Volume 304, number 2,3, 1I9-123 F E B S 11130 © 1992 Federation of European Biochemical Societies 00145793/9~$5.00

June 1992

Characterisation of a chloroplast-encoded sec Y homologue and a t p H from a chromophytic alga Evidence for a novel chloroplast genome organisation Carol D. Scaramuzzi, H a r o l d W. Stokes and R o g e r G. Hiller School o f Biological Sciences, Macquarie University, Sydney, N S W 2109, Australia

Received 17 March 1992; revised version received 21 April 1992 sec Y is a prokat~otic gone that encodes the SeeY protein, an integral membrane component of the prokaryotic protein translocation apparatus. A chloroplast-encoded see Y homologue has been identified in the unicellular, chromophytic alga, Pavlova lutherii. The gone predicts a protein composed of ten membrane-spanning regions, that is approximately 25% homologous and 50% similar to bacterial and plastid S¢¢Y proteins. The secl' gone from P. lutherli is independent of the ribosomal protein (rp) gone cluster to which it is closely linked in other organisms. In P. Iutherii see}" is located 5' to atpl and atpH. Since, in higher plants the atplHFA gone cluster and the rp gen0 cluster are s~paratcd by approximately 50 kb, we conclude, this indicates a novel chloroplast gone arrangement in P. lutherii. Chloroplast genom¢; Chromophyta: Pavlova lutherii; see Y; atpH; Protein translocation

1. INTRODUCTION The chloroplast genome of land plants consists of a circular double-stranded molecule of DNA, ranging from 120 to 150 kb [1], The circular molecule contains an inverted repeat which separates a small copy and a large copy region. This arrangement of the chloroplast genome, thought to confer evolutionary stability, is almost universal among the higher plants [1]. The chloroplasl genomes of algae show a greater diversity of size, ranging from 84 kb to 600 kb [2] whereas those of the chromophytic algae for which there is much less information available, fall within the range of t00 to 160 kb [2]. There are indications, based on nucleotide sequence data for the Rubisco genes, rbcL and rbcS, that the chromophytic alga contain several genes that are similar to prokaryotic genes but which are not present in higher plant and green algal chloroplast DNA [3]. Some genes involved in protein translocation have also been retained on the chloroplast genome of eukaryotic algae outside of the chlorophyta [4-6]. The sec Y gone is found in both Escherichia cell [7] and Bacillus subtilis [8] where it forms part of the spc operon of the ribosomal protein (rp) gone cluster and in Cyanophora paradoxa [9] where Correspondence address: C, Scaramuzzi. School of Biological Sciences. Macquarie University, S),dney, NSW, 2109, Australia. Fax: (61) (2) 8058245. The nucleotide sequences of the aelaes l.eported here have b ~ n filed in the EMBL database under the accession numbers X64731 (atpH); and X64732 (sec Y).

Published b), Elsevier Sc&nce Publishers B.V.

it is located immediately adjacent to the final gone of this operon. Recently, a chloroplast-encoded sec Y homologue from a cryptophytic algae has also been located within the spc operon [6]. The bacterial rp gone cluster is composed of three operons, the SI0, spc and alpha operons collectively containing 28 genes. Only I1 of these genes are found in the analogous rp cluster of higher plants [10,11]. It is presumed that the remainder have been lost or transferred to the nucleus. Among these is sec Y, of the spc operon the product of which is involved in prokaryotic protein translocation [8]. We report here the nucleotide sequence of two chloroplastencoded genes, seeY and atpH, from the chromophytic alga, Pavlova lutherii and show that they are linked on the chloroplast genome but positioned independently of the rp cluster. We conclude that the chloroplast genome arrangement in this organism is novel.

2. MATERIALS AND METHODS P. haherii was cultivated in Provosoli's enrichment media [12]. Harvesting, pr0paration of chloroplast DNA, construction of clone banks and hybridisation techniques have been previously described [4]. Manipulations of DNA were performed according to standard protocols [13] or, when using DNA-modifying en2ymes, to the manufacturers instructions. DNA fragments for subeloning into M13 or for use in hybridisation analysis were gel-purified using GoneClean II (BIO 101, La Joila° CA, USA). The host strains JM 101 1"!4]and HBI01 [I 3] were us~l for all manipulations. A chtoropl~t DNA clone bank was generated using Hindlli chloro. plast DNA fragments and the plasmid vector, pUCl9 [15l. In the course of investigating the gone organisation of the P. lutherii chloro-

