ELSEVIER Biochimica et Biophysics Acta 1263 (1995) 45-52 et Biophysics Acta

Heterologous expression of the highly conserved acidic ribosomal phosphoproteins from Dictyosteliumm discoideum in Saccharomyces

cerevisiae

Blanca Bermejo a, Jesus Prieto b, Miguel Remacha a, Antonio Coloma b, Juan P.G. Ballesta a-* a Cenirv de Biologia Molecular Sever0 Ochoa, CSIC and UAM, Canto Blanco, 28049 Madrid, Spain

b Department of Biochemistry, Facultad de Ciencias, UAM, Arzobispo Morcillo s/n, 28029 Madrid, Spain

Received 27 December 1994; revised 11 April 1995; accepted 13 April 1995

Abstract

The genes encoding the acidic ribosomal phosphoproteins DdPl and DdP2 from Dictyosfelium discoideum have been cloned into yeast plasmid vectors under the control of the inducible GAL1 promoter. These constructions have been used to transform S. cerevisiue strains D45 and D67 lacking the equivalent ribosomal components. The D. discoideum genes are properly transcribed when cells are grown in the presence of th’e inducer galactose and the mRNAs incorporated into polysomes. However, the heterologous ribosomal proteins are not able to rescue the growth deficiency in S. cerevisiue caused by the absence of their own ribosomal proteins. When the heterologous proteins are analyzed using specific antibodies, only protein DdPl is found in the ribosomes of the transformed S. cerevisiue D67 strain. No other heterologous protein is found in any other transformed strain, suggesting that the heterologous acidic ribosomal components are rapidly degraded when they are not bound to the ribosomes. The results indicate that D. discoideum DdPl protein is able to interact with the yeast ribosome, though the interaction is functionally inefficient. Protein DdP2, in spite of having a higher sequence similarity to its yeast counterparts, is completely inactive in S. cereuisiae. Since the P proteins from both organisms have extensive amino acid sequence similarity ranging from 60% to 70%, these results warns about establishing a direct relationship between the extent of amino acid sequence similarity and the capacity of heterologous proteins to be functional in host species. Moreover, our data suggest that evolution affected the interacrion of the acidic proteins with the ribosome rather than the structural features responsible for their primary functions.

Keywords: Ribosome structure; Acidic ribosomal phosphoprotein; Heterologous expression; (S. cereuisiae); (D. discoideum)

1. Introduction

The cellular protein synthesis machinery is accepted to have been well conserved during evolution and the basic features of the different components, ribosomes, tRNAs, soluble factors, are very similar among eukaryotic species. There are, however, differences among the components forming the translational apparatus that have been used as evolutive marks for establishing phylogenetic relationships [l]. Most of these comparative studies are based on struc- tural analysis, but little is known about how these differ- ences and similarities relate to the function of the protein synthesis constituents. Thus, data concerning the in vivo

* Coresponding author. Fax: t-34 1 3974799.

0167-4781/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0167-4781(95)00081-X

function of translational apparatus components in eukary- otic heterologous systems are rather scarce although this type of information may be important in establishing the functional significance of the structural variations. Heterol- ogous expression of plastid ribosomal protein genes in bacteria has been shown to functional in some cases [2-41. In eukaryotes, the mouse initiation factor eIE4E has been shown to be able to substitute for the equivalent protein in yeast [5]. Also yeast ribosomal protein L29 can be replaced in vivo by mouse protein L27’ [6] and a similar ribosomal protein replacement has been reported for human and hamster ribosomal protein S14 [7].

The acidic ribosomal proteins form a group of poly- peptides with pl in the 3.5 to 4.5 range that are found in the ribosomes of species from all kingdoms [8] forming a typical protuberance in the large ribosomal subunit [l]. These proteins participate in the interaction of the cyto-

46 B. Bermejo et al. / Biochimica et Biophysics Acta I263 (1995) 45-52

plasmic factors with the bacterial ribosome (see [9] for a review), and it is accepted that they have a similar role in archaebacteria [ 101 and eukaryotes [l II. In addition, the acidic proteins are found phosphorylated in eukaryotic organisms, and they seem to be part of a regulatory mechanism coupling the ribosome activity to the metabolic activity of the cells [12,13].

