The predicted amino acid sequence of a centrosphere protein in dividing

sea urchin eggs is similar to elongation factor (EF-1a)

RYOKO KURIYAMA1'*, PAUL SAVEREIDE2, PAUL LEFEBVRE2 and SANTANU DASGUPTA3

1 Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis Minnesota 55455, USA2 Department of Genetics and Cell Biology, University of Minnesota, St Paul, Minnesota 55108, USA3 laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

* Author for correspondence

Summary

Monoclonal antibody (SU5), prepared from isolatedmitotic spindles of sea urchin eggs, stained centro-spheres preferentially and recognized a 50K(K=103Mr) polypeptide on immunoblots. Threepositive clones were isolated by screening a AgtllcDNA expression library prepared from sea urchinegg mRNA with SU5. One clone containing a 1.8-kb(1 kb= 103 base-pairs) insert was selected for furthercharacterization. The /J-galactosidase fusion pro-tein encoded by the cDNA clone had an apparentrelative molecular mass of 150K, indicating that theinserted cDNA produced an estimated 34K of poly-peptide. A single 2.2-kb RNA transcript was

detected in sea urchin embryos using the cDNAclone as a probe. The cDNA fragment wassequenced and the nucleotide sequence was used topredict the amino acid sequence of the open readingframes in the clone. The putative gene productshows striking similarity to the peptide chainelongation factor (EF-la) from yeast, fungus,shrimp, insect, mouse and human.

Key words: centrosphere, sea urchin eggs, mitotic spindles,monoclonal antibodies, lambda g t l l , recombinant DNA,sequence analysis.

Introduction

Mitotic spindles formed in dividing eucaryotic cells aretransient structures responsible for equidistribution ofgenetic material into two daughter cells. In an effort toelucidate the mechanism of mitotic spindle organizationand its regulation, we have attempted to identify spindlecomponents by raising monoclonal antibodies (mAbs)against spindles isolated from dividing sea urchin eggs(Kuriyama and Borisy, 1985). One such antibody, SU5,reacted with microtubule-containing structures in the eggduring the course of development. It preferentiallystained the centrosphere, an area in mitotic poles fromwhich spindle microtubules originate. A protein of anapparent relative molecular mass of 50K (K = 103 Mr)was recognized by this antibody on immunoblots ofwhole cell lysates as well as isolated spindle fractions(Kuriyama and Borisy, 1985). To gain more informationabout this component, SU5 was further used to isolatecDNA sequences corresponding to the centrosphereprotein by screening a Agtll expression library. Here wereport the isolation and characterization of a cDNA cloneencoding a polypeptide immunoreactive to SU5. Theputative amino acid sequence shows striking similarity tothe peptide chain elongation factor (EF-la) from severalspecies.

Journal of Cell Science 95, 231-236 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

Materials and methods

Isolation ofcDNA clonesA cDNA expression library cloned in Agtll from isolatedmRNA of sea-urchin blastulae was kindly provided by Dr T.Thomas. 106 clones were screened with the monoclonal anti-centrosphere antibody, SU5 (Kuriyama and Borisy, 1985),following the method of Young and Davis (1984). A mouseperoxidase-anti-peroxidase complex (ICN Biochemical., Lisle,IL) was used to visualize immunoreactive colonies after colorreaction with 4-chloro-l-naphthol as the enzyme substrate(Sellitto and Kuriyama, 1988). Phage from plaques givingpositive signals were further purified, the recombinant insertsfrom pure phage clones were excised from purified DNA andsubcloned further into M13mpl9 vector using standardmethods (Maniatis et al. 1982).

RNA blot and immunoblot analysisTotal RNA was isolated from sea-urchin eggs at the blastulaestage using the guanidine thiocyanate procedure, and thepoly(A)+ RNA fraction was prepared using oligo(dT) columns(Maniatis et al. 1982). After fractionation on formaldehyde-containing 1.75% agarose gels, RNAs were transferred tonitrocellulose filters, and hybridized with the cloned cDNAinsert as described (Maniatis et al. 1982).

Proteins prepared from bacterial cell lysates were run on7.5% acrylamide gels, transferred to nitrocellulose filters andblots were probed with SU5 or anti-/?-galactosidase antibody

231

(Promega, Madison, WI) as previously described (Sellitto andKuriyama, 1988).

Nucleotide sequence analysis and comparisonNucleotide sequence was determined by the dideoxy chaintermination method (Sanger et al. 1977) using a librarygenerated by random insertion of Mu transposons into bacterio-phage Ml3 carrying the cloned fragments as developed byAdachi et al. (1987). Sequence analysis was carried out usingthe IntelliGenetics Software Package through the MolecularBiology Computing Facility at the University of Minnesota.

