ELSEVIER Biochimica et Biophysica Acta 1305 (1996) 29-33

ii

BB Biochi~& et Biophysica A~ta

Short sequence-paper

Identification and sequence analysis of a rap gene from the true slime mold Physarum polycephalum

Piotr Kozlowski *, Joanna Trzcinska-Danielewicz, Kazimierz Toczko Institute of Biochemistry, Warsaw UniversiO,, Zwirki i Wigu~' 93, 02 089 Warsaw, Poland

Received 19 July 1995; revised 26 September 1995; accepted 18 October 1995

Abstract

A member of the ras gene superfamily, belonging to the rap family and designated Pprapl, was isolated from a cDNA library from the true slime mold Physarum polycephalum by plaque hybridization in combination with 5'-RACE. The assembled nucleotide sequence of Pprapl (1062 bp) has an open reading frame coding for a protein of 188 amino acids of a calculated M r of 21 035. This protein exhibits: (i) a highly conserved GTP binding domain containing a putative effector domain, with the threonine-for-glutamine substitution characteristic of rap proteins, (ii) a hypervariable domain, and (iii) the CAAX motif. Analysis of the C-terminal amino acid sequence of Pprapl shows that it presumably undergoes geranylgeranylation but is not palmitoylated; however, it contains a lysine-rich domain which might serve as the second membrane localization signal. Pprapl exhibits significantly high amino acid homology within the GTP binding domain with its homologues: Ddrapl from Dictyostelium discoideum (92%) and human RaplA (83%), and relatively low homology (59%) with the Saccharomyces cerevisiae homologue, RSR1. It has also 59% and 61% homology with the P. polycephalum Pprasl and Ppras2 proteins, respectively. This gene is the third member of the ras gene superfamily identified in P. polycephalum so far.

Keywords: rap gene; ras-related gene; GTP binding protein; Geranylgeranylation; Lysine-rich domain; (P. polycephalum)

Rap genes are members of the ras gene superfamily coding for low molecular weight GTP binding proteins. They are highly conserved and have been identified in diverse organisms ranging from protoctists and fungi to humans [ 1,2]. Rap proteins share approx. 50% amino acid identity with protein products of true ras genes. The major conserved region of these proteins is the domain impli- cated in the binding of guanine nucleotides; however, rap proteins possess a threonine-for-glutamine substitution in this domain (at the 61st residue within a highly conserved DTAGQE sequence found in the human H-rasl protein, a model protein of the ras superfamily) [ 1,2]. Another highly conserved region is a putative effector domain (YDPTIEDSY sequence found at positions 32-40 of hu- man H-rasl) [1,2]. The strict conservation of the effector domain suggests that members of both families may inter-

* Corresponding author. Fax: + 48 22 232046; e-mail: koziol@asp.bio- geo.uw.edu.pl.

1 The nucleotide sequence reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number U15594.

0167-4781/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSD1 0167-4781 ( 9 5 ) 0 0 2 0 7 - 3

act with the same effector molecule to regulate common cellular processes, e.g., transduction of signals controlling both proliferation and differentiation of eukaryotic cells [1,2]. The ability of human raplA to cause reversion of the transformed phenotype of v-K-ras transformed NIH 3T3 cells and the ability of the human raplB protein to block the H-rasl(Vall2)-induced germinal vesicle breakdown in Xenopus laevis oocytes led to the hypothesis that in some cells rap proteins are capable of antagonizing the function of activated ras, possibly by competing for factors such as ras GTPase activating protein and serine/threonine kinase c-Rafl [1-3].

The cellular slime mold Dictyostelium discoideum has at least six members of the ras superfamily, one rap gene (Ddrapl) and five ras genes (DdrasB, DdrasC, DdrasD, DdrasG and DdrasS). Each of these genes displays a distinct pattern of expression during development [4-8]. The Ddrapl protein is proposed to be involved in the regulation of cell morphology [9]. We believe that, by analogy with D. discoideum, rap and ras proteins may be involved in differentiation programmes of another slime mold, Physarum polycephalum [10]. In previous communi- cations two ras genes, Pprasl and Ppras2, were identi-

30 P. Kozlowski et al. / Biochimica et Biophysica Acta 1305 (1996) 29-33

fled in P. polycephalum [11,12]. Here we describe the cloning and sequencing of the rap cDNA from P. poly- cephalum designated as the Pprapl gene, analyse the structure and putative posttranslational modifications of the Pprapl protein, and compare it to rap proteins from other organisms and to the P. polycephalum ras proteins.

