1995, 15(9):4754. Mol. Cell. Biol. 

E Guarinos and J P BallestaM Remacha, A Jimenez-Diaz, B Bermejo, M A Rodriguez-Gabriel, Saccharomyces cerevisiae.the pattern of protein expression inare not required for cell viability but regulate Ribosomal acidic phosphoproteins P1 and P2

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MOLECULAR AND CELLULAR BIOLOGY, Sept. 1995, p. 4754–4762 Vol. 15, No. 90270-7306/95/$04.0010Copyright q 1995, American Society for Microbiology

Ribosomal Acidic Phosphoproteins P1 and P2 Are NotRequired for Cell Viability but Regulate the Pattern of Protein

Expression in Saccharomyces cerevisiaeMIGUEL REMACHA, ANTONIO JIMENEZ-DIAZ, BLANCA BERMEJO,

MIGUEL A. RODRIGUEZ-GABRIEL, ESTHER GUARINOS, AND JUAN P. G. BALLESTA*

Centro de Biologia Molecular ‘‘Severo Ochoa,’’ Universidad Autonoma de Madrid-Consejo Superiorde Investigaciones Cientıficas, Canto Blanco, 28049 Madrid, Spain

Received 4 May 1994/Returned for modification 15 June 1994/Accepted 2 June 1995

Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivatedgenes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1a, YP1b, YP2a, andYP2b, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, thosefrom the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they showa cold-sensitive phenotype, growing very poorly at 23&C. Cell extracts from the quadruple-disruptant strain areabout 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidicP proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains,and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicatingthat the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns ofprotein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteinsfrom the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. Inaddition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upontransformation with one of the missing genes. These results indicate that acidic proteins are not an absoluterequirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation ofcertain mRNAs differently.

The presence of a set of strongly acidic proteins is a univer-sal feature of ribosomes from all organisms. These proteinshave been involved in interactions with different supernatantfactors during protein synthesis in prokaryotic as well as eu-karyotic systems (see reference 16 for a review).In eukaryotic organisms, the acidic ribosomal proteins,

called generically P proteins, are capable of being phosphory-lated, and this modification drastically affects the interaction ofthe polypeptides with the ribosome in vitro (11, 14). In fact,this process might be part of a control mechanism that, byregulating the amount of bound protein, can affect the activityof the particles in relation to the metabolic requirements of thecell (24).In Saccharomyces cerevisiae, four acidic ribosomal proteins

have been reported and characterized; according to structuraland functional considerations, they can be classified in twogroups, YP1a/b and YP2a/b, corresponding to the two types ofacidic polypeptides present in mammalian ribosomes (35). Us-ing gene disruption techniques, we previously found that theindividual elimination of each polypeptide, and even the dou-ble disruption of any two of the four proteins, is not a lethalevent for the cell, although the generation time is affected todifferent extents (20, 21). The effect is especially importantwhen the genes encoding the two members of the same groupare disrupted, since in this case the remaining acidic proteinspresent in the cell are unable to bind to the ribosome, decreas-ing the efficiency of the particles in the polymerizing process(21). These results clearly demonstrate that a stable interactionof the acidic proteins with the ribosomes is not a prerequisite

for the involvement of the particles in translation; however,they did not exclude an implication of the free or loosely boundacidic proteins in the process.To test whether the presence of acidic proteins is indeed an

absolute requirement for eukaryotic protein synthesis, we haveprepared disruptant strains in which the four acidic proteingenes have been simultaneously disrupted, showing that theyare also viable but that they present an altered pattern ofprotein expression.

MATERIALS AND METHODS

Strains and growth conditions. Either S. cerevisiaeW303 (a/a leu2-3,112 trp1-1ura3-1 his3-11,15 ade2-1 can1-100) or S. cerevisiae W303-1b (a leu2-3,112 trp1-1ura3-1 his 3-11,15 ade2-1 can1-100) was used as a wild-type strain in the exper-iments performed in this study. S. cerevisiae MT552/36c (a sal4-2 SUQ5 ade2-1ura3-1) was used as a positive suppressor control for in vivo readthrough tests.Strains carrying either a single acidic protein gene disruption (D4, D5, D6, andD7) or a double disruption (D45, D46, D47, D56, D57, and D67) were obtainedas previously reported (20, 21). Yeast strains were grown in either YEPD me-dium or minimal SD medium (22) supplemented with the appropriate nutritionalrequirements.When radioactive labeling of proteins was required, the cells were grown in SD

medium, and [35S]methionine (10 mCi/ml) was added when the A600 of theculture reached 0.2. Cells were allowed to grow for one doubling-time period andwere then collected.Escherichia coli JM83 and E. coli DH5 were used for handling of cloning

vectors and were grown in LB medium. Bacteria were transformed according tostandard procedures (10).Genetic manipulations. S. cerevisiae mating, sporulation, and tetrad analyses

were performed according to published methods (22). Transformation was car-ried out by the lithium acetate method as described by Schiestl and Gietz (30).Plasmids. pFL-46CYH was derived from the centromeric plasmid pFL38 (3)

by inserting the complete rpYP1b gene as a 1.0-kb PstI-HindIII fragment fromplasmid pMRH46 (19). The original URA3 auxotrophic marker in pFL38 wassubsequently removed by digestion with BglII and replaced by the cycloheximideresistance gene CYH2 as a 2.3-kb BamHI-BamHI fragment from plasmid pRC20(5).pUKC series plasmids were constructed as indicated elsewhere (32).