Results

Isolation and characterization of the SU5-antigencDNA clone and its fusion proteinWe have screened a Agtll cDNA expression library,prepared using RNA from sea urchin blastulae, using themAb SU5 as a probe. Three positive clones wereidentified out of 10 recombinant phage screened. One ofthese clones contained a 1.8-kb (lkb=103 base-pairs)cDNA insert and was selected for further characteriz-

A B C D E

ation. Immunoblot analysis of the fusion protein pro-duced by the clone is shown in Fig. 1. Lysates of bacteriaexpressing the fusion protein contain a 150K(K = 103Mr) polypeptide immunoreactive to both SU5(lane B) and anti-/3-galactosidase antibody (lane D).Since the molecular mass ofy3-galactosidase is 116K (laneE), the 1.8-kb cDNA insert encodes approximately 34Kof the fusion protein. Lysates of control bacterial cells donot contain any polypeptides immunoreactive to SU5 asshown in lane C. The mAb SU5 was further affinitypurified using fusion proteins bound to nitrocellulose.The affinity-purified antibody reacted with the same 50Kband (lane A) seen in fractions of whole mitotic cells andisolated spindles using the original SU5 antibody (datanot shown), indicating that the Agtll recombinant phagecontained at least part of the SU5 antigen codingsequence.

In order to characterize the RNA species correspond-ing to the SU5 antigen, total RNA and poly(A)+ RNAfrom sea urchin embryos were separated on formal-dehyde-agarose gels and transferred to nitrocellulose. Asshown in Fig. 2, a single 2.2-kb mRNA was detected byhybridization using the 32P-labeled cDNA insert as aprobe (lane B). Intense hybridization can also be seenusing total RNA (lane A).

B

15OK— —

— 200 K

— 116K

— 93K— 66K

4.2

2.0 —

50 K —

— 45 K

Fig. 1. Identification of the SUS antigen in isolated seaurchin spindles and bacterial fusion proteins by immunoblotanalysis. Proteins were run on 7.5 % acrylamide gels,transferred to nitrocellulose filters, and immunoblotted witheither SU5 (A-C) or anti-/3-galactosidase antibody (D, E).Lane A: mitotic spindles isolated from sea urchin eggs,showing a 50K band immunoreactive to SU5. Lanes B andD: proteins prepared from bacterial cell lysates expressing a150K fusion protein encoded by the 1.8-kb cDNA fragment.Lanes C and E: proteins of control bacterial cell lysates.

Fig. 2. Hybridization of radiolabeled 1.8 kb insert fragmentto a Northern blot of total RNA (lane A) and poly(A)+ RNA(lane B). Lanes A and B contain 10 fig and 2 fig of RNA,respectively.

232 R. Kuriyama et al.

200 400I

600I

800 1000 1200 1400 1600 1800

B10 19 28 37 46

a AAT TCC GGT GCT ACA CCT TGG GGT GTC AAG CAG CTC ATT GTT GCT GTC AACN S G A T P W G V K O L I V A V N

55 64 73 82 91 100AAG ATG GAC AGC GCA CAA TAC AAT GAA GCC CGT TTC AAA GAA ATC GTC AGG GAGK M D S A Q Y N E A R F K E I V R E

109 118 127 136 145 154GTC TCT GGC TAC ATC AAG AAG GTT GGC TAC AAC CCA AAG GCT GTG CCC TTC ATCV S G Y I K K V G Y N P K A V P F I

163 172 181 190 199 208CCA ATC TCA GGA TGG GTT GGT GAC AAC ATG ATG GAG GCA GCC ACA ACG ACT ATGP I S G W V G D N M M E A A T T T M

217 226 235 244 253 262CCA TGG TTC AAG GGG TGG TCA ATT GAA CGC AAG GAT AAT AAT GCC AGT GGA GTGP W F K G W S I E R K D N N A S G V

271 280 289 298 307 316ACC CTG TTG AAT GCC CTT GAT GCT ATC ATG CTC CCT AAG AGG CCA CAC GAT AAGT L L N A L D A I M L P K R P H D K

325 334 343 3S2 361 370CCT CTT CGT CTA CCA CTC CAG GAT GTC TAC AAA ATT GGA GGT ATT GGA ACT GTGP L R L P L Q D V Y K I G G I G T V

379 388 397 406 415 424CCA GTG GGC CGT GTT GAA AGT GGA ACA ATC AAG GCC GGT ATG ATC GCT AGG TTTP V G R V E S G T I K A G M I A R F