The structure of these proteins has been highly con- served during evolution among the eukaryotes. Two types of acidic ribosomal proteins have been reported in eukary- otic ribosomes, named Pl and P2 [ 141. The sequence of the proteins from both groups is very similar at the carboxyl region but diverge more at the amino terminal end. Thus, in yeast the P2 proteins are incorporated unprocessed into the protein synthesis machinery while the amino end of Pl proteins is blocked and results from the removal of the terminal methionine followed by the acetylation of the following serine residue [ 151.

In lower eukaryotes, contrary to mammalian organisms, more than two acidic proteins have been found associated with the ribosomes. Four acidic phosphoproteins have been well characterized in Succharumyces cerevisiae [ 151 and in Schizosacchuromyces pombe [ 161, and the number is even larger in protozoans like Trypanosoma cruzi [ 17- 191

Based on structural and functional homologies [20-221, two yeast acidic proteins have been included into the Pl group and the other two into the P2 group. Consequently, they have been called YPl (Y and YPl /3 in the first case and YP2o and YP2p in the second [14]. We have previ- ously referred to proteins YPl p, YP2 (Y and YP2p as L44’, L44 and L45, respectively [23].

The slime mold Dictyostelium discoideum, in spite of being considered older that the ascomycete 5. cerevisiae [24], has only two acidic ribosomal proteins, DdPl and

DdP2, resembling in this respect to the higher eucaryotes. The genes encoding these proteins have been cloned and the encoded polypeptides [25,26] show a high similarity with the yeast proteins (Fig. 1).

We have previously obtained different S. cerevisiae strains deprived of one or more acidic ribosomal proteins by gene disruption [21,22]. These mutants allow an analy- sis of the in vivo functional and structural interchangeabil- ity of equivalent acidic proteins from different sources, which could be expressed in the disrupted strains. We have attempted this approach using the D. discoideum acidic protein genes. Since there is only one protein from each group in this organism, this study can illustrate the role of the two yeast proteins of the same group and at the same time provide information on how the evolutive changes have affected the functionality of the acidic proteins.

2. Materials and methods

2.1. Bacterial and yeast strains

Escherichia coli JM83, TGl and DH5o were used as host for the routine maintenance and preparation of plas- mid used in these studies and were grown in LB medium. S. cerevisiae W303-la (a; leu2, trpl, ura3, his 2, ade2, canl) was used as the parental strain for D45 (rpYP2cx::URA3, rpYP2 p ::HIS3) and D67 (rpYPla::LEU2, rpYPlp::TRPI) disruptant strains used in this report. Disruptants were obtained as described previously [22]. Yeasts were grown in either rich YEP medium (1% yeast extract, 2% peptone, 2% glucose) or minimal SD medium [27] supplemented with the required nutritional requirements.

YP2B 1 MKYLAAYLLLVQGGNAAPSAADIKAWASVGAEVD!ZARINELLSSLEGKGSLEE IIIIIIIII :lll .ll.:. ::.

DdP2 I I I. I I I. I I&&cKE~=~;.;;A

1 MKYLAAYLLASLSGNA..NAASVTKILQSV- IIIIIIIII . .I1 :I..:. (I:IIl:l::..:I.Il ..l:Il. :.

yP2a 1 MKYLAAYLLLNAAGNl'.PDATKIK&ILESVGIEIEDEKVSSVLSALEGKS..DE

IIAEZGOKKFATVFTGGASSAAAGAAGAAAGGDAAEEEKEEEAKEESDDDMGFGLFD

II..I..I::.I :II:II.I.:II:I.:.II:... II lIIIIlII:IIII LITEGNJZKLAAV.PAAGPASAGGAUA SGDAAAFaEEKE!EEXA.EEhSDDDMGFGLFD

110

106

106

YPlC 1 MSD.....SIISFAAFILADAGL?ZITSDNLLTITKAAGANVDNVWADVYAKALE II . .l.l:lll.l.: 1: .lI. .I.. l:::ll:.(.

DdPl 1 MSEIKTEiACiY&~~QDDGIEITADKIKTLLEAANITVASHWPGLYARSLA 1) _I[ 1 I.:l:I.I.:III..:I: 11 :(Il:.I.. (::::[:.I.