The P. polycephalum eDNA library ML6A containing approx, one million of independent clones, constructed in the Agtl0 vector from poly(A) + RNA from a mixture of amoebae and flagellates using Amersham cDNA synthesis system (for details see [13]), was kindly provided by Dr. T. Burland. The library was divided into 32 sublibraries, each containing approx. 30000 sequences. From each of the sublibraries a DNA miniprep was digested with EcoRI, Southern blotted and screened by low-stringency hy- bridization using as a probe the 760 bp EcoRI-BgIII restriction fragment of the human raplA gene [14]. One sublibrary exhibited significant hybridization with the raplA probe giving a band of approx. 800 bp. In order to

isolate the positive clone three rounds of screening by plaque hybridization were performed. The cDNA insert of approx. 800 bp excised with EcoRI was subcloned into pUC19 resulting in the p0.8 plasmid and was sequenced with dideoxynucleotide Sanger method [15] (Sequenase Version 2.0 kit, USB). In order to sequence the cDNA insert of the p0.8 plasmid completely multiple primers were used. The nucleotide sequence (813 bp) of this insert contained an open reading frame (ORF) coding for 184 amino acids truncated at the N-terminus and a 3' untrans- lated sequence. The ORF began with an EcoRI restriction site which was the putative internal EcoRI site at the 5' end of the protein-coding sequence of the original cDNA. In order to recover the 5' end of the original cDNA, which was probably lost during bank preparation, a 5'-RACE reaction was performed with total RNA from amoebae employing primer PI (5'-TTCCAACATGCATTGTTGTC- 3') for cDNA synthesis and primer PII (5'-CAAAGATAC- CCTGTACGAAT-3') for nested PCR [16,17]. The 5'-

TTACGAATGAGGAGATTCGAGCTCGCACGAGCTCGCGAA

40 CAGTCGAGATATGGTT~GGACAAATGGATTCG~TGAATTGATTGGATTGTCGGAGAACCA~GGGATCCTCCTGCGCCCAGCCAAACAAACCATCTT

139 •CCGTTTCT•CCTGCTGCTTGCTGGAGCTCTGCTTTTCGTATTGACAACGCTCAGAGCTTGAAGCCTTGTAATTTACTGTTGCCTACTATTATTCTATA

238 1

ATG CCT CTT AGA GAA TTC AAG ATT GTG GTG TTG GGG TCT GGA GGT GTT GGC AAG AGT GCG TTA ACC GTA CAA TTC Met Pro Leu Arg Gtu Phe Lys I re Val Va[ Leu GLy Ser GLy GLy Val Gty Lys Sen Ala Leu Thr Vat Gln Phe

313 GTA CAG GGT ATC TTT GTT GAG AAG TAT GAC CCA ACC ATC GAA GAT AGC TAC 26 Vat Gln GLy I l e Phe Val Glu Lys Tyr Asp Pro Thr I l e Glu Asp Ser Tyr

. . . . . . . . . . . . . . . . . . . . . PI I pr imer

388 CAA CAA TGC ATG TTG GAA ATT TTG CAT ACT GCT GGA ACG GAA CAG TTT ACC 51 Gin Gln Cys Met Leu Gtu I r e Leu Asp Thr Ata GLy Thr Gtu Gin Phe Thr

. . . . . . . . . . . . . . . . . . . . . . . PI pr imer

463 AAT GGT CAA GGT TTT GTT CTT GTG TAC AGT ATT ATT GCA ATG TCG ACT TTC 76 Asn GLy Gtn Gty Phe Vat Leu Vat Tyr Set I r e I re Ala Met Ser Thr Phe

5]8 CAA ATT CTG AGA GTG /tAG CAT TGC CAT GAT GTC CCC ATG GTG TTG GTG GGC 101 Gln [ l e Leu Arg Val Lys Asp Cys Asp Asp Vat Pro Met Vat Leu Vat Gty