* Corresponding author. Mailing address: Centro de Biologıa Mo-lecular, Canto Blanco, 28049 Madrid, Spain. Phone: 34 1 3975076. Fax:34 1 3974799.

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pUKC815 was derived from YCp50 by cloning the phosphoglycerate kinase PGKpromoter and the PGK 59 region encoding the N-terminal 33 amino acids fusedto the lacZ gene. pUKC817, -818, and -819 are like pUKC815 but carry thenonsense codons UAA, UAG, and UGA, respectively, at the beginning of theb-galactosidase sequence. Only when these codons are suppressed in the trans-formed strain is the enzymatic activity expressed.Southern (DNA) blots. Yeast DNA was prepared as described previously (22).

DNA, after digestion with restriction enzymes, was resolved by electrophoresis in0.8% agarose gels and blotted to nylon membranes (Amersham Corp.). Hybrid-ization was performed according to standard procedures (25).Northern (RNA) blots. Total yeast RNA, extracted with phenol from intact

cells, was prepared basically as described by Warner and Gorenstein (34). RNAwas fractionated in 1.0% agarose gels and transferred to nylon membranes(Hybond, Amersham). Specific DNA probes were labeled by random hexanucle-otide priming with [a-32P]dCTP and Klenow enzyme. Hybridization was per-formed according to standard procedures (25).Cell fractionation. Cells were broken with glass beads in 20 mM Tris-HCl (pH

7.4)–80 mM KCl–10 mM MgCl2 including a cocktail of protease inhibitors (0.5mM phenylmethylsulfonyl fluoride, 10 mg of aprotinin per ml, 2 mg of leupeptinper ml, 2 mg of pepstatin per ml). The extracts were centrifuged for 15 min at15,000 rpm in a Sorvall SS34 rotor, producing an S-30 fraction.The S-30 fraction was centrifuged to obtain the ribosomes and supernatant

fractions as previously described (26). As a source of supernatant factors for invitro protein synthesis, the fraction precipitated between 20 and 50% saturationof ammonium sulfate, called S-100, was used. The acidic P proteins (SP0.5fraction) were extracted from the ribosomes by ammonium-ethanol treatment(26); the extracted fraction was dialyzed against 10 mM N-2-hydroxyethylpipera-zine-N9-2-ethanesulfonic acid (HEPES; pH 7.4)–0.5 mM phenylmethylsulfonylfluoride and concentrated by filtration through Centricon SR3 membranes (Ami-con).Immunological methods. The presence of the acidic proteins in cell fractions

was estimated by using specific monoclonal antibodies (33) in inhibition orindirect enzyme-linked immunosorbent assays (ELISAs) as previously described(33).Electrophoretic methods. Proteins from total cell extracts were resolved by

two-dimensional (2D) polyacrylamide gel electrophoresis as described byO’Farrell (18), with some modifications (27). Gels were processed for fluoro-graphy, dried, and exposed at 2708C for various periods of time. Approximately106 trichloroacetic acid-precipitable cpm were routinely applied to each gel.Activity tests. (i) Polyphenylalanine synthesis. The reaction was performed in

50-ml samples containing 10 pmol of 80S ribosomes, 5 ml of S-100, 0.5 mg oftRNA per ml, 0.3 mg of polyuridylic acid per ml, 40 mM [3H]phenylalanine (120cpm/pmol), 0.5 mM GTP, 1 mM ATP, 2 mM phosphocreatine, and 40 mg ofcreatine phosphokinase per ml in 50 mM Tris-HCl (pH 7.6)–15 mM MgCl2–90mM KCl–5 mM b-mercaptoethanol. After incubation at 308C for 30 min, sam-ples were precipitated with 10% trichloroacetic acid, boiled for 10 min, andfiltered through glass fiber filters. When required, 5 ml of a 0.1-mg/ml SP0.5fraction were incubated with the ribosomes in 20-ml samples at 378C for 15 min,and then the remaining components of the reaction mixture were added.The misreading capacity of the ribosomes was tested in conditions similar to

those used for the poly(U)-dependent synthesis but substituting [3H]phenylala-nine by a mixture of [3H]serine, [3H]tyrosine, and [3H]isoleucine at the sameconcentration.The transpeptidation reaction was measured by the fragment reaction (17),

using N-acetyl-tRNA. The reaction mixture, in 150 ml of 20 mM Tris-HCl (pH7.3)–270 mM NH4Cl–13 mM MgCl2, contained 40 pmol of ribosomes, 2 mMpuromycin, and 1 pmol of N-[acetyl-3H]tRNA. The reaction was initiated by theaddition of 1 volume of methanol (33%, final concentration), allowed to proceed

at 08C for 30 min, and then stopped by the addition of 100 ml of 0.3 M sodiumacetate (pH 5.5) saturated with MgSO4. The samples were extracted with 1.5 mlof ethyl acetate, and 1 ml of the organic phase was checked for radioactivity.A yeast cell-free translation system dependent on exogenous mRNA (8) was

