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~Si 1991 Ieetlerationof [~urop(an Biod~¢mlea| SO~NIIeSt10141~79~!19|:$21,~0 A DONI,~ 0014~'/9~19ItiO,~¢aM

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subunit of the chloroplast ATPase is plastid-encoded in the diatom Odontella sinens s Peter G.. Pantie t. Heinrich S t r o t m a n n z a n d K l a u s V. Kowallik:

zh)stina fi'& Biorhcml¢ dcr Pflan:en and ahrsttmt fiTr Bomnik der Hcim'lt'h.Jt¢inc.Univ~rsitiit Dfisaghtorf. Universitiltx.~tr~tlt¢ I, D 4000 Diissehlorf, Germm)y

Received 14 January 1991 A 5,2 kb P.~tl re~trietion trallment eontainlnll the atpA tt©necluster or' the plastic I~nom© of th~ centrl~: diatom Od.nrella ,~/.¢))sts wa~ cloned, $¢qu~acin~ revealed a readin$ frame o1" 561 hp separatin~ the ~cne~atpF at~d I~,tpA,which is preceded by a putative ribosome bindin8 site, The third nueleotide or the codon for the last aminn acid of atpF is the first nucleotid¢ of the initiation codon of the 561 bp reading I'ram¢, The amino acid sequcnc~ deduced from the nueleotlde ~¢qu~nce of this Ilene (atpD) is ¢olinear with t~subunits of different l~,F).ATPases and shows an overall ~equcnce homolol~y of up to .~5~ when compared with the sequenexs ofcyanobacteria and C,vanophor~ parmloxo. Tl~e results are (li~usscd in context with the evolution of chloroplasts of tl~echlorophyll.a + b and -a + c lineages, respeetlvely, CF, CF~,ATPa~e: 6 S~lbunit: Nucleotide sequence; Amino acid sequence; Chloroplast i~enome: Diatom; Odantella ~#wnsis

1. INTRODUCTION Fo-Frtype ATPases of the plasma membrane o f eubacteria [1], the inner membrane of mitochondria [2], and the thylakoid membrane of cyanobacteria and chloroplasts [3] couple transmembrane proton translocation with the reversible formation of ATP from ADP and phosphate, Structure, subunit stoichiometry and composition as well as amino acid sequences of the subunits are quite similar in Fo-FI-ATPases from different sources [4], The multimeric complex is subdivided into the peripheral F~ part, containing the catalytic centers for ATP formation [5] and the transmembrane sector Fo which acts as a proton channel. F~ is composed of 5 subunits (u, ~3, 3', 8 and ~), Fo consists of 3 subunits in eubacteria, 4 subunits in chloroplasts, and at least 6 subunits in mitochondria [4,61. In E. colt the genes for all subunits are tightly linked and constitute a single transcriptional unit [7]. In cyanobacteria they are arranged in two clusters, the atpB gene cluster containing the genes for subunits/3 (atpB) and e (atpE) and the atpA gene cluster including all Fo genes together with atpA and atpD, coding for subunits ~ and 8, respectively. The gene for subunit 3' (atpC) may be attached to the atpA gene cluster or isolated, dei~ending on the organism [8-10]. For eukaryotic organellar FoF1-ATPases a dual genetic origin has been found. In chloroplasts of higher Correspondence address: P.G, Pancic, Institut ffir Biochemie der Pflanzen der Heinrich-Heine-Universitfit Dfisseldorf, Universit~tsstraBe 1, D 4000 Dfisseldorf, Germany

Published by Elsevier Science Publishers B, V.

plants the subtinits % 8 and II are transcribed from nuclear genes and translated on cytoplasmatic ribosomes. Subsequently, the products are imported into the plasmid compartment [11]. T h e genes for these three subunits are also missing in the chloroplast genomes of the chlorophyll-a+b-containing algae Euglena gracilis [12], Chlamydomonas reinhardtii [ 13] and Chlamydomonas moewusii [14], However, in Chlamydomonas the linear array of the chloroplast ATPose genes is scrambled due to extensive intramolecular rearrangements. Little is known about the organization of ATPase genes in other algal lineages. Among those the Chromophyta deserve particular interest because of the origin of their chloroplasts. In contrast to the Chlorophyta, chromophytic plastids are considered to have evolved from eukaryotic rather than prokaryotic cells [15,16]. As the nucleus of the putative eukaryotic endosymbiont has completely disappeared in most chromophytes, a secondary (eukaryotic/eukaryotic) transfer of genes must have occurred, including those that have previously been transferred from the genome of the evolving chloroplast to the nucleus of the first host cell. The extensively rearranged plastid genomes of chromophytic algae may result from these secondary endocytoses [17]. In this context we were particularly interested in the location of the coding sites for subunits % 8 and II of chromophytic chloroplast ATPases. Here we report on a plastid gene of the diatom Odontella sinensis, which shows sequence similarity to atpD coding for subunit 8. As in eubaeteria including cyanobacteria this gene is located in the atpA gene cluster and flanked by the genes atpF and atpA.

