us tocompare the effects of PPZ1 overexpression in strainscarrying normal alleles and deleted or mutant allelesthat block phosphorylation of these proteins.

Tef5p, a yeast analog of EF1Bα translation elonga�tion factor, was the most probable candidate protein tomediate the effect of Ppz1p phosphatase on transla�tion [30]. Biochemical experiments revealed thatPpz1p was able to dephoshorylate EF1Bα at serine res�idue in 86 position. tef5�S86A mutation that resulted inserine substitution for alanine completely preventedEF1Bα phosphorylation in vivo [11]. If we assume thatEF1Вα indeed relates Ppz1p activity to translation,the antisuppression effect observed under PPZ1 over�expression should be absent in the [PSI+] strain with amutant tef5�S86A allele.

We obtained a deletion of TEF5 gene in the 5V�H19[PSI+] strain, carrying a centromeric plasmid with a

wild�type allele TEF5 that rescues the lethal effect ofthe deletion. Moreover, using consecutive substitutionof plasmids, we obtained a 5V�H19�tef5Δ [PSI+] straincarrying a pRS315�TEF5�S86A plasmid with amutant TEF5 allele. The efficiency of suppression inthis strain was reduced compared to a strain with wild�type TEF5 allele (Fig. 3a). Therefore, the suppressiondepended on the Ser86 phosphorylation of EF1Bα.Dephosphorylation of EF1Bα at this position resultedin antisuppression. These data confirm the assump�tion that EF1Bα links at least partially the activity ofPPZ1 and the efficiency of nonsense suppression.

At the same time, the dependence of phenotypicmanifestation of PPZ1 overexpression on EF1Bαphosphorylation was rather insignificant. We concludethis considering the fact that the strains carrying wild�type and mutant TEF5 and transformed with multi�copy YEplac195�PPZ1 and YEplac195�PPZ1�R451LΔplasmids exhibit antisuppression effect of PPZ1 andallosuppression effect of ppz1�R451L in the presence ofeither TEF5 allele (Fig. 3b). Thus, the reduction ofantisuppression effect of PPZ1 overexpression in thestrain carrying a mutant tef5�S86A allele is insignifi�cant, and therefore, can not be detected by visual esti�mation of nonsense suppression efficiency.

SMM SMM�Ade

wt

ppz1Δ

ppz2Δ

ppz1/2Δ

HAL3

HAL3

HAL3

ppz1Δ

ppz2Δ

ppz1/2Δ

(a)

(b)

Ø

Ø

Ø

Fig. 2. The influence of PPZ1 and PPZ2 deletions on themanifestation of HAL3 gene overexpression. (a) deletionsof PPZ1 and PPZ2 genes result in allosuppression in[PSI+] strain. (b) HAL3 overexpression induces allosup�pression in the presence of single PPZ1 and PPZ2 deletions(ppz1Δ and ppz2Δ) but not in the presence of a double dele�tion of these genes (ppz1/2Δ). Ø and HAL3 refer to thederivates of 5V�H19 [PSI+] strain transformed withYEplac195 and YEplac195�HAL3 plasmids, respectively.

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Hence, we assume that the antisuppression effectobserved under Ppz1p overexpression in the [PSI+]strain depends not only on EF1Bα, but also on someother phosphoproteins involved in translation. Andthe role of such proteins is more significant comparedto EF1Bα elongation factor.

The Search for Other Ppz1p Substrates

Translation termination factor eRF1 (Sup45p),encoded by SUP45 gene, is phosphorylated by CK2kinase at serine residues in 421 and 432 positions. Adouble sup45�S421/432A mutation that causes serinesubstitution for alanine, completely prevents Sup45pphosphorylation in vivo. On the contrary, the substitu�tion of serine for aspartate mimics the constitutivelyphosphorylated state of Sup45p [15]. If Sup45p is atarget of Ppz1p, the antisuppression effect induced byPPZ1 overexpression will be reduced in strains con�taining Sup45p with serine substituted for alanine at421 and 432 positions.