prepared as described by Feinberg and McLaughlin (7). After treatment withRNase to eliminate the endogenous mRNAs, the system was complemented withtotal RNA extracted from exponentially growing S. cerevisiae W303-1b cells.When required, the cell-free reaction samples were supplemented with 4 pmol ofacidic ribosomal proteins (SP0.5 fraction) per pmol of ribosomes in the reactionmixture, estimated from the A260 of the sample.(ii) Determination of b-galactosidase activity. Tests were performed as de-

scribed by Miller (15). Two-milliliter aliquots of cells growing exponentially (A5505 0.5) were centrifuged and resuspended in 400 ml of 0.1 M potassium phosphate(pH 7.0) and 400 ml of buffer Z (0.065 M Na2HPO4, 0.04 M NaH2PO4, 10 mMKCl, 1 mM MgSO4, 40 mM 2-mercaptoethanol [pH 7.0]). Then, 10 ml of 1%sodium dodecyl sulfate, 10 ml of chloroform, and 10 ml of 40 mM phenylmeth-ylsulfonyl fluoride in dimethyl sulfoxide were added, and the reaction was initi-ated by the addition of 200 ml of 2-nitrophenyl-b-D-galactopyranoside at 4 mg/mlin 0.1 M potassium phosphate (pH 7.0). The reaction was stopped by the additionof 500 ml of 1 M Na2CO3, and the A420 was measured.

RESULTS

Preparation of S. cerevisiae haploid strains carrying threedisrupted acidic protein genes. Strains of opposite mating typecarrying either one (single disruptants) or two (double dis-ruptants) disrupted acidic protein genes were mated as indi-cated in Table 1, yielding the corresponding diploid strains. Allof the triple-disruptant heterozygote strains grew with normalgeneration times and were able to sporulate, producing fourviable spores per tetrad. After sporulation and tetrad separa-tion, spores having the expected genetic markers for all fourpossible triple disruptants were selected and yielded slowlygrowing colonies.Construction of quadruply disrupted S. cerevisiae from dou-

ble heterozygote diploid strains. Quadruple acidic protein dis-ruptants were obtained from diploid strains which have onlytwo of eight acidic protein genes intact (Table 2). These strainsare therefore heterozygous for only two of the acidic proteingenes, which thus makes it feasible to select the quadrupledisruptant by tetrad analysis.Four diploid heterozygous strains were prepared, but only

two of them, Dd56 and Dd47, which have one acidic proteinfrom each type, YP1 and YP2, were able to sporulate (seebelow). Diploid Dd47, expressing YP1b and YP2b, was sporu-lated, and 23 tetrads were dissected. Fifteen tetrads segregatedas tetratype, seven segregated as nonparental ditype, and onesegregated as a parental ditype with respect to the auxotrophicmarkers; consequently, 29 spores were Ura1 Leu1 Trp1 His1

and therefore expected to be quadruply disrupted cells. The

TABLE 1. S. cerevisiae strains mated to obtain disruptant cells lacking three acidic proteinsa

Disrupted strain Disrupted genes encoding:Characterization of parental strains

Strain Genotype

D456 YP1b (L449) D4 a trp1-1 leu2-3,112 his3-11,15 ade2-1 rpYP2a (L44)::URA3YP2a (L44) D56 a ura3-1 leu2-3,112 rpYP1b (L449)::TRP1 rpYP2b (L45)::HIS3YP2b (L45)

D457 YP1a D4 a trp1-1 leu2-3,112 his3-11,15 ade2-1 rpYP2a (L44)::URA3YP2a (L44) D57 a ura3-1 trp1-1 rpYP1a::LEU2 rpYP2b (L45)::HIS3YP2b (L45)

D467 YP1a D46 a leu2-3 his3-11,15 rpYP2a (L44)::URA3,rpYP1b (L449)::TRP1YP1b (L449) D7 a ura3-1 trp1-1 his3-11,15 ade2-1 rpYP1a::LEU2YP2a (L44)

D567 YP1a D7 a ura3-1 trp1-1 his3-11,15 ade2-1 rpYP1a::LEU2YP1b (L449) D56 a ura3-1 leu2-3 rpYP1b (L449)::TRP1 rpYP2b (L45)::HIS3YP2b (L45)

a Homologs in E. coli are given in parentheses.

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four spores were viable in 15 tetrads. Only one of the eightnongrowing spores in the remaining tetrads corresponded to aUra1 Leu1 Trp1 His1 quadruple disruptant.A Southern analysis of DNA from the four haploid strains

derived from tetrad 10, which by selection with auxotrophicmarkers included one quadruple disruptant, is shown in Fig. 1.As controls, the original diploid strain, the wild-type strainW303-1b, and an independently obtained quadruply disruptedstrain were also included. As expected, the hybridization pat-tern confirmed the disruption of YP1a and YP2a in all sam-ples except those from strain W303-1b. In addition, the YP1bgene is disrupted in spores 10a and 10b (Fig. 1, lanes 2.1 and2.2), and the YP2b gene is disrupted in spores 10b and 10d(lanes 2.2 and 2.4). Consequently, spore 10b in the analyzedtetrad corresponded to a quadruple disruptant, which wascalled D4567-10b. The results of the Southern analysis wereconfirmed by ELISA, using specific antibodies to detect thepresence of the proteins (data not shown).In two tetrads, an independent segregation of the His1 char-

acter and the YP2b disruption took place (Table 3). In thiscase, the YP2b protein is detected in His1 spore 3c but not inHis2 spore 3b. There is no obvious explanation for this anom-alous segregation, which is being analyzed further, but it doesnot affect the conclusions of this report.Characterization of triple- and quadruple-disruptant strains.