433 442 451 460 469 478GCC CCT GCC AAC TTT ACC ACT GAG GTG AAG TCT GTG GAG ATG CAC CAT GAA ACTA P A N F T T E V K S V E M H H E T

487 496 505 514 523 532TTG GAA AAG GCA TTG CCA GGT GAC AAT GTC GGC TTC AAT GTC AAG AAC GTC TCTL E K A L P G D N V G F N V K N V S

541 550 559 568 577 586ATC AAG GAT ATC AGG CGT GGT ATG GTT TGC GGA GAA TCC AAG GAC AAT CCC CCAI K D I R R G M V C G E S K D N P P

595 604 613 622 631 640ATG GCT GCC AAG TCC TTC CAG GCT CAG GTC ATT GTC ATG AAC CAC CCC GGT GAGM A A K S F Q A Q V I V M N H P G E

649 658 667 676 68S 694ATC CAT GCT GGC TAC ACC CCG GTT TTG GAT TGT CAC ACA GCT CAC ATT GCC TGCI H A G Y T P V L D C K T A H I A C

703 712 721 730 739 748AAG TTC GCA GAA CTC AAG GAG AAG CTC GAC CGT CGA TCA GGC AAG AGT CTT GAGK F A E L K E K L D R R S G K S L E

757 766 775 784 793 802GAT AAC CCC AAG TTT GTG AAG AGT GGT GAT GCC GTC ATT GCC AAA CTG ATT CCCD N P K F V K S G D A V I A K L I P

811 820 829 838 847 856TCC AAG CAG ATG GTG GTT GAG AGT TAC ACA GAT TTT GCT CCT CTT GGA CGT TTTS K Q M V V E S Y T D F A P L G R F

865 874 883 892 901 910GCT GTC CGT GAC ATG AGG CAG ACC GTA GCC GTC GGT GTG GTG AAG TCT GTC GAAA V R D M R Q T V A V G V V K S V E

919 928 937 946 955 964AAG GAT GAA GCA TCT ACA GGC AAG GTT ACC AAG TCT GCC CAG AAG GCT GGC AAGK D E A S T G K V T K S A Q K A G K

973 982 991 1000 1010 1020 1030AAG AAA TGA TGG CGA TGG CAT TCT GCA TCC CAAGGCTACA ACATGTACCC ACTCCCCTTCK K

1040 1050 1060 1070 10B0 1090 1100ACTAATGAGT TCAAAGGAAT CACTGCAAGC CTTCGTTTAC CTATGCTTCT TGAAAAAATG AAAACCCAAA

1110 1120 1130 1140 1150 1160 1170GAATATCACA CTAATTCTTC CTCCGCAGAT AGCAGAGGAA GATAGTGTTG CTATTATATC GGCAGCATGT

1180 1190 1200 1210 1220 1230 1240TTTTATCAAG TCTATTCTAA ACAAGACTGT TACAGCAATG TGCTGAGAGT AAAACAAAAA TAAAAGAAAC

1250 1260 1270 1280 1290 1300 1310GCTTAATATT GCTCTAAATA TGTACCACCC ATGAAAGACG AATGGGGTTA TAAGAAGTGA CATTTCTTTG

1320 1330 1340 1350 1360 1370 1380AAATTTTAAA ATAAGATATG TCTGTATCAA GGTATAATCA AAAAGCTTAT CACACACTGT GTCTGCACCA

1390 1400 1410 1420 1430 1440 1450AGTTTTATTT AACCCTGAAA GCAGGGGTTG TTTGACATAC AGCACTGGGA GCTCACCCAT GATCTCGATT

1460 1470 1480 1490 1500 1510 1520TAGTTAACAC AAAAAAAAGA AAAGAGAAGT CATCTTTTTT CAACACGGTA AACATGCAGG CAAAATGTGG

1530 1540 1550 1560 1570 1560 1590TCCCTCATGG GTTAATCATT CATCAGATAT GTTTTTACAT CTGTGTTGCA TGTAAGCAAA TTGATTTTAG

1600 1610 1620 1630 1640 1650 1660AAATCCTTAG TTCAAGTGGC AATTGCAACT AAATGTGCTC AAGTGAACTA CTTGTATGGA CAGAGCTGGT

1670 1680 1690 1700 1710 1720 1730CCTTAATTTT GGAAAATGTA ATTTCTTGGT GACTTAACAA TTTATTTGTA TGTAAATGGA AACAAGTGTA

1740 1750 1760 1770 1780TGCCTTGGAA ATAAATATTA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAA

Fig. 3. DNA sequence of the 1.8-kb cDNA insert.The sequencing strategy is shown in A. The arrowpointing to the left represents the sequence obtainedusing an oligonucleotide primer (NorthernBiosciences, Minneapolis, MN) predicted from thesequence of the first strand. B. The sequence isshown in the S'-to-3' direction of the niRNA strand.Bold-type letters indicate the putative polyadenylationsignal. The predicted amino acid sequence is shownon the second line.