We obtained the deletion of SUP45 gene in trans�formants of 5V�H19 [PSI+] strain carrying pDB800,pDB902, and pDB858 plasmids that rescue a lethaleffect of the deletion. The phenotype of these strainsindicated the absence of a significant effect of sup45 on

the manifestation of [PSI+] factor (data not shown).Thus, phosphorylation of eRF1 termination factorhad no influence on reading of stop�codons not onlyin wild�type strains, as was shown previously [15], butalso in [PSI+] strains with the reduced accuracy oftranslation termination. Therefore, phosphorylationstatus of Sup45p protein had no effect on the manifes�tations of PPZ1 overexpression. The conclusion wasconfirmed by the fact that overexpression of PPZ1 andppz1�R451L was observed both in the presence of wild�type SUP45 allele and of mutant alleles of this gene(data not shown).

Cytoskeletal phosphoprotein Sla1p interacts invivo both with Ppz1p phosphatase [31] and translationtermination factor eRF3 (Sup35p) [32]. Thus, thisphosphoprotein can be implicated in the effects ofPpz1p overexpression. If so, the effects should beinhibited in the presence of SLA1 deletion. Weobtained a 5V�H19 [PSI+] strain with SLA1 deletion. Itturned out that the deletion of this gene resulted inallosuppression of [PSI+] factor (Fig. 4a). Thus, wedemonstrated for the first time that the absence ofSla1p protein might affect the efficiency of nonsensesuppression. Nevertheless, overexpression of PPZ1and ppz1�R451L caused effects both in the originalstrain without deletion and in the strain carrying SLA1deletion (Fig. 4b). Therefore, Sla1p protein has noeffect on overexpression of Ppz1p, and its role in theprocess is insignificant.

SMM SMM�Ade(a)

(b)

TEF5wt

tef5�S86A

PPZ1

ppz1�R451L

PPZ1

ppz1�R451L

TEF5wt

tef5�S86A

Ø

Ø

Fig. 3. The effect of PPZ1 overexpression is independent ofTEF5 gene. (a) expression of a mutant tef5�S86A alleleresults in antisuppression of [PSI+] factor. (b) antisuppres�sion effect of PPZ1 overexpression and allosuppressioneffect of ppz1�R451L allele is observed in the presence ofboth wild�type TEF5 allele, and mutant tef5�S86A allele.TEF5wt and tef5�S86A refer to the derivates of 5V�H19�tef5Δ [PSI+] strain carrying pRS315�TEF5 and pRS315�TEF5�S86A plasmids, respectively. Ø, PPZ1, and ppz1�R451L here and in Figs. 4⎯6 refer to the strains trans�formed with YEplac195, YEplac195�PPZ1, andYEplac195�PPZ1�R451L plasmids, respectively.

SMM SMM�Ade(a)

(b)

PPZ1

ppz1�R451L

SLA1wt

sla1Δ

sla1Δ

Ø

Fig. 4. The effect of PPZ1 overexpression is independent ofSLA1 gene. (a) deletion of SLA1 gene results in allosup�pression of [PSI+] factor. (b) effects of PPZ1 and ppz1�R451L overexpression are detected in the presence ofSLA1 deletion. SLA1wt and sla1Δ refer to the original 5V�H19 [PSI+] strain carrying a wild�type SLA1 allele and toits derivate 5V�H19�sla1Δ [PSI+] with the deletion ofSLA1 gene, respectively.

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PROTEIN COMPLEX Ppz1p/Hal3p 911

Ssb1p and Ssb2p chaperones can be considered asputative proteins involved in the manifestation ofPpz1p overexpression. Ssb1p and Ssb2p proteinsexhibit 99% identity and fulfill similar functions insidethe cell. Ssb1p interacts in vivo with Ppz1p [33] andwith eRF3 termination factor [34]. Moreover, the effi�ciency of nonsense suppression was shown to dependon the level of Ssb1p [35, 36].

We obtained single SSB1 and SSB2 deletions, and adouble deletion of these genes in 5V�H19 [PSI+]strain. The double deletion of SSB1 and SSB2 resultedin allosuppression of [PSI+] factor, while single muta�tions had no effect on its manifestation (data notshown). These data indicate that functions of Ssb1pand Ssb2p overlap during the regulation of nonsensesuppression. The strains with deletions were trans�formed with plasmids carrying PPZ1 alleles. Antisup�pression effect of PPZ1 overexpression was developedin all analyzed strains (Fig. 5). Therefore, Ssb1p andSsb2p proteins are not involved in the effect of wild�type PPZ1 overexpression.