Ypla 1 MS.. .TE.SALSYAALILADSEIEISSEKLLTLTNAANVP VENIWADIFAKALD

GKDLKEIL..SGFHNAGPVAGAGAASGAAAAGGDAAAEEEKEEEAAEESDDDMGFGLFD 106 ::.I:[ .I .l:..l..:Il.:IIl:::.I I..III IIIIIIII:IIII

KVNIPELLLNAGSSGAAGAAPVAAATSAAAPAAAAKK ETKKE-ESDDDMGMGLFD 113 l:.:/I:I :.::[[.I:..::..::. I..I.l.II II IIIIIIIII:IIII

GQNLKDLLVNFSAGAJhAPAGVAGGVAGGEAG-.. .EEEAKEzESDDDMGFGLFD 106

Fig. 1. Comparison of amino acid sequences of S. cereoisiae acidic ribosomal proteins YPI cz//? and YP2 a/P, with D. discoideum proteins DdPl and DdP2, respectively.

B. Bermejo et al. /Biochimica et Biophysics Acta 1263 (1995) 45-52 47

2.2. Cell transformations

Bacterial transformations were performed according to the procedure of Hanahan [28]. Yeast transformation was carried out using either spheroplasts [29] or intact cells. In this case either the LiCl method [30] or electroporation in a Gene pulser (Bio-Rad) following the maker instructions, were used.

2.3. Plasmids

The pPL series of plasmids are derived from pUC19 by addition of yeast auxotroghic markers, replication origins (ARS element) and the centromere from chromosome III [31]. pPL30 was obtained by removing the ARS and centromere from pFL38 and inserting a 750 bp EcoRI-XhoI

fragment from pCG542 [32], including the upstream acti- vating sequence (UAS) regulated by galactose, the TATA box, and the transcription start site of GALl-GAL10 [33,34]. pYactI1 contains the S. cerevisiae actin gene [35].

2.4. Construction of specific plasmids

pBG-DI U A 450 bp EcoRI-EcoRI fragment obtained from

pUC19-Pl [26], containing the cDNA encoding D. dis- coideum acidic protein Pl(DdPl), was inserted into the MCS of Bluescript resulting in plasmid BS-Dl. Then, a 450 bp PstI-Hind111 fragment, containing the same DdPl encoding sequence, was taken from BS-Dl and together with an EcoRI-PstI 770 bp fragment from pFL30-GAL, which contains the GAL1 promoter, were inserted between

pFL39U EcoRI-Hindlll (4,692 kb)

pBG-D 1 U (5,9kb)

A

pFL-GAL puc19-P2

pFL39U (4,692kb)

I

\/

EcoRI-Hindlll Hincll EcoRI-EcoRI

(Klenow) (466 bp)

E Bn N i ATG P PH TAA

t I . . . . 4 - pBG-DZU .,::., ::::::s .-: ‘.’ : ,, B#’

pFL-GALP2 EcoRI-Hindlll (6 W (1,22kb)

B Fig. 2. Scheme of the construction of plasmids pBG-DlU (A) and pBG-D2U (B). Details of construction are given in Material and Methods. B, BamHI; Bn, BanII; Cl, ClaI; D, DraI; E,, EcoRI; H, HindIII; Hc, HincII; P, PstI; S, SalI.

48 B. Bermejo et al. /Biochimica et Biophysics Acta 1263 (199.5) 45-52

the EcoRI and Hind111 sites in the multiple cloning sites (MCS) of the centromeric vector pFL39U, as indicated in Fig. 2A.

pBG-D2U A 466 bp EC&I-EcoRI fragment from pUC19-P2 [25],

after filling up with DNA polymerase Klenow fragment, was inserted in the H&II site of pFL30-GAL obtaining pFL-30-GALP2 and then, a 1.2 kb EcoRI-Hind111 insert from this plasmid, containing the DdP2 gene and the GAL1 promoter was subcloned in the corresponding sites of the MCS of pFL39U yielding pBG-D2U (Fig. 2B).

pBG-DIL and pBG-D2L These plasmids were obtained upon removal of the

URA3 genetic marker from pBG-DlU and pBG-D2U, respectively, by BglII treatment. Afterwards, a B&II frag- ment obtained from the vector YEpl3, carrying the LEU2 marker, was inserted in the same site.

RNA blots (Northern tests) Yeast RNA, extracted by phenol from either intact cells

or polysomes was prepared basically as described [36]. RNA was fractionated in 1 .O% agarose gels and transferred to nylon membranes (HybondN, Amersham). Hybridization was performed using specific DNA probes as indicated in the text, according to standard procedures 1371.