A Northern analysis of the RNA from three quadruple-dis-ruptant strains confirmed the absence of the mRNA corre-sponding to the four acidic proteins but the presence of themessenger for ribosomal protein P0, used as a control (Fig. 2).The presence of proteins YP1b, YP2a, and YP2b in dis-

ruptant cells was directly tested in total S-30 extracts by inhi-bition ELISA. The lack of an antibody specific for YP1a didnot allow testing for this protein. Quadruple-disruptant strainswere negative for the three antibodies, and only the proteinwhose gene had not been disrupted was detected in the tripledisruptants, namely, YP1b in D457, YP2a in D567, and YP2bin D467 (Table 4). However, the acidic protein detected in the

FIG. 1. Southern analysis of HindIII-restricted DNA from the four haploidstrains derived from spores 10a (lane 2.1), 10b (2.2), 10c (2.3), and 10d (2.4),dissected from tetrad 10 after sporulation of diploid S. cerevisiae Dd47. DNAfrom Dd47 (lane 1), from the parental wild-type S. cerevisiae W303-1b strain(lane 3), and from one independently obtained quadruple disruptant (lane 4) wasalso included. Probes specific for the four acidic P proteins, YP1a, YP1b, YP2a,and YP2b, were used as indicated in the upper right corner of each panel. Aphysical map of the original insert containing the corresponding P-protein geneand of the deleted construct used to transform the cells and to isolate therespective singly disrupted strains (20, 21) is shown at the top of each panel; anopen bar represents the gene coding region, a dotted bar indicates an intron, anda filled bar corresponds to the auxotrophic marker. In the cases of YP1a, YP1a,and YP2b, the upstream HindIII site in the hybridizing genomic DNA fragmentis not present in the original cloned fragment and consequently is not shown inthe physical maps. As probes, the following DNA fragments from the originalclones were used: EcoRI (E)-PvuII (Pv) from YP1a, HindIII (H)-HincII (Hc) fromYP1b, EcoRI-PvuII from YP2a, and HincII-PstI (P) from YP2b. The positions ofsize markers are indicated at the left in kilobase pairs. B,BamHI; Sl, SalI.

TABLE 2. Genotypes of diploid double disruptants

Strain Missingproteins Parental strains Genotype

Dd45 YP2a,YP2b

D456 3 D457 a/a rpYP1a::LEU2/rpYP1a rpYP1b/rpYP1b::TRP1 rpYP2a::URA3/rpYP2a::URA3rpYP2b::HIS3/rpYP2b::HIS3

Dd56 YP1b,YP2b

D456 3 D567 a/a rpYP1a::LEU2/rpYP1a rpYP1b::TRP1/rpYP1b::TRP1 rpYP2a/rpYP2a::URA3rpYP2b::HIS3/rpYP2b::HIS3

Dd67 YP1a,YP1b

D467 3 D567 a/a rpYP1a::LEU2/rpYP1a::LEU2 rpYP1b::TRP1/rpYP1b::TRP1 rpYP2a/rpYP2a::URA3rpYP2b::HIS3/rpYP2b

Dd47 YP1a,YP2a

D467 3 D457 a/a rpYP1a::LEU2/rpYP1a::LEU2 rpYP1b/rpYP1b::TRP1 rpYP2a::URA3/rpYP2a::URA3rpYP2b::HIS3/rpYP2b

TABLE 3. Anomalous genetic marker segregation in a strainDd47 tetrad

MarkerSpore phenotypea

3a 3b 3c 3d

HIS 1 2 1 2TRP 2 2 1 1YP1b 1 1 2 2YP2b 2 2 1 1

a 1 and 2 represent growth and no growth in the absence of the respectiveauxotrophic nutritional requirement (HIS and TRP) or presence or absence ofthe indicated proteins detected by ELISA (YP1b and YP2b).

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total extracts is not found in the ribosomes derived from them,indicating that the polypeptides are unable to bind to particleswhich are deprived of all tested P proteins (Table 4), as pre-viously found for the doubly disrupted strains D45 and D67(21).Growth characteristics of triple and quadruple disruptants.

The triply (D456, D457, D567, and D467) and quadruply(D4567) disrupted strains form small colonies in YEPD agarplates at 308C but show a cold-sensitive phenotype, growingvery poorly at 238C (Fig. 3). The estimated doubling times ofthese strains in rich and minimal media, together with thosepreviously reported for singly and doubly disrupted strains (20,21), are shown in Table 5. The four triple disruptants and thequadruply disrupted strain grow with doubling times of around4 to 5 h and 6 to 7 h in rich and minimal media, respectively.These values are not very different from the doubling times ofdisruptants D45 and D67 and are considerably greater thanthose of the single disruptant and the remaining doubly dis-rupted strains. A very long lag period before resuming growthin rich medium after a prolonged period at stationary phase isalso a peculiarity of the quadruply and triply disrupted strainsas well as strains D45 and D67. The lag is probably related tothe fact that the number of viable cells decreases notably in thecultures of these mutants when stored at room temperature forextended periods of time.