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Volume 280, number 2 2, M A T E R I A L S

AND

Mur¢l~ 1991

L2. CVo.~nX . n d sequen¢In~ q[ lh¢ Odot~l¢llu arpD.xe.¢ Southern hybridi~Hon e,~periments regaled lllal an at pA gear probe from spinach chloroplasts cross-reacted with a :L2 kb P~tl restriction frallmem (P$). This rra~me.t was de~lro-eh.ed from allaros¢ i~elsand~ionrd into pUC.lil vc~(or 0sinl E, ¢oli s;ra4n DI-IJ¢~, as I~ost. The clones were screened by Psd disc=lion. ~nd the insert checked b~ $onthern hybriditation usinlt the spinach atpA Ilene Its ~4 probe, The resuhinll clone (pOsP~,}wa~ digested wi|h p#ti~Kbai and

METHODS

2. I. /~olatlon of pie=rid DNA Plaslid DNA was isolated from purified chloroplasts el' Otlo.tWla s/ne.s/s cultivated n...described [18l, The cells were harvested by fihra. zion |hrough a .~0/~m ilauxe, wasl~edseveral times widt isotonic NaCI solution (3,$e/~,w/v) and sabsequcndy disrupted in ice.cold isol~ttlon medium (0.4 M mannttol; SO mM Tris.HCl pH ~l.0; 30 mM Na=EDTA, 4 mM MgCI=; 0.1~,~ BSA; t m M mercaploethanol) uslnil a glass potter homoBenizer, The homo~enat¢ was filtered throu~,h ~ 20 ;=m ilauz~and the filtrate centrifuged rot 3 mln az !~00 x ~f in order to remove cell debris, The supernntant which essentially cofllain©d morpholo~ically intact plasdds was titen centrifuged for 10 rain at 3500 x ~. The pelleted plas(ids were wasited in modified isolation medium lackinli BSA and DTT and centrifuged agitin, The plastids were transferred into lysis burrer (~0 mM Tris.HCI, pH 8.3; 100raM Nat EDTA; ~0 mM NaCI; 0,$% SD$; 0,"/e/0 lauroyl saree=inure; I mg/ml Proteinase K), and tl~e lysate was phcnolized according to standard protocols, The D N A was precipitated by ~thanol, dried, re. dissolved in Tris.EDTA buffer (i0 m M / l raM, pH "/,5) and mtrificd in a CsCl/ethidium bromide density gradient using a vertical rotor at 70 000 rpm for .~ h. The DNA was stored in Tris.EDTA buffer Itt a final concentration of O.S 1,8/~d,

yielded t~o subfralmen|s or 2.4 and 2.8 kb, respectivei~.The 2,4 kb

frajment (pOsPgX2} contained most or tl~e atpa ~Jene,Nested delc. lions or this fra$mem were performed using the exonuclease

III/munll bean nuclense enzymes from Boehrinjer, Klenow fill.in reactions prior tO relijalion enhanced tlt,¢ number of deleted clones, The clones were sequenced accordlnit to the dideoxy chain termi.~. lion method [igJ using the Pharmacia T,~sequenci.= kit, For DNA sequenceanalysis and translation we used the computer program 'DM' [20}, Multiple aliimmems were carried ou! usinll the program 'CIo~I=I' [2i]. The pro=ram 'SOAP' [22]served to calculate hydrophohichy plots.