613 AGA GTT ATC TCG ACG GAA CAA GGG GAC GAG CTC GCC CGC AAG TTT GGA GGG 126 Arg Vat l l e Ser Thr Gtu Gtn GIy Asp Gtu Leu Ata APg Lys Phe GLy GLy

688 AAG AAC AAG ATC AAT GTG GAG CAA ATC TTC TAT GAC CTC ATC CGC CAA ATC 151 Lys Asn Lys I l e Asn Val Glu Gln I l e Phe Tyr Asp Leu I l e Arg Gln l l e

763 AAC AAG AAA GAG AAG AAA AGT GGC GGC TGC ATC TTG CTG TAG 176 ASh LYs Lys Glu Lys Lys Ser Gly GLy Cys Ile Leu Leu ter

848

CGC AAG CAG GTA GAG GTT GAC GGA Arg Lys Gin Vat Gtu Vat Asp GLy

4--

GCC ATG AGA GAC CTC TAC ATG AAG Ata Met Arg Asp Leu Tyr Met Lys

AAT GAC CTC CCC GAT TTG AGG GAG Asn Asp Leu Pro Asp Leu Arg Gtu

/~T /~G TGT GAC TTG GCC GAA CAG Asn Lys Cys Asp Leu ALa Gtu Gtn

TGT GCA TTC CTT GAG GCC TCC GCC Cys Ala Phe Leu Gtu Ata Ser Ata

AAC CGCAAG AAC CCG GGA CCA ACA As~nAr9 Lys ASh Pro Gly Pro Thr

AAGAACCCAGCTCCCCCCAGCATTCCCTGGAGCTTTCTCTATT

TGCATTGCGGAATAA•CCCCTCCCCTCCCTCAAATTGTATTGATAGTCCACTGCGATCCCCTCCTACCTTATGTCCAACTTACAGGGTAATATTCTTCT

947 TCATT•ATTGTTGTAAAGGTTA•ATGTAAATAGTGACATCTTATGGTTCAAGGCGACCGACGTGTTGTCTTGACTGTTGTGCATAGCAAAAATATATAT

1046 AAATTTCCAGGAATCCC

Fig. 1. The assembled nucleotide sequence and the predicted amino acid sequence of the Pprapl cDNA. The positions of the PI and PII primers employed in the 5'-RACE reaction are indicated. The EcoRI restriction site and the putative polyadenylation signal are underlined. The termination codon is denoted as ter. The threonine-for-glutamine substitution, the glycine and asparagine insertions, the lysine residues of the putative lysine-rich domain, the potentially phosphorylated serine residue, and the cysteine and leucine residues of the CAAX motif are indicated by double underlining.

P. Kozlowski et al. / Biochimica et Biophysica Acta 1305 (1996) 29-33 31

RACE product contained a fragment exactly matching 79 bp of the 5' end of the truncated ORF and, in addition, 249 bp upstream of this fragment comprising a leader and the missing part of the ORF.

The assembled nucleotide sequence (1062 bp) and the deduced amino acid sequence of the studied cDNA are presented in Fig. 1. The nucleotide sequence contains a leader of 237 bp, an ORF of 567 bp coding for a protein of 188 amino acids of a calculated M r of 21 035 and ending with a TAG codon, and 258 bp in a 3' untranslated sequence. It is likely that the 3' region of this cDNA does not contain the complete 3' untranslated sequence of the full-length mRNA since neither a poly(A) tail nor a con- sensus AATAAA motif for transcript termination and polyadenylation are present within the sequenced cDNA. However, there is an adenine- and thymine-rich region of 12 bp at the 3' end of the cDNA which perhaps could serve as a substitute for an AATAAA motif. The nu- cleotide sequence of the ORF was used to search the NCB] Library using the BLAST program [18]. The most signifi- cant similarity detected was with members of the rap gene family. This ORF exhibits three features characteristic of ras/rap proteins: (i) a highly conserved GTP binding domain (amino acids 4 through 171) containing the puta-

tive effector domain YDPTIEDSY (amino acids 34 through 42), (ii) a hypervariable domain (amino acids 172 through 184), and (iii) the CAAX motif (where C is cysteine, A is an aliphatic amino acid and X is any amino acid) at the COOH terminus (Figs. 1 and 2) [1]. The GTP binding domain possesses the threonine-for-glutamine substitution at the 63rd residue of the ORF (corresponding to the 61st residue of the human H-rasl protein) characteristic of rap proteins and two amino acid insertions, glycine at the 142nd and asparagine at the 168th residue [1]. Based on all these features we conclude that the isolated cDNA codes for a true Physarum rap protein and designate it as Pprapl.