At the same time, we revealed that overexpressionof mutant ppz1�R451L allele entailed allosuppressionin the presence of single deletions of SSB1 and SSB2genes, while the effect was not observed in the pres�ence of double deletion of these genes (see Fig. 5).Thus, the manifestation of mutant ppz1�R451L allelerequires at least one Ssb proteins, indicating that phos�phatase Ppz1p is coupled with translation not only

through its phosphatase activity but also through theinteraction with Hsp70 chaperone proteins: Ssb1p andSsb2p.

DISCUSSION

The level of Hal3p/Ppz1p expression is known toinfluence the efficiency of nonsense suppressioncaused by the mistakes of translation termination [11–13]. However, the mechanisms of Hal3p/Ppz1p inter�action with the translation machinery remain obscure.Here we have tried to elucidate the possible mecha�nisms.

First of all, we verified if the influence of Hal3pprotein on the level of nonsense suppression wascaused only by the inhibition of Ppz1p phosphatase. Itturned out that Hal3p inhibited the activity of not onlyppz1p but also of a homologous phosphatase Ppz2p.Moreover, we have shown that the absence of Ppz2pmight also influence the efficiency of nonsense sup�pression; therefore, the activity of yeast phosphatasescoupled with translation is controlled by the same reg�ulatory protein.

Then we searched for a protein target of ppz1pphosphatase activity responsible for the manifestationof overexpression effects. A number of proteinsinvolved in translation are phosphorylated in vivoand/or interact with Ppz1p phosphatase and can beconsidered as putative substrates of this enzyme. We

SMM SMM�Ade(a) (b)

PPZ1

ppz1�R451L

wt

SMM SMM�Ade

SMM SMM�AdeSMM SMM�Ade

PPZ1

ppz1�R451L

(c) (d)ssb1Δ

ssb2Δ ssb1/2Δ

Ø

Ø

Fig. 5. Deletions of SSB1 and SSB2 genes influence the manifestation of ppz1�R451L overexpression. the effects of PPZ1 over�expression are detected (a) in the original 5V�H19 [PSI+] strain and in the presence of single (b) SSB1 and (c) SSB2 deletions.(d) Allosuppression effect of ppz1�R451L overexpression is not observed in the presence of a double deletion of SSB1 and SSB2genes.

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analyzed a possible role of the following proteins in theeffects of ppz1p overexpression: translation elongationfactor EF1Bα (Tef5p), translation termination factoreRF1 (Sup45p), cytoskeleton protein Sla1p, andSsb1p and Ssb2p chaperones.

We previously demonstrated that Ppz1p dephos�phorylates translation elongation factor EF1Bα in vivo[11]. Therefore, it was suggested that antisuppressioninduced by PPZ overexpression was caused by theincrease of EF1Bα dephosphorylation; however, thehypothesis has not been confirmed with experimentaldata [11]. It remained obscure whether the level ofEF1Bα phosphorylation depended on the efficiency ofnonsense suppression. Our data indicate that constitu�tively dephoshorylated mode of EF1Bα is caused bytef5�S86A mutation, and leads to antisuppression (seeFig. 3a). Thus, antisuppression effect of PPZ1 overex�pression might be at least partially mediated by EF1Bαdephosphorylation.

The fact that the effects of Ppz1p overexpressionare observed in the presence of a mutant tef5�S86Aallele indicate that they result not only from EF1Bαdephosphorylation (see. Fig. 3b). Therefore, Ppz1pphosphatase regulates functional activity of at leasttwo proteins involved in translation—translationelongation factor EF1Bα and some other unknownprotein. Moreover, the role of this protein that medi�ates Ppz1p effects might be more significant than therole of EF1Bα since we could not detect a reliabledecrease of antisuppression effect in the presence oftef5�S86A mutant allele (see Fig. 3b). Available dataindicates only that this protein is neither a translationtermination factor eRF1 (since mutant alleles ofSUP45 gene encoding nonphosphorylable eRF1 haveno effect on the efficiency of nonsense suppression)nor a cytoskeletal Sla1p protein (since the effects ofPpz1p overexpression were observed in the strain car�rying SLA1 deletion (Fig. 4b). If the effects of Ppz1pon the efficiency of nonsense suppression are caused

simultaneously by the translation elongation factorEF1Bα and Sla1p protein, a significant decrease ofantisuppression effect should be observed in the pres�ence of SLA1 deletion, since the impact of EF1Bα onPpz1p effects is negligible.