2.5. Enzymes and reagents

Restriction endonucleases were purchased from Boehringer-Mannheim, New England BioLabs, and Amer- sham and were used as recommended by the suppliers. T4 DNA ligase, calf intestinal alkaline phosphatase, and DNA polymerase I Klenow fragment were from Boehringer- Mannheim, and DNA polymerase I and T4 DNA poly- merase were from New England BioLabs. [ CU-~*P]~CTP (3000 Ci/mmol) was obtained from Amersham.

2.6. Cell fractionation

Yeast cells, resuspended in 20 mM Tris-HCl, pH 7.4, 80 mM KCl, 10 mM MgCl, were usually broken with glass beads in the presence of a cocktail of proteinase

inhibitors (0.5 mM phenylmethylsulfonyl fluoride (PMFS), 10 pg/ml aprotinin, 2 ,ug/ml leupeptin, 2 pg/ml pep- statine) and the extracts were centrifuged for 15 min at 15 000 i-pm in a Sorvall SS34 rotor resulting in a S-30 fraction.

Ribosomes and supematant fractions (S-100) were pre- pared from S-30 as previously described [ll]. The acidic proteins were extracted from the ribosomes by ammonium-ethanol treatment and acetone precipitation [ill.

Polysomes were obtained from exponentially growing cells that were inhibited with cycloheximide (100 pg/ml) 5 min before harvesting and broken as previously indicated but including 100 pg/ml cycloheximide in all buffers. Polysomes were pelleted from the S-30 fraction by centrif- ugation for 2 h at 40000 rpm in a Beckman Ty65 rotor. In these conditions about 80% of the ribosomal particles are sedimented.

2.7. Immunological methods

The presence of the acidic proteins in cell fractions was estimated using specific monoclonal antibodies [38] by inhibition enzyme-linked immunosorbent assay (ELISA) as previously described [38].

2.8. Electrophoretic methods

Proteins from total cell extracts were analyzed by SDS polyacrylamide gel electrophoresis. Proteins in the gel were electrophoretically transferred to Immobilon-P mem- brane (Millipore) using LKB Novablot buffer (39 mM glycine, 48 mM Tris-HCl, 0.0375% SDS, 20% methanol) as both anode and cathode electrode solutions. Elec- trophoresis was performed at 0.8 mA/cm* for 1 h. Treat- ment with antibodies was performed as described by Tow- bin et al. [39].

2.9. Comparison of amino acid sequences

Comparisons were made using the Bestfit program of the Genetics Computer Group (GCG) Sequence Analysis Software Package, Wisconsin University Biotechnology Center.

Table 1 Saccharomyces cerevisiae strains used in this study

Strain Parental strain Genetic markers Disrupted genes Transforming plasmid Plasmid encoded protein

Plasmid marker

D67-D 1 D67-D2 D45-D 1 D45-D2

D67 D67 D45 D45

ura3,his3 ura3, his3 trpl, leu2 trpl,led

YPla,YPl~ YPl CY,YPl p YP2ff,Yp2p Ynff,YnP

pBG-DlU pBG-D2U pBG-DlL pBG-D2L

DdPl DdP2 DdPl DdP2

uRA3 uRA3 LEU2 LEU2

B. Bermejo et al. / Biochimica et Biophysics Acta I263 (1995) 45-52 49

B

Fig. 3. Estimation of D. discoideum Pl/PZ protein mRNA in trans- formed yeast strains. (A) RNA -From the indicated S. cereuisiae strains, grown in YEP medium in the presence of either glucose or galactose, was run in agarose gels, transferred t’o nylon membranes and hybridized with DNA probes to either DdPl (D45-Dl and D67-Dl) or DdP2 (D45-D2 and D67-D2) genes. Probe to the DdPl gene corresponds to a 450 bp EcoRI-EcoRI fragment from pIIS-Dl. For DdP2 gene a 272 bp XhoI- PauII fragment from pFL30-GAI.P2 was used. The position of 26s rRNA and 18s rRNA used as size markers is indicated. (B) RNA from total cell extracts (T) and polysome fraction (P) from the indicated S. cereuisiae strains, was separated and processed as in the previous experiment.

3. Results

Saccharomyces cerevisiae strains D4.5 and D67, lacking the acidic phosphoproteins YP2a//3 and YPI a/P, re- spectively, were transfomled with plasmids carrying the genes encoding the equivalent proteins DdPl and DdP2 from Dictyostelium discoideum placed under the control of the inducible GAL1 promoter. The different transfor- mants obtained are summarized in Table 1.