Cloning of centrosphere protein 233

Sequence of the 1.8-kb cDNAThe cDNA fragment was sequenced using a sequencestrategy as summarized in Fig. 3A, and the resultingnucleotide sequence of the 1.8-kb clone is shown inFig. 3B. A 35-nucleotide poly(A)+ stretch is found at the3' end of the clone, probably representing a portion of thepoly(A)+ tail of the mRNA. A potential polyadenylationsignal (AAATAAA) appears 11 nucleotides upstreamfrom the first A of the polyadenosine tract (bold typeletters). One open reading frame extends from nucleotide1 to nucleotide 978; the other two possible reading framesare interrupted by stop codons. The insert is thuscomposed of the coding region of 978 translated nucleo-tides, and 806 untranslated nucleotides at the 3' end. Thecorresponding amino acid sequence is shown below thenucleotide sequence in Fig. 3B. Since the coding se-

quence in the 1.8-kb fragment accounts for only 34K ofthe 50K polypeptide recognized by the SU5 antibody,and since the 3 -end is included in the cloned fragment, itis estimated that an additional 400 base-pairs must beadded to the 5' end of this clone to encode the completesequence. This is supported by the observation of a 2.2-kb mRNA on Northern blots (Fig. 2). It is possible that,during cloning of the cDNA, theiscoRI site in the middleof the complete message was not methylated, resulting inloss of the 5' end of the message (T. Thomas, personalcommunication).

Sequence similarity to genes coiresponding to theelongation factor I aA computer search of the NIH-Genbank and the EMBLDNA sequence database was performed to find DNA se-

S. purpuratusDrosophilaArtemiaHumanMouseMucorS. cerevisiae

S. purpuratusDrosophilaArtemiaHumanMouseMucorS. cerevisiae

S. purpuratusDrosophilaArtemiaHumanMouseMucorS. cerevisiae

S. purpuratusDrosophilaArtemiaHumanMouseMucorS. cerevisiae

S. purpuratusDrosophilaArtemiaHumanMouseMucorS. cerevisiae

******WGVKQLIVAWKMDS**AQYNEARFKEIVREVSGYIKKVGYNPKAVPFIPISGWVGDNMMEAALLAFTL

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ATTTMPWFKGWSIERKDNNASGVTLLNALDAIMLPKRPHDKPLRLPLQDVYKIGGIGTVPVGRVESGTPS N

SDRL YPS ANPS ANES N

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EG D K VDEGK D K D

G T EGS VAP E

AGSKT K EAGWK K E

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V

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IKAGMIARFAPANFTTEVKSVEMHHETLEKALPGDNVGFNVKNVSIKDIRRGMVCGESKDNPPMAAKSL P

PL PL P

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W TW TW NW T

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Fig. 4. Comparison of the predicted amino acid sequence of the SU5-antigen clone (S. pwpuratus) with predicted amino acidsequence of the female factor from Drosophila (Walldorf et al. 1985) (lane 2), and with the predicted amino acid sequences ofEF-lcv from Drosophila (Hovemann et al. 1988) (lane 2), Artemina salina (brine shrimp) (van Hemert et al. 1984) (lane 3),Homo sapiens (Brands et al. 1986) (lane 4), Mus musculus (mouse) (Lu and Werner, 1989) (lane 5), Mucor racemosus (mycelialfungus) (Linz et al. 1986) (lane 6), and Saccharomyces cerevisiae (Schirmaier & Philippsen, 1984; Nagashima et al. 1986) (lane7). Asterisks mark locations where deletions were used to align the amino acid sequences. The S. putpuratus sequence is on thetop line, and amino acids are noted on the lower lines only at positions that differ from the S. putpuratus sequence.