Both the cysteine residue of the CAAX motif and the cysteine residue(s) of the hypervariable domain of mem- bers of the ras superfamily, including rap proteins, undergo posttranslational modifications (prenylation and palmitoy- lation, respectively) which are necessary as two signals for membrane targeting of these proteins [1]. The terminal amino acid (X) of the CAAX motif determines whether the cysteine is modified by a farnesyl group (X = Ser, Cys, Met or Gin) or by a geranylgeranyl group (X = Leu) [1]. The Pprapl CAAX motif terminates with a leucine and, therefore, we suppose that its cysteine residue undergoes geranylgeranylation rather than farnesylation (the first

GDP/GTP binding domain . 1 0 .20 .30 .40 . 50 . 60 . 7 0 .80

P p r a p l NPLREFK[VVLGSGGVGKSALTVQFVQG! FVEKYDPT]EDSYRKQVEVDGQQCNLE[LDTAGTEQFTANRDLYHKNGQGFVLVYS

Ddrap l NPL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

R a p 1 A N . . Y . L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . k . . . .

R S R 1 N . D Y . L . . . . A . . . . . . C . . . . . . . . VYLDT . . . . . . . . . . . X l . I .NKVFD . . . . . . . . IA . . . . . . E . . I . S . H . . L . . . .

P p r a s l N T . Y . L . I V . G . . . . . . . . . I . L I . N H . I D E . . . . . . . . . . . . . T I . E E T . L . D . . . . . . Q.EYS . . . . Q . .RT . . . . L C . . .

P p r a s 2 N A Q L . Y . L . I V . G . . . . . . . . . I . L I . N H . I D E . . . . . . . . . . . . . V I . E E T . L . D . . . . . . Q.EYS . . . . Q . .RT . . . . . H . . ,

GDP/GTP b i n d i n g domain . 90 . 100 .110 . 120 .130 . 140 . 150 . 160 . 170

P p r a p ] ] [ANST FNDLPOLREQ l LRVI(DCDDVPt4VLVGNKCD LAEQRVI STEQGDELARKFGGCAFLEASAKNK ] NVEQ ] FYDL [ RQ ]NRKN

] ~ r a p l . .SN . . . . E . . . . . . . . . . . . . . E . . . . . . . . . . . . . HD . . . . . . . . . E . . . . . . . D . Y . . . . . . . . . V . . . . . . . N . . . . . . . . .

R a p l A . T A Q . . . . . . Q . . . . . . . . . . . r E . . . . I . . . . . . . . EDE. .VGK. . .QN. . . ( I ICCN . . . . . S . . . S . . . . NE . . . . . V . . . . . . T

R S R 1 VTDRQSLEE.NE . . . . V . . I . . S . R . . . . . I . . . A . . INE . . . . V . E . l .VSS.W.RVP.Y . T . . L L R S . . D E V . V . . V . . . l .NE

P p r a s l .TSR.S .DE IASF . . . . . . . . . K . K . . . IV . . . . . . . E G E . Q V T . G E . Q D . . . S . . - . P . H . T . . . S R V . . . E S . . Q . V . E . - . . D

Ppl"'ilS2 .TSR.S .OEINAF . . . . . . . . . K.T . . . . . A . . . . . . . SE.QVT.NE.Q . . . . A . . - . P . V . T . . . A R L . . . E C . . G . V . E . - . . E

• 18o

P p r a p l PGPTNKKEKKSGGCI LL 188 aa D d r a p I PVGPPSKAKSKCALL 186 ea

RaplA PVEKKKPKKKSCLLL 184 aa

R S R ] ( A 90 ~m) KKKKKNASTCTIL 272 aa

P p r a s l SRTDTKGPGGKGGKKTLKCLLL 189 aa 1~11"aS2 V l GDKKGGGGKKKKLNGI DRCKLL 191 aa

Fig. 2. The pairways amino acid sequence comparisons between Pprapl and rap proteins from other organisms (D. discoideum-Ddrapl, human-RaplA and S. cerevisiae-RSR1), and the ras proteins from P. polycephalum (Pprasl and Ppras2). The first 90 amino acids of the RSRI hypervariable domain are not shown. Numbering is according to the Pprapl sequence. Identical amino acids within the GTP binding domain (amino acids 4 through 171) are denoted with a dot, and gaps introduced to maximize homology are denoted with a dash; no attempt was made to align sequences outside the GTP binding domain. The putative effector domain YDPTIEDSY and the DTAGQ/TE sequence are underlined. The threonine-for-glutamine substitution, and the glycine and asparagine insertions are indicated by double underlining.