3, R E S U L T S Sequence analysis of the fragment pOsPSX2 revealed 1452 nucleofides with a strikingsimilarityto atpA genes

ATTATCCTATTAOTTTTAAAACGTACAGTAGCTCGC G CTCAGCAAACTTTTGGTC CAAAAGAAAG I I L L V h K R T V A R A Q (~ T F G P K E R AGCAACAGCATTAATTACTGAAACAATTAATAAATTAGAACLQAGATTTGTTATGRGTATAAATCC A T A L I T E T Z N K L E---U- D L L Te~ a C p F aepD --> M S z N

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AAAAATAATACAATGATAAATATTCGTCCAGACGAAATTAGTAGTATTATCCGTGAACAAATTGA atpA --> M I N I R P D E I S s I I R E Q

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ACAATATGATCAAGATGTTAAAGTAGATAATATTGGTACTGTATTACAAGTAGGTGATGGTATTO Q Y D Q D V K V D N ~ G T V L Q V G D G I

Fig, 1, Nucleotide sequence and deduced amino acid sequence of the gene atpD of the plastid genome of Odontella sinensis together with the upstream (atpF) and downstream (atpA) sequences, Numbers above the atpD sequence start from t . e presumptive initiation codon. A putative ribosome binding site is underlined, start and termination codons are in italics,

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Fig. 3. Sequence similarity matrix of c5subtinits from different sources, In addition to tile sequences listed in Fig. 2 the ~5subunits of Rhodospirillum rubrum [30], PS3 [31], Bacillus megaterium [23] and of mitochondria o f sweet potatoe [32] are included, Similarity is given in percentage identical amino acids.

389

FEBS I..I~TTERS

Volume 280, number 2

from different sources (data not sllown). About 70-8007o of the deduced amino acid resldu¢s are identical with those from c~ subunits o f land plant chloroplasts and cyanobaeteria, 5' to the atpA gene, anti separated by 47 bp, is a reading frame of 561 bp with a coding capacity for 187 amino acids. The start eodon 5' ATG3' of this reading frame overlaps with the codon for the last amino acid, and the termination codon 5 ' T G A 3 ' of a Bene that was identified as atpF coding for CFe subunit I, A G + A,rich sequence presumably containin~ a ribosome binding site was found around 10 bp upstream of the start codon o f the 56] bp reading frame. Fig. 1 shows the nucleotide sequence o f this reading frame together with the adjacent 5'atpA and 3 ' a t p F sequences and the deduced amino acid sequence. The molecular mass of the 561 bp gene product was calculated as 21.1 kDa, Comparison of the amino acid sequence with known sequences of ATPase polypeptides revealed that the gene product resembles subunit ~ from different sources (Fig. 2). The sequence of the Odontella gene product is co-linear with other prokaryotic sequences and does not contain the three amino acid insertion near the Cterminus of the pinach protein, :lo., 1

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390

March 1991

Although the overall sequence homology nmong these proteins is relatively poor (Fig, 3). The N- and Cterminal regions exhibit some degree of sequence con. servation. Seven out of nine amino acid residues that are identical in oliver 6 subunits are also present in the deduced sequence of Odontella. Sequenc¢ conservation is increased when only the polypeptldes of ortanisms per formingoxygenic photosynthesis are compared. The overall sequence homology is highest between tile gone product of Odottr¢ll~ and the 8 subunits of cyanobacteria and Cyanophom parado.x'a (Fig. 3), The hydropathy plots of the Odontella gone product and of ¢Ssubunits from spinach chloroplasts [11] and the blue-green alga Amzbaena PCC "/I20 [9] are compared in Fig. 4. They indicate that the Odontella protein - - as the c5subunits of other ATPases ~ most likely is not a membrane-anchored protein, This view is confirmed by calculations of secondary structures using published computer programs (data not shown), Nevertheless, several hydrophobic regions can be discerned which a r c located in similar positions of the three protein se, q uences. 4, DISCUSSION In chlorophyll-a + b.containing eukaryotes examined so far, the genes for subunits v, 8 and II of the chloroplast ATPase were shown to reside in the nuclear genuine. Accordingly, these genes are missing in the corresponding region of the plastid chromosome when compared with the eubacterial operons [10]. In the chromophyte Odontella sinensis, however, at least the investigated part of the atpA gone cluster including the gone atpD resembles the eubacterial gone order (Fig. 5), The identity o f the 561 bp reading frame in the plastid genuine of Odontella as the gone coding for subunit 8 is based on (1) its coding capacity which is equivalent to other prokaryotic and eukaryotic atpD genes, (2) its sequence similarity with ~ subunits of ATPases from other sources, especially from cyanobacteria, and (3) its hydrophobicity pattern showing similarities to those of other ~ subunits. In addition, the atpD reading frame of Odontella overlaps in the same way with atpF as in Cyanophora paradoxa (D.A. Bryant, V.L. Stirewalt and M.B. Annarella, unpublished results), Anabaena PCC 7120 [9] and Bacillus megaterium [23] (Fig. 6). Such overlapping genes are extremely rare in cyanobacteria and chloroplasts. A well-known example are the genes atpB and atpE which overlap in certain land plants by l b p , but are separated in others [3]. This atpBE overlap which apparently resembles a late evolutionary event in land plant lineages, does neither exist in cyanobacteria [8,24] nor in Odontella (Pantie, etal., unpublished results) or Dictyota (Leitsch and Kowallik, in preparation). A similar prokaryotic feature is found in the psbDC operon of another chromophyte, Vaucheria bursata, exhibiting a 14 bp overlap (Kowallik etal.,