234 R. Kuriyama et al.

quences with similarity to the SU5-antigen clone. Oursearch recovered sequences coding for translationalelongation factor lor (EF-1 a) from six eucaryotic species:Homo sapiens (Brands et al. 1986), Mus musculus(mouse) (Lu and Werner, 1989), Drosophila melanogas-ter (Hovemann et al. 1988), Artemina salina (brineshrimp) (van Hemert et al. 1984), Mucor racemosus(mycelial fungus) (Linz et al. 1986), and Saccharomycescerevisiae (Schirmaier and Philippsen, 1984; Nagashimaet al. 1986) (Fig. 4). Similarity was also observed to asequence coding for a 'female-specific' factor in Dros-ophila melanogaster (Walldorf et al. 1985). This factorwas later identified as cytoplasmic elongation factor-lainDrosophila (Hovemann et al. 1988; Henikoff and Wal-lace, 1988). The predicted amino acid sequences from thehuman EF-1 a clone was identical to the Stronglyocentro-tus purpuratus cDNA clone at 231 of 321 positions. TheEF-lcv sequences of the other four showed comparablesimilarity to the SUS-antigen sequence (223/321 forArtemina, 228/321 for mouse, 227/321 for Mucor, and226/321 for Saccharomyces). In the same region, theamino acid sequences of the SU5 antigen and theDrosophila factor were identical at 228 of 321 positions.

Discussion

The homology between the predicted amino acid se-quence of the SU5 antigen and EF-1 a from other speciesis surprising because the SU5 antigen specifically immu-nolocalized at the centrosphere in sea urchin eggs (Kur-iyama and Borisy, 1985). One possibility is that the 50Kprotein immunoreactive to SU5 is indeed EF-1 a, whichhappens to be located at the centrosphere in sea urchineggs, because of the numerous ribosomal particles foundin association with spindle microtubules (Rustad, 1959;Zimmerman, 1960; Harris, 1975; Tempero and Supre-nant, 1988). Sea urchin eggs are filled with yolk granulesand the mitotic spindle is a region free of those large yolkgranules. Major soluble cytoplasmic components, such asEF-lo- (Lenstra and Bloemendal, 1983; Slobin, 1980;also see Fig. 2), would, therefore, tend to be localizedaround yolk-free regions, like mitotic spindles. Suchpassive accumulation around the spindle region has beendemonstrated from the accumulation of microinjectedfluorescently tagged bovine serum albumin around mi-totic spindles in cleaving sea urchin eggs (Wang andTaylor, 1979).

SU5 recognizes the centrosphere not only in wholemitotic cells, but also in isolated forms. It is thus highlylikely that the 50K protein recognized by the SU5antibody is an intrinsic centrosomal component and thesimilarity of the SU5 antigen to EF-lci'would be morethan accidental. Apparently unrelated proteins can, anddo, share amino acid homologies as has recently beenshown from the amino acid sequence identity betweenvarious crystallins, highly specialized structural proteinsfrom lens of animal eyes, and common cellular enzymessuch as lactate dehydrogenase and alpha enolase (Wistowand Piatigorsky, 1988). The presence of a single polypep-tide with multiple active sites has also recently been

reported in the cases of a mannose 6-phosphate receptorand an insulin-like growth factor II receptor (Tong et al.1988), the neurotrophic factor neuroleukin and phospho-hexose isomerase (Chaput et al. 1988), and oligosaccharyltransferase and three other luminal endoplasmic reticu-lum proteins (protein disulfide isomerase, the /3-subunitof prolyl hydroxylase and thyroid hormone bindingprotein) (Geetha-Habib et al. 1988). Those observationsgave rise to the concept of gene sharing, where a genemay acquire and maintain more than one functionwithout loss of its original role (Piatigorsky and Wistow,1989).

There is also another possible explanation for thesequence homology of the SUS-antigen with EF-lo". Maoand Wang (1988) have shown that statin, a protein foundonly in nonproliferating cells, is 80 % homologous tohuman elongation factor la 'a t the nucleotide sequencelevel. A gene product of ts mutant gstl in Saccharo-myces, which is defective at the Gpto-S transition, is alsoshown to contain consensus sequences for a target site ofcyclic AMP-dependent protein kinase(s) and for GTPasewith extensive homology to polypeptide chain elongationfactor EF-lo- (Kikuchi et al. 1988). It is thus possible tospeculate that EF-lcv represents a multi-gene family inwhich the 50K centrosphere-specific SU5 antigen isincluded.

We thank Dr K. Mizuuchi (Laboratory of Molecular Biology,NIDDKD, NIH) and all the members of his laboratory fortheir kind help and support in analysis of nucleotide sequences;Ms T. Brandt (University of Minnesota) for technical assist-ance; Dr T. Thomas (Texas A & M University) for the gift ofthe cDNA library of sea urchin embryos. One of us (R.K.)thanks Drs F. Gonzalez and S. Kimura (NCI, NIH) forinitiating the work and providing the technique of screeninglambda gt l l libraries. This work was supported by NSF grantDCB8S101S1 and NIH grant GM413S0 to R.K. and by a grantfrom the University of Minnesota Graduate School to P.L.

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(Received 18 August 1989 -Accepted 18 October 1989)

236 R. Kuriyama et al.