Table 2 Estimation of acidic protein present in ribosomes by ELISA

Ribosomes Protein expressed from strain in 5 train

3.1. Expression of the heterologous genes

The presence of the mRNAs corresponding to the het- erologous genes was tested in Northern blots of total cell RNA from the transformants using DNA probes corre- sponding to the coding region of D. discoideum acidic protein genes. It was possible to show (Fig. 3A) that mRNAs transcribed from each one of the heterologous genes are present when strains are grown in the presence of galactose. No hybridizing RNA was found, however, in extracts from cells grown in glucose. The results clearly indicated, therefore, that the expression of the plasmid-en- coded genes responded properly to the inducer and tran- scription of the heterologous genes did take place.

3.2. Presence of the heterologous mRNAs in polysomes

The capacity of the heterologous DdPl and DdP2 mR- NAs to enter the translational machinery was tested by checking their presence in polysome fractions from strains D67-Dl and D45D2 by Northern blots. Fig. 3B clearly shows that the mRNAs were found in polysomes. An estimation of the hybridizing band intensity indicated that roughly 70-80% of the messenger in the total extracts seems to be involved in protein synthesis appearing in the polysome fraction.

3.3. Acidic protein in the ribosomes from transformed S. cerevisiae strains

It has been previously shown that in order to bind to the ribosomes the acidic protein complex has to be formed by at least one member of each one of the two groups, Pl and P2 [22]. Consequently, the ribosomes from the non-trans- formed D4.5 and D67 strains, in which both members of the Pl and P2 groups are respectively missing, are de- prived of all acidic proteins and those expressed from the intact genes accumulate free in the cytoplasm [22].

A 450

Antibody specific to:

YPlP YP2a YP2P DdPb

D-45 D45-Dl D45-D2 D67-Dl D67-D2 D67 W303-la D. discoideum

Via/P 0.09 0.08 0.06 YPl a//3, DdPl 0.09 0.08 0.07 YPl o//3, DdP2 0.09 0.08 0.08 YP2[Y/p, DdPl 0.10 0.09 0.51 YP2 o/P, DdP2 0.10 0.08 0.10 YP2 o/p 0.10 0.12 0.13 YPla/P, YP2a/ ‘P 0.52 0.84 0.80 Ddl'l , DdP2 0.06 0.09 0.10

nt 0.14 0.14 0.55 0.19 nt 0.21 0.77

Equivalent amounts of 0.5 M NH,Cl- 50% ethanol ribosomal extracts were used in each case. Proteins were tested by indirect ELISA. Monoclonal specific antibodies to proteins YPl p, YP2o and YP2p [38] and a rabbit antiserum to D. discoideum acidic proteins Pl, P2 and PO [26] were used in the assay. The A,, is proportional t,o the amount of the specific protein. nt, not tested.

50 B. Bermejo et al./ Biochimica et Biophysics Acta 1263 (1995) 45-52

To test wether the D. discoideum proteins were able to affect the interaction of the yeast proteins present in the cells with the ribosomes, an estimation of acidic proteins in ribosomal extracts from the transformed D45 and D67 strains was performed by ELISA using monoclonal anti- bodies specific to yeast proteins YPl p(L44’), YP2 cr(L44) and YP2p(L45) [38]. Antibodies to YPl cr are not avail- able. The rabbit antibody to D. discoideum proteins was also included in the test.

The results shown in Table 2, in addition to confirm the absence of the yeast acidic proteins in ribosomes from the non-transformed D45 and D67 strains, as expected, indi- cate that these polypeptides are also missing in the parti- cles from all the transformants, except D67-Dl. In this strain, protein YP2p(L45) is found and an anti-D. dis- coideum cross-reacting protein, obviously protein DdPl, is also detected. The presence of YP2p(L45) in the ribo- somes from D67-Dl was confirmed by isoelectrofocusing of ribosomal extracts (not shown).

3.4. Detection of heterologous proteins in the transformed strains

The failure to detect D. discoideum acidic proteins in the ribosomes from most transformed strains led us to test directly the presence of the heterologous proteins in these cells.

The strains were grown in rich medium in the presence of galactose to induce the expression of the heterologous proteins. The presence of these proteins in total cell ex- tracts and S-100 fractions of induced cells was detected by immunoblotting using antiserum to the D. discoideum acidic proteins [26]. The antibody recognizes specifically the acidic proteins, as well as the 38 kDa non-acidic protein PO which contains the same carboxyl end [40-421, in extracts of D. discoideum used as control (Fig. 4).