Volum~ 280, number 2

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unpublished results) as in Synechococcus P C C 7942 [25], where these genes s h a r e 50 bp in land plant chloroplasts [26], These results suggest a close relatton. ship between cyanobacteria and chromophytic chloroplasts, T h e unexpected gone c o m p o s i t i o n manifested in the a t p A gene cluster o f Odontella ~oints to the question h o w c h r o m o p h y t i c and c h l o r o p h y t i c plastids have evolved. T h e r e is now do d o u b t that both types o f plastids originated f r o m c y a n o b a c t e r i a l ancestors a l t h o u g h c h r o m o p h y t e s m a y h a v e experienced different kinds of s e c o n d a r y ( ¢ u k a r y o t i c / e u k a r y o t i c ) e n d o c y toses [27]. T h e existence o f a gone for subunit 8 in the plastid c h r o m o s o m e o f Odontella at the same position as in c y a n o b a c t e r i a and the cyanelles o f C y a n o p h o r e paradoxa suggests that the t r a n s f e r o f this gone into the nuclear g e n o m e has o c c u r r e d within the c h l o r o p h y l l a + b lineage only. This suggestion is s u p p o r t e d by the finding that the same gene a r r a n g e m e n t o f the Odontel/a a t p A gene cluster was f o u n d in the b r o w n alga Dictyota dichotoma (Kuhsel et al., in p r e p a r a t i o n ) . T h e identification of a plastid gone in the C h r o m o p h y t a that is nuclear in c h l o r o p h y l l - a + b - c o n t a i n i n g organisms n o w renders the s e a r c h for the c o d i n g sites o f a t p C and a t p G in c h r o m o p h y t e s attractive. W e m a y then perhaps answer the q u e s t i o n as to w h e t h e r the transfer o f the genes atpC, a t p D , and atpG results f r o m a single event which m a y be u n i q u e for the c h l o r o p h y l l -

Ackno)v/¢gem#nts: The authors thank Prof, D,A, Bryant, Pennsylvania State University, for the kind permission to shiny unpublish. ed data and Prof. R.G. Herrmann, University of Munich, for pro. riding the spinach atpA gone probe, Further~ore, we tl~ank Thorsten Fricdrich, University of Dtisseldorf, for help in secondary structure calculations, This work was supported by the Deutsche Forsehungsge. meinschafi (Sonderforschungsbereieh 189) and the Fonds der Chemiscl~en lnduslrie,

REFERENCES [I] Senior, A,E, (1988) Physiol. Roy. 68, 177-132. [2] Walker, J,E,, Foamily, I.M., Gay, N.J., Gibson, B,W., Northrop, F,D, Powell, S.J.. Runswick, M,J., Sarast¢, M. and Tybulewicz, V,L,J. (1985) J, Mol, Biol, 184, 677-701. [3] Hudson, G.S. and Mason, J.G. 0988) Photosynth. Res. 18, 205-222. [4] Walker, J.E., Cozens. A,L., Dyer, M,R,, Fearnley. [,M.0 Powell, S,J, and Runswick, M.J. (1987} Biochem, Soc, Trans, 15, 104-106. [5] Xue, Z,, Zhou, J,M., Melese, T,, Cross, R,L and Boyer, P.D. (1987) Biochemistry 26, 3749-3753, [6] Strotmann, H, and BickeI-Sandk6tter, S. (1984) Annu. Rev, Plant Physiol, 35, 97-120.