However, the strain carrying a deletion of SLA1gene and a multicopy plasmid with PPZ1 gene doesnot exhibit significant growth on adenine�freemedium (see Fig. 4b). The list can be completed withUpf1p and Upf2p phosphoprotein, which are notdephosphorylated by Ppz1p, according to our data [6].

At the same time we have obtained remarkable dataconcerning the role of Ssb1p and Ssb2p chaperoneproteins in the manifestation of PPZ1 overexpressioneffects. Although these proteins are not dephosphory�lated by Ppz1p, the allosuppression effect of ppz1�R451L overexpression depends on at least one of func�tional Ssb proteins (see Fig. 5d).

These apparently controversial data might have thefollowing explanation. The overexpression of Ppz1pphosphatase might influence the efficiency of non�sense suppression in two ways (Fig. 6a). On the onehand, it enhances the level of target protein dephos�phorylation inducing antisuppression. On the otherhand, the overexpression of Ppz1p might increase thenumber of its substrate Ssb molecules, reducing thepool of Ssb proteins that interact with ribosomes, andresulting in the increase of nonsense�suppression,since the deletion of SSB genes are known to causenonsense�suppression effect [35].

The nonsense suppression effect induced by theinteraction of Ppz1p and Ssb proteins can be maskedby a more pronounced antisuppression effect thatresults from the increase of Ppz1p phosphatase activityunder the overexpression of wild�type PPZ1 allele (seeFig. 6a). In the case of mutant ppz1�R451L allele over�expression, the antisuppression effect is hindered by amutation disrupting phosphatase function. The latter

(a) (b)PPZ1 ↑ ppz1�R451L ↑

Phosphataseactivity ↑

Interaction withSsb1/2p in vivo

Dephosphorylationof target protein ↑

ANTISUPPRESSION

Functional poolSsb1/2p ↓

Allosuppression

Phosphataseactivity

Interaction withSsb1/2p in vivo ↑

Dephosphorylationof target protein

Antisuppression

Functional poolSsb1/2p ↓

ALLOSUPPRESSION

Fig. 6. A putative mechanism explaining the influence of (a) wild�type PPZ1 and (b) mutant ppz1�R451L overexpression on theefficiency of nonsense suppression.

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PROTEIN COMPLEX Ppz1p/Hal3p 913

reveals the allosuppression caused by the interaction ofPpz1p phosphatase and Ssb proteins (Fig. 6b).

If Ssb�dependent allosuppression is hindered by adouble deletion of SSB1 and SSB2 genes, the overex�pression of a mutant ppz1�R45L allele does not affectthe efficiency of nonsense suppression (see Fig. 5d).Therefore, the influence of Ppz1p overexpression onnonsense suppression is confined by these two mecha�nisms.

Summing up our results, we conclude that themechanism relating Hal3p/Ppz1p complex to thetranslation machinery is evidently more complicatedthan it seemed in the beginning. Apparently there areat least two phosphoproteins that mediate the effect ofPpz1p on the efficiency of nonsense suppression.Moreover, there should be another mechanism thatlinks Ppz1p with nonsense suppression, which doesnot involve the phosphatase activity and is indepen�dent of Ssb proteins. We have also shown that thetranslation of stop�codons depends not only on Ppz1pphosphatase but also on Ppz2p, which is regulated byHal3p protein. A complex functional dependencebetween serine�threonine phosphatases and transla�tion in yeasts requires further research.

ACKNOWLEDGMENTS

We are grateful to Dr. D. Bedwell (University ofAlabama, Birmingham, United States), Dr. J. Ariño(Autonomous University of Barcelona, Spain), and toDr. V.V. Kushnirov (Cardiology Research ComplexMH RF, Moscow, Russia) for generous gifts of yeaststrains and plasmids.

The work was financially supported by the RussianFoundation for Basic Research (08�04�00353) and byState Contract P799 with the Federal Agency of Edu�cation of Russian Federation.

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