Changes in the pattern of protein expression in acidic pro-tein disruptant strains. A possible explanation for the meta-bolic changes displayed by these disrupted strains could be analteration in the pattern of protein expression due to the acidicprotein-deficient ribosomes. To confirm this possibility, resultsof 2D gel electrophoresis of total [35S]methionine-labeled cel-lular extracts from the different disruptant strains were com-pared with results obtained for extracts from wild-type cellsgrown in the same conditions. Clear differences are noticed inthe protein patterns obtained from the different disruptantstrains and that of the parental cells. As an example, Fig. 4shows autoradiographs of 2D gels from three quadruple dis-ruptants and strain W303-1b labeled with [35S]methionine. Theprotein patterns of the mutant strains are very similar. A num-ber of protein spots in the wild type are either absent or clearlydiminished in comparison with the mutants; more interest-ingly, some proteins are more highly expressed in the dis-ruptant cells.Effect of acidic protein disruption upon sporulation. As

indicated above, the double heterozygous diploid strains lack-ing genes encoding both P proteins of the same group, eitherDd67, lacking the P1 group, or Dd45, lacking the P2 family,were unable to sporulate. This defect could be a consequenceof the alteration in the protein expression pattern caused bythe acidic protein-depleted ribosomes present in these strains.To test this possibility, Dd67 was transformed with the centro-meric plasmid pFL-46CYH, carrying the wild-type gene encod-ing YP1b and a cycloheximide resistance marker.Antibiotic-resistant transformants were grown and analyzed

for the presence of acidic proteins as well as for the ability to

FIG. 2. Northern blot of RNA extracted from S. cerevisiae W303-1b (wildtype [wt]) and three quadruple disruptants, D4567-2b (lanes 1), D4567-5d (lanes2), and D4567-8a (lanes 3). Total RNA was extracted, resolved by agaroseelectrophoresis, blotted to nylon membranes, and hybridized with probes specificfor proteins P0 (A), YP2a (B), YP2b (C), YP1b (D), and YP1a (E).

FIG. 3. Growth on YEPD agar plates of S. cerevisiae W303-1b (wild type[wt]), triple disruptants D456, D457, D467, and D567, and quadruple disruptantsD4567-2b (D4567A) and D4567-5d (D4567B) at 23 and 308C. Plates were incu-bated for 5 days.

TABLE 4. Detection of proteins YP1b, YP2a, and YP2b in total cell extracts by ELISAa

Cellextractfromstrain:

Antibody specificity

YP2b YP2a YP1b

S-30, %inhibition

Ribosomes,A492

S-30, %inhibition

Ribosomes,A492

S-30, %inhibition

Ribosomes,A492

Wild type 87 1.61 81 1.50 69 0.380D456-5b 0 0.17 0 0.08 0 0.04D457-7c 0 0.10 0 0.08 61 0.04D467-1a 87 0.15 0 0.10 0 0.03D567-3a 0 0.11 65 0.12 0 0.06D4567-2b 0 0.10 0 0.10 0 0.04D4567-5d 0 0.09 0 0.09 0 0.02

a Estimation of the acidic proteins was performed by inhibition ELISA (for total S-30 extracts) or by indirect ELISA (for ribosomes) (see Materials and Methods).The amount of the corresponding protein in the sample is proportional to the percent inhibition obtained in the first method and to the A492 in the second method.

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FIG. 4. 2D gel electrophoresis of total [35S]methionine-labeled proteins extracted from S. cerevisiaeW303-1b (A), D4567-2b (B), D4567-7c (C), and D4567-10b (D).Some significant spots either absent (open arrows) or strongly diminished (filled arrows) in the disruptant strain are marked in the gel for the wild-type strain (A).Similarly, some spots present in the disrupted cells and either absent or diminished in the parental strain are marked in one of the gels for disruptants (B). IEF,isoelectric focusing; SDS, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

TABLE 5. Growth rates of strains carrying all possible acidic protein gene disruptions

Strain Missing Pprotein(s)

Doubling time (min) in:Reference

YEPD YNBD

W303 0 90 135D4 2a 95 160 22D5 2b 125 185 22D6 1b 110 170 23D7 1a 145 220 23D45 2a, 2b 235 290 23D46 2a, 1b 122 175 23D47 2a, 1a 160 225 23D56 2b, 1b 160 215 23D57 2b, 1a 165 230 23D67 1a, 1b 255 310 23D467-1a 2a, 1a, 1b 285 325 This reportD567-3a 2b, 1a, 1b 330 380 This reportD457-7c 2a, 2b, 1a 285 370 This reportD456-5b 2a, 2b, 1b 260 300 This reportD4567-2b 2a, 2b, 1a, 1b 285 430 This report