[7] Futai M, and Kanazawa, H, 0983) Microbiol. Roy. 47, 285-312, [8] Cozens, A,L. and Walker, J,E, (1987) J. Mol. Biol, 194, 359-383, [9] McCaru, D,F., Whitaker, R,A,, Alum. J .0 Vrba, J,M. and Curtis, S,E, (1988) J, Bact. 170, 3448-3458, [lO] Palmer, I.D, in: Cell culture and Somatic Cell Genetics in Plants, Vol, 7 (VasiI, I,K,, ed) in Dress, [il] Herrnans, J,, Rother, C., Steppuhn, J. and Herrmann, R.G, (1988) Plant. Mol. Biol. 10, 323-330, [12] Hallick, R.B, and Buetow, D.E. 0989) in: The Biology of Euglena (Buetow. D.E., ed) Vol IV, pp. 351-414, Academic Press, New York, [13] Woessner, J.P., Gilham N.W, and Boynton, J,E. (1987) Plant Mol. Biol. 8, 151-158. [14] Turmel, M,, Lemieux, B, and Lemicu×, C. (1988) Mol, Germ, Genet 214, 412-419, [15] Gibbs, S,P. (1981) Ann, NY Acad, Sci. 361, 193-208. [16] Whatley, J,M, and Whatley, F.R. (1981) New Phytol, 87. 233-247, 391

Volume 2#0, number 2

FE[I$ LETTEES

[l?l Kowalllk, K,V, (19J9) In: Tl~e Chromophyle Aline. Probl~m~ and Per=pecdv¢~, The Syllematl*.~, As,odallon. Voh 31~tGreen, J.C,, Leadb¢ater, B,$,C. and Diver, D.L,, Ida) pp. 101=124, Clarendon, Oxford, 1181 Partite, P,G,, Kowallik, K,V, a~td $1ro~mann, H, (1990) I]ol, Ac~a 103,274=2#O, [191 $anller, F,, Nicklen, $, and Coul~on, A.R. (1977) Proc. Nail, Acad, $¢1, USA 74, $463-~467, [201 Wilbur', W.J, and Lippman, D,J. (1911~)Pro¢. Nail. ,&cad, $¢1, USA 80, 726-730, [21l Flillllins, D,G. =nd Sharp, P,M. (19811)C3ene"/3, 23?-244. [22] Kyle, J, and Doolitzle, R,F, (1982) J, Mol, Blol, I$7, 10:5-13L (23) Brusliow, W.S,A., Searpetla, M,A,, Haw|home. C,A. anti Clark, W,P, (1989) J. Biol, Chem. 264/3, i.~28=153]. [241 Curzis, S,E, (1987)J. Bac~t, 169, 80-86. [2~1 Golden, S,S, and Stearns, G,W, (19118)¢.3ene 67.11:5-96,

392

M~reh 1991

[261 All, J,, MorriL J,, we~lhorr, P, and Herrmaml, R.G. (19W4] Curr. Genel, il, $97=606. [271 Whatley, J,M. and Whatley, F.R. (19~4) in: Encyclopedia or Plant Phy~tololly (LiniMent, tt.F. ~nd H~llop.Harrl,~on. J., ed+l pp, 18-~8. Sprinilcr, Berlin, [28] T)tbulewk~, V.L,L, Falk, G. cad Walker, J.E. (1984) J. Mol. Biol, 1"/9, 185-214, [29) WRlkcr, J,E,, Gay, N,J., Sara~le, M, and Eberle, N, (1984) Biochem, J, 224, 799-#1 ~, [30} Falk, G,, Hampc, ^ , and Walker, ,t,E, (19~5) Btochem, J. ;~28, 391-409, 1311 Ol~la, $., Yohda, M,, I~ht~uka, M,, Hirula, H,, Hamamom, T,, Olawara.Hamamoto. Y., Ma~uda, K. and Gawa, Y. (19~8) Blnehim, Biophys. Acta 93~, 141=15~, [321 Kimura, T,. Takeda, S., A~:thl, T, and Nakamara. K. (1990) J, Biol, Chem, 26S, 6079-6085.