32 P. Kozlowski et al. / Biochimica et Biophysica Acta 1305 (1996) 29-33

membrane-targeting signal) [1]. The relatively short hyper- variable domain contains no additional cysteine residue required for the second modification, palmitoylation [1]. However, it contains four lysine residues (the 177th, 178th, 180th and 181st residues) forming a lysine-rich domain which might serve as the second signal [1]. Thus this third member of the ras superfamily from P. polycephalum possesses the same types of membrane localization signals as the two true ras proteins, Pprasl and Ppras2, identified so far in this organism [11,12]. Some of rap proteins undergo yet another posttranslational modification, phos- phorylation by cyclic AMP-dependent protein kinases at a serine residue adjacent to the lysine-rich domain (e.g., at serine residues within the sequences K K K P K K K S C and GKARKKSSC of human rap lA and raplB, respe~ively), which may-also regulate their membrane association [1,2]. Pprapl exhibits a similar motif, KKEKKSGGC, at its C-terminus.

The comparison of the amino acid sequence of the Pprapl protein with rap proteins from other organisms and with ras proteins from P. polycephalum is shown in Fig. 2. Pprapl exhibits within the GTP binding domain the highest amino acid homology (92%) with its relative from D. discoideum, Ddrapl [8]. It also exhibits a significantly high homology (83%) with human R a p l A [14] and a rather low homology (59%) with RSR1 (also called BUD1), a rap protein from Saccharomyces cerevisiae [19,20], which is only distantly related to all known rap proteins [1,2]. This comparison shows also that Pprapl has 59% and 61% homology with P. polycephalum Pprasl [11] and Ppras2 [12], respectively.

1 2 3 4

4 . 3 -

2 . 8 -

4 . 3 -

2 . 8 -

Fig. 3. Southern blot analysis of the Pprapl gene. DNA purified from P. polycephalum strain M3CIV (lanes 1 and 2) or MCC (lanes 3 and 4) was digested with EcoRI (lanes 1 and 3) or HindlII (lanes 2 and 4) and hybridized with the cloned Pprapl cDNA insert of the p0.8 plasmid under high-stringency conditions. Bands are indicated by their respective sizes in kbp.

Southern blot hybridization of restriction endonuclease digested genomic DNA isolated from diploid microplas- modia of two P. polycephalum strains shows unique bands hybridizing at high stringency with the cloned Pprapl cDNA insert of the p0.8 plasmid both in the HindlII digests and the EcoRI digests (Fig. 3). These results suggest the existence of a unique copy of the Pprapl gene in the P. polycephalum genome.

On the basis of Northern blots we established that, similarly to the D. dictyostelium Ddrapl gene [8], expres- sion of Pprapl is developmentally regulated - its single transcript (approx. 1050 nt) was detected in all P. poly- cephalum developmental stages examined (amoebae, plas- modia, spherules and fruiting bodies) reaching the maxi- mum level in fruiting bodies and the minimum level in plasmodia (details to be published elsewhere). This obser- vation confirms our assumption that slime molds might be interesting models to study functions and mechanisms of action of rap proteins in processes of cell growth and differentiation in lower eukaryotes.

We thank Drs. E. Paul, W. Dove and T. Burland for the ML6A library, Dr. V. Pizon for the raplA clone, R. Konopinski and P. Bieganowski for primer synthesis, NCBI for sequence analysis and Dr. J. Fronk for critical review of the manuscript and stimulating discussion. This research was supported by grants PB 2 2 1 3 / 4 / 9 1 from KBN, Bi- mol 3 7 / 9 3 from FNP and in part by BW 1203/7 and BW 1300/9 from UW.

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