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sporulate. As can be seen in Table 6, when Dd67 expressesprotein YP1b, the growth rate is increased to that of disruptantD7, the ability to sporulate is recovered, and acidic proteins arepresent in the ribosome.Suppressor capacity of disruptant strains. Changes in the

ribosome structure are known to be able to alter the transla-tional accuracy of the protein synthesis apparatus. To testwhether the absence of ribosome-bound acidic proteins hasthis effect, the capacity of each of the disruptant strains tosuppress different nonsense codons was estimated. pUKC se-ries plasmids carrying either a UAA, UAG, or UGA stopcodon at the beginning of the coding region in a lacZ genecloned under the control of the PGK promoter (32) were usedto transform the cells. The b-galactosidase activity of the trans-formed strains provides an accurate estimation of their sup-pressor capacity.The experiment was carried out on strain D67, since the

absence of available genetic markers did not allow the trans-formation of the quadruple disruptant. S. cerevisiae MT552/36c, carrying the temperature-sensitive allosuppressor sal4-2mutation, was also transformed with the pUKC plasmids andwas used as a positive control for suppression. At 308C, thesuppressor activity in strain MT552/36c is mostly due to sal4-2.As shown in Fig. 5, the disruptant strain expresses a level ofb-galactosidase activity slightly higher than that of the parentalstrain but much lower than that of a strain carrying an allo-suppressor mutation.In vitro polymerizing activity of quadruple-disruptant ex-

tracts. To confirm the activity of the translational machinery instrain D4567A, ribosomes and S-100 fractions were preparedand tested for protein synthesis activity in a poly(U)-depen-dent polymerizing system. As shown in Table 7, the disruptantextracts show about 30% of the control polymerizing activity,and this activity can be substantially stimulated by the additionof exogenous acidic proteins. Moreover, the disruptant ribo-somes are able to carry out the transpeptidation reaction to anextent similar to that of the wild-type particles (Table 7).The decreased polymerizing activity of the disruptant ex-

tracts did not, however, affect the fidelity of the translationprocess as tested in a misreading assay. As shown in Table 7,the extract derived from the quadruple disruptant incorpo-rated a level of miscoded amino acids similar to that of thewild-type control.Cell extracts derived from disruptant D4567-2b are also able

to translate exogenously added mRNA. The activity of thequadruple disruptants in this test changes with the preparation,ranging from 20 to 40% of the control level, and it is stronglystimulated by the addition of exogenous acidic proteins, asshown in Table 8.Pattern of proteins translated in cell-free systems. To test

whether the alteration of the protein expression pattern in

quadruply disrupted strains found in vivo is also detected invitro, the proteins translated by mutant and control extracts,using a total mRNA population from exponentially growingwild-type cells, were resolved by 2D gel electrophoresis (Fig.6). The lower specific radioactivity of the in vitro samples thanof the in vivo preparations made it necessary to apply a largeramount of protein to the gels, which resulted in higher back-grounds. Despite this fact and the obviously different transla-tion conditions, some of the most relevant spots coincide in thein vivo and in vitro protein patterns. Moreover, differences inthe proteins translated by the mutant and wild-type extracts areclear (Fig. 6A and B). A number of proteins are expressed atthe same level in both extracts, while expression of others isdrastically reduced or even abolished, and a few seem to beexpressed only in the mutant extract. These differences are due

FIG. 5. In vivo readthrough activities of different S. cerevisiae strains, deter-mined by measuring relative b-galactosidase activities of S. cerevisiae W303-1b(f), D67 ( ), and MT552/36c ( ) transformed with the indicated plasmids.pUKC815 carries a lacZ gene expressed through a PGK promoter and is used asa b-galactosidase activity standard. Expression of b-galactosidase activity frompUKC817, pUKC818, and pUKC819 requires readthrough of the UAA, UAG,and UGA codons, respectively (see Materials and Methods). Activity is given aspercentage of that of the respective controls (strains transformed withpUKC815). b-Galactosidase activities of strains W303-1b, D67, and MT552/36ctransformed with pUKC815 were 192.5, 254.7, and 87.2 arbitrary units (15),respectively.

TABLE 6. Characteristics of disruptant diploid strains

Strain Transformingplasmid Sporulation Generation time

(min)

Acidic protein inribosomes detectedby indirect ELISA(A492) using antibody

specific to:

YP2a YP2b YP1b

W303 1 100 1.50 1.60 0.380Dd67 2 255 0.11 0.21 0.03Dd67 pFL-46CYHa 1 150 1.81 1.65 0.50

a Carries the rpYP1b gene.

TABLE 7. Activities of cell extracts derived from disruptant strains

Strain used assource for: Additiona

Polyphenyl-alanineactivityb

(%)

Misreadingc

(cpm)

Peptide bondformationd

(%)Ribosomes S100

D4567-2b D4567-2b 31 467 89D4567-2b D4567-2b SP0.5 48 NTe NTD4567-2b W303-1b 50 NT NTW303-1b W303-1b 100 477 100W303-1b W303-1b SP0.5 101 NT NTW303-1b D4567-2b 85 NT NT

a An extract of acidic ribosomal proteins (SP0.5 fraction) from S. cerevisiaeW303-1b was added to a final concentration of five molecules of proteins perribosome. This concentration was shown in previous experiments to be optimalfor maximum stimulation of activity.b In the poly(U)-dependent polymerization test, the control sample (100%)

polymerized 8.6 mol of phenylalanine per ribosome.c Trichloroacetic acid-precipitable radioactivity incorporated from the mis-

coded amino acids (see Materials and Methods).d Tested by the fragment reaction (see Materials and Methods).e NT, not tested.