A B

1 234Dl 234

Fig. 4. SlOO (A) and S-30 (B) cellular extracts from S. cereuisiae D67-Dl (11, D67-D2 (2). D45-Dl (3) and D45-D2 (4). were resolved in SDS-polyacrylamide gel electrophoresis, blotted to nylon membranes and incubated with rabbit antiserum to the D. discoideum acidic ribosomal proteins. A D. discoidem ribosomal extract was used as control (D). The position of the 34.5 kDa, 18.4 kDa, and 14.3 kDa molecular weight markers is indicated.

Table 3 Effect of heterologous acidic ribosomal proteins on cell growth

Strain protein

W303-la none W303-D 1 DdPl W303-D2 DdP2 D45 none D45-YP2P a none D45-Dl DdPl D45-D2 DdP2 D67 none D67-YPlfi b none D67-D 1 DdPl D67-D2 DdP2

Generation time (min) on:

glucose galactose

85 120 85 120 85 180 215 360 95 120 215 420 215 660 235 240 145 230 235 240 235 240 a

a Strain D45 transformed with a plasmid carrying the gene encoding protein YP2p(L45) including its own promoter. b Strain D67 transformed with a plasmid carrying the gene encoding protein YPl p(L44’) including its own promoter.

However, only the total extracts from D67-Dl show the presence of one specific anti-D. discoideum immuno- reactive protein of around 12 kDa which must correspond to protein DdPl. In none of the remaining yeast prepara- tions was possible to find the heterologous D. discoideum proteins (Fig. 4).

3.5. Biological effects of heterologous acidic proteins

The disruption of yeast acidic protein genes is not lethal for the cell but causes a variable negative effect on the growth rate depending on the number and type of proteins eliminated [21,22]. Elimination of both proteins of the same type, either Pl (YPl LY and YP1/3(L44’)) or P2 (YP2 a(L44) and YP2 p&45)) as in strains D67 and D45, respectively, causes a significant increase in the cell dou- bling time [22].

The biological activity of the heterologous protein can be verified by testing their capacity to induce the recovery of the negative growth effect caused by the elimination of the equivalent yeast polypeptides. As shown in Table 3, the transformation of D45 and D67 with each one of the D. discoideum proteins, DdPl and DdP2, has no positive effect on the doubling time when cells are grown in either inducing (galactose) or non-inducing (glucose) conditions. Moreover, transformation of D45 with the heterologous P2 protein seems to be toxic for the cells since a two-fold increase in the doubling time is observed in the trans- formed strain under induction conditions. Transformation with the same plasmid has a lower negative effect, a 50% increase in the doubling time, on wild-type W303-la cells and no effect at all on strain D67. On the other hand, transformation with the DdPl encoding plasmids have a slight harmful effect on strain D45 only.

B. Bemejo et al. /Biochimica et Biophysics Acta 1263 (1995) 45-52 51

4. Discussion

Our results show that the D. discoideum genes encod- ing the acidic ribosomal protein DdPl and DdP2, inserted into centromeric plasmids under the control of an inducible promoter, are expressed when introduced into yeast cells. As expected, the genes are transcribed only when cells are in the presence of the inducer, and the transcribed mRNAs are found associated to polysome fractions indicating their capability to enter the protein synthesis machinery and, consequently, to be transl,ated. In most cases, however, the heterologous proteins have not been detected in total cell extracts using specific antibodies. Only extracts from D67- Dl showed the presence of a D. discoideum protein and, in this case, the protein was found only in the ribosomes. These results, therefore, oeem to indicate that the heterolo- gous acidic ribosomal proteins are probably degraded in yeast unless they are bound to the ribosome. The sensitiv- ity of the acidic proteins to degradation when not bound to ribosomes seems to be higher in D. discoideum than in yeast. In fact, the specific rabbit antibodies raised against Dictyostelium P proteins a.re unable to detect these proteins in the soluble fractions from this organism (Prieto, J., unpublished results). However, it cannot be excluded that this antiserum is unable to react with the proteins in the soluble fraction.