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to the absence of the acidic ribosomal proteins, since the wild-type pattern is basically restored when the mutant extracts aresupplemented with exogenous P proteins (Fig. 6C).

DISCUSSION

The viability of the S. cerevisiae D4567 quadruple-disruptantstrains, lacking all four ribosomal acidic proteins, clearly indi-cates that the presence of the P proteins in the cell is not aprerequisite for cell viability and consequently for protein syn-thesis.Previous results have shown that in the absence of both

members of one of the ribosomal acidic protein types, eitherP1 or P2, the remaining proteins from the other group areunable to bind to the ribosomes in a stable manner (21).Despite this fact, the cells, although having reduced growthrates, are viable, indicating that a stable interaction with theribosome is not required for the ribosomes to participate intranslation (21). Nevertheless, it could not be excluded that theacidic proteins in the cytoplasm would allow translation to takeplace by interacting in a nonpermanent way with the ribo-somes. However, the D4567 strain, lacking the four acidicproteins, grows with doubling times similar to those of thedouble- and triple-disruptant strains. Moreover, the in vitroactivity of the cell extracts derived from the D4567 strainproves that these ribosomal components are not necessary forribosome activity and protein synthesis in vivo as well as invitro. Bacterial ribosomes deprived of acidic proteins can beactive in certain specific in vitro conditions (1, 9, 13); however,there is no evidence indicating that proteins L7 and L12 arenot required for E. coli viability. In fact, although numerousviable bacterial mutants lacking 1 or 2 of up to 17 individualribosomal proteins have been described, none of them hasbeen shown to be deficient in the acidic proteins (4).Nevertheless, it has to be kept in mind that eukaryotic ribo-

somal protein P0, equivalent to bacterial protein L10 in theformation of the large ribosomal subunit stalk, contains a car-boxyl-end extension that is structurally similar to the acidic Pproteins (31). This extension contains a carboxyl region typicalof the P proteins, including the highly conserved DDDMGFGLFD end peptide. Although the complete P0 protein is es-sential for cell viability (28), the C-terminal domain is notrequired for activity when acidic P proteins are bound to theribosome; however, in the absence of bound P proteins, the P0C terminus is absolutely required for protein synthesis (29).These data indicate that yeast ribosomes require at least onebound copy of this conserved carboxyl-end peptide to be func-tional. Therefore, like their bacterial counterparts, ribosomestotally depleted of this structure are inactive.The absence of the acidic proteins is, however, not without

an effect on yeast ribosome activity as well as cell metabolism.The most visible consequence of the absence of acidic proteins

FIG. 6. Autoradiograms of 2D electrophoresis gels of [35S]methionine-la-beled proteins synthesized in exogenous mRNA-dependent cell extracts derivedfrom S. cerevisiaeW303-1b (A), D4567-2b (B), and D4567-2b supplemented withexogenous acidic P proteins (C). The same preparation of total mRNA fromexponentially growing S. cerevisiae W303-1b was added to the three systems.Spots either absent or strongly diminished in the disruptant strain extract aremarked in the gel for the parental strain (A). Similarly, spots present in thedisrupted cells and absent in the parental strain are marked in the gel for thedisruptant (B). Most spots absent in the sample from the disrupted strain (B) arepresent when the extract is complemented with P proteins (C). IEF, isoelectricfocusing; SDS, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

TABLE 8. Exogenous mRNA-dependent activity ofS. cerevisiae extracts

Extract derivedfrom:

[35S]methionine polymerized

2Acidic proteins 1Acidic proteinsa

cpm/A260

% cpm/A260

%

W303-1b 166,721 100 173,913 104D4567-2b 57,397 34 128,876 77

a Acidic ribosomal proteins (SP0.5 fraction) extracted from S. cerevisiaeW303-1b ribosomes were added to the samples (see Materials and Methods).

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is a cold-sensitive phenotype and a reduction in the cell growthrate, indicating a decrease in the protein synthesis rate. Theextent of this effect depends on the missing proteins. Compar-ison of the growth rates of the different single disruptantsindicates that elimination of protein YP2a has little effect,while the absence of YP1a seems to be the most damaging forribosome function. Among the doubly disrupted strains, thereis a clear difference between cells carrying one protein fromeach group, namely, D56, D57, D46, and D47, and strains D45and D67, lacking both members of the same group. The lattertwo strains have doubling times similar to those of the tripleand quadruple disruptants, indicating that the absence of ri-bosome-bound P proteins is the determinant in causing themaximum negative effect on cell growth. The relatively smalldifferences in doubling times among these seven slowly grow-ing disruptants may not be significant, but an effect due to thepresence of the different free acidic proteins in the cytoplasmcannot be excluded. Supporting this possibility is the negativeeffect that an overexpression of YP2b has been reported tohave on the growth of strain D45 (2).The effect of the absence of acidic proteins on the growth

rates of disruptant strains can be related to a reduced efficiencyof the protein synthesis machinery in translating mRNAs. Thisreduction is mainly associated with the ribosomes, as is clearlyshown in the in vitro protein-synthesizing systems derived fromthe disruptant strains.It is important to note that the alteration caused by the

absence of ribosome-bound P proteins, although reducing theefficiency of the translational machinery, does not seem tonotably affect the accuracy of the system. The in vivo misread-ing capacity of the extracts derived from the quadruple dis-ruptant is similar to that of extracts from the parental strain.Similarly, the termination codon readthrough tests indicatethat the disruptant strain does not have an in vivo suppressingcapacity much higher than that of the parental strain.The absence of ribosome-bound acidic proteins also affects

the pattern of protein expression. A direct confirmation of thisalteration is obtained in vivo as well as in a cell-free translationsystem derived from the disrupted strains. A number of pro-teins translated by the wild-type cells and extracts are eithermissing or dramatically reduced in the 2D electrophoresis gelof proteins expressed by the disruptant sample; more interest-ingly, there are a few proteins which are expressed in thedisruptant and not in the control. The recovery of these effectsby the exogenous addition of P proteins to the mutant cellextract clearly confirms that they are caused by the absence ofthese components from the ribosome. It seems, therefore, thatthe presence of the acidic proteins is able to affect both posi-tively and negatively the translation of different mRNAs.The alteration of the pattern of expression may explain the

phenotypic effects of some disruptant strains shown in thisreport. The disruption probably affects the expression of pro-teins required for spore formation, abolishing in this way thesporulating capacity of yeast strains carrying acidic protein-freeribosomes. Sporulation is recovered upon transformation withone of the missing acidic proteins that allow the formation ofa ribosome-bound P-protein complex, thus restoring the stan-dard translating capacity of the particles. In the same way, thechange in protein expression may determine the cold sensitiv-ity of the disrupted strains.How can the large ribosomal subunit affect the pattern of

protein expression in the cell? First of all, the reduced effi-ciency of the P-protein-deficient ribosomes may differentiallyaffect the rate of translation, depending on the mRNA second-ary structure. The deficient ribosomes might be less efficient indisrupting the double-stranded RNA regions, and therefore

they will stall on those mRNA having a complex structure,which would be translated at a lower rate than in the wild typeor not translated at all. This mechanism explaining the de-crease and even the absence of some proteins in the disruptantstrains cannot be very specific and would affect families ofmRNAs having peculiar structures rather than specific mes-sengers, which also accounts for the relatively large number ofproteins affected.However, the presence of some proteins only in the dis-

ruptant strains is not compatible with the previous hypothesisand suggests that the presence of acidic proteins may somehowrepress the translation of some mRNAs. There is experimentalevidence supporting a role for 60S subunits in the control oftranslation initiation (23), and the absence of the P proteinsmay affect the 60S function by making it dependent on somespecific structural features of the mRNA. Moreover, a role ofthe acidic proteins in the initiation step of protein synthesis canalso be considered; in fact, a dependence of initiation factoractivity on acidic proteins L7 and L12 has been previouslyreported in in vitro bacterial model systems (6, 12). A morespecific effect of the free acidic proteins on the expression ofsome proteins cannot be ruled out, and some experimentaldata support this possibility. Thus, it has been previously shownthat the absence of the acidic P1-type proteins strongly stim-ulates the expression of YP2b, and conversely, the eliminationof the P2 family represses the expression of YP1b (2). Thismutual effect seems to take place at the level of translation (2).The strong acidic character of these polypeptides precludes adirect interaction with the mRNA, and therefore their regula-tory activity must be effected through interactions with otherpolypeptides, perhaps some initiation factors.In fact, the two mechanisms proposed to explain the differ-

ential effects of P protein on translation of some mRNAs arenot incompatible and may work simultaneously in the cell. Toexplore the different alternatives further, we are presentlyidentifying some of the proteins differently affected by thepresence of the P proteins; our final aim will be to characterizethe elements in their mRNAs responsible for the responsive-ness to the P proteins.The translating activity of P-protein-free ribosomes is not

simply an interesting oddity of the disruptant yeast strains.Previous results have shown that the amount of acidic proteinsfound in the ribosomes is linked to the metabolic activity of thecells. Thus, ribosomes from stationary-phase cells containabout half the amount of P protein in the particles from ex-ponentially growing cells (24). It seems, therefore, that a pop-ulation of acidic protein-deficient particles normally exists inthe cells and that they may be a majority at certain stages of themetabolic cycle. The results in this report open up the possi-bility that these particles are not just resting ribosomes but thatthey can be implicated in the translation of some of the specificproteins which are expressed by the resting cells and not by thegrowing cells. This may be an additional mechanism that eu-karyotic cells have developed to regulate more carefully thetranslation process that is so extensively used in higher organ-isms to control metabolic processes.

ACKNOWLEDGMENTS

We thank M. C. Fernandez Moyano for expert technical assistance.We thank M. F. Tuite (Biological Laboratory, University of Kent,Canterbury, Kent, England) for his gift of the pUKC plasmid seriesand S. cerevisiaeMT552/36c, J. Fernandez Santaren for his help in the2D gel electrophoresis system, and A. Jimenez (Centro de BiologıaMolecular, Canto Blanco, Madrid) for plasmid pRC20.This work was supported by grant PB91-0011 from the Direccion

General de Polıtica Cientıfica (Spain) and by an institutional grant to

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the Centro de Biologıa Molecular from the Fundacion Ramon Areces(Madrid).

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