D. discoideum protein DdPl is, nevertheless, able to bind to the yeast ribosome forming part of the Pl-P2 protein complex required for the interaction with the parti- cle [22]. However, DdPl forms a complex with only one member of the yeast P2 family, namely YP2p(L45), since YP~cx(L~~) is not found in the ribosomes from strain D67-Dl, suggesting a closer functional homology of DdP2 to YP2p(L45) than to YP2 a(L44). This functional ho- mology is not, certainly, endorsed by a closer sequence similarity which is comparable in both cases (Table 4) but indicates the existence of functional differences between the two members of the yeast protein P2 group. The interaction of the hybrid complex DdPl-YP2P(L45) with the particle is, nevertheless, insufficient to rescue the cell growth rate, indicating thlat DdPl is not functional even though it is able to bind to the yeast ribosome.

Table 4 Amino acid sequence homologies among S. cereuisiae and D. dis- coideum acidic ribosomal proteins

Protein DdPl DdP2

complete first GE complete first 30 aa sequence sequence

YPlff 43.2 38.4 39.4 36.8

W1P 39.6 29.1 35.3 21.7 YP2ff 38.9 31.8 51.4 58.6

yp2P 35.8 35.0 50.9 57.1

Amino acid sequence of D. discoideum and yeast acidic proteins was compared using the GCG Bestfit program. The scores obtained for identity are shown.

The absence of protein DdPl from ribosomes of trans- formed D45-Dl is not surprising since this protein should be unable to form the ribosome interacting complex with yeast YPl (Y and YPl p, present in this strain.

On the other hand, D. discoideum protein DdP2 is not found in the D45 or D67 transformed strains. Probably, either this protein is unable to form a complex with the yeast Pl proteins, or the complex is ill-suited to bind to the ribosome. The first alternative is obvious in strain D67 that totally lacks YPl proteins, but is also possible in D45 that express them at a very low level (Table 2, [43]).

These results suggest the existence of more precise structural requirements for P2 proteins, which, in opposi- tion to Pl proteins, can not be replaced by the heterolo- gous polypeptides at any level. This is again unexpected since the sequence similarity between the yeast and D. discoideum P2 proteins is higher than between the corre- sponding PI polypeptides (Table 4). Moreover, the similar- ity among P2 proteins is specially notable, almost 60% amino acid sequence identity (Table 4, Fig. l), at the ribosome-binding amino terminus.

The inactivity of D. discoideum protein DdP2 in yeast may otherwise lay with the phosphorylation requirement for functional activity shown by the eukaryotic acidic proteins in vivo [12], and in vitro [23]. D. discoideum acidic proteins are also phosphorylated [44] but the yeast specific kinase may not correctly modify the D. dis- coideum DdP2 protein affecting the binding of the polypeptide to the ribosome. In this sense, it must be noted that the protein kinase involved in the phosphorylation of the Pl and P2 proteins is probably not the same [ 131.

In any case, our results indicate that, in spite of the close structural similarity, the acidic proteins from a rela- tively close eukaryotic organism like D. discoideum are not functional in S. cereuisiue. Neither protein DdPl nor DdP2 is able to rescue the cell growth deficiency caused in yeast by the deprivation of the equivalent endogenous ribosomal acidic proteins. On the contrary, in strain D45 the effect of transformation with plasmids carrying the heterologous P2 protein, and to a lesser extent the Pl protein, is harmful and causes an additional decrease in the cell growth rate. This negative effect is not due to a direct interaction of the proteins with the ribosome, as indicated above, and must be caused by some other process, perhaps a degradation product since the intact proteins are not detected in the cell lysates. Supporting this possibility, the conserved carboxy terminal decapeptide of the acidic P proteins has been shown to inhibit an in vitro reticulocyte protein synthesizing system [45].

As a final remark it must be stressed that the functional inactivity of the D. discoideum P proteins in yeast clearly indicate that a high amino acid sequence similarity does not warrant the capacity of cellular components to function in a different, though relatively close organism. In fact, a partial functionality has been found in the case of DdPI, the polypeptide showing lower structural similarity with

52 B. Bermejo et al. / Biochimica et Biophysics Acta 1263 (1995) 45-52

the yeast equivalent. Interestingly, the amino acid se- quence of mouse initiation factor eIF-4E, that has been shown to substitute for the equivalent factor in S. cere- uisiue [5], is only 33% identical to its yeast counterpart.

Acknowledgements

We thank M.C. Femandez Moyano for expert technical assistance. This work has been supported by grant. PB91- 0011 from DGICYT (Spain) and by an institutional grant to the Centro de Biologia Molecular from the Fundacion Ramon Areces (Madrid).

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