119

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plast l~enome we identified a sec Y clone, pMAQ804, containin~ a 807-bp DNA l'ragment internal to the sec Y gent (+735 to + 1542) (Fi~, I ) and a~l atpl/tf clone, pMAQS06, containing a 264-bp D N A fragment incorporating the 3' coding region of atpl and the 5" codinl~ region ofatpH. Inserts from pMAQS04 and pMAQS06, were used to screen the chloroplast DNA clone bank previously described [4], 13oth hybridised to pMAQS03 which contains a 8,0-kb Bglll fragment of chloroplast DNA, DNA sequencing was perlbrmed by the chain t~rmlnation method [16] using a Sequenase kit (United States Biochemical Corporation). Sequencing of both single and double-stranded templates was performed according to the manufacturers protocols. Prim. ers used were either the universal primer supplied or synthetic cligonucleotldes synthesized on a Pharmacia Gene Assembler Markli, OIigonucleotides were designed to provide extensive overlap between gels and. where necessary, compressions were resolvt.~d by the substitution o f d l T P lbr dGTP. PCR reactioris were prepar~ using tl Gene. Amp Kit (Perkin Elmer Cetus) in a 50/sl volume using 10 ng of chloroplast DNA and oligonucleotide primers (16-mers) at 200 nM. An !,'aitial cycle of 3 rain at 94~C. 1 mi~ at 37"C and 3 rain at 65~C was followed by 29 cycles of i m!n at 94~C. 1 rain at 37~C and 3 rain at 65~C.

3. RESULTS The sec Y gene is composed of 1257 nucleotides (Fig. 1) which predicts a protein of 419 amino acids with a molecular weight of 46,563. The predicted protein sequence of P. haherii sec Y is shown in Fig. 2 and compared with the predicted sec Y protein of Cryptomot~as [6] and C. paradoxa [9]. The percent identity/similarity of organelle encoded SecY proteins are compared with each other and with SecY from E. coli and B. subtilis in Table I. The five SecY protein sequences are approximately 50% conserved, with regions of honaology confined to ten hydrophobic regions and several charged regions. These domains correspond to the ten putative hydrophobic, membrane-spanning regions (MSR) and four cytosolicaily exposed charged loops consistent with the structure of other SecY proteins [6,8,9,17]. However, P. lutherii SecY shows considerable variation at both termini, in particular at the amino terminus, which contains an additional 7 and 39 amino acid re.~idues in the E. coil and C. paradoxa SecY proteins respectively. The chloroplast-encoded atpH gene of P. lutherii is composed of 249 nucleotides (Fig. 3) and predicts a protein of 83 amino acids, which is highly conserved. P. luthevii AtpH is 87% identical to AtpH from the eyanobacteria [18,19], 88% identical to its counterpart in Odontella sinensus [20] and spinach [21], 90% identical

June 1992

to the wheat AtpH [22] and 91.3% identical to AtpH of Marchantia polymorpha [23]. pMAQ803 DNA was mapped by restriction endonuclease analysis involving digestion with XholI, XholI/ EcoRl, EcoRl, Pstl and PstI/EcoRl and hybridisation with secY and atpl/H DNA inserts of pMAQS04 and pMAQ806. Both probes hybridised to a 6.0-kb XholI fragment. From the pattern of fragments in the digests that hybridised to each of the two probes, a physical map of this XholI fragment was constructed (Fig. 4). The orientation and position of the 2.3-kb EcoRl fragment containing the sec Ygene relative to the atpl/H genes was deduced from PCR experiments. Outward facing primers were made to the 3' and 5' sequenced non-coding regions surrounding sec Y and to the 3' and 5' sequenced regions surrounding the atpH gene. The relative positions of the PCR primers (A, B, C, D) are indicated in Fig. 4 and the nucleotide sequence of each is overlined in Figs. I and 3. All four combinations of outward facing primers were cycled with chloroplast DNA. An amplified product of 1050 bases was generated only with primers B and C (Fig. 4). Combinations without template DNA failed to sylathesise a product. No open reading frames (ORFS) greater than 100 bases were identified in either of the 500 bases 5' or 3' to the secY coding sequence. Nucleic acid searches of each of these regions with sequences in the genbank database failed to reveal any significant matches. As the primers B and C were inset by 93 and 188 bp, respectively, from the end of the sequenced regions, we conclude that the unsequenced region between the secY EcoRl fragment and atpl/H Xholl/EcoR1 fragment is 780 bases, As atpl is highly conserved and encompasses approximately 725 bp [20] we determine from the nucleotide sequence and PCR data that there is 600 buses of non-coding sequence between the end of the sec Y and the start ofatpL The results also show that the sec Y and atpl"/H genes are transcribed in the same direction. 4. DISCUSSION The presence of a sec Y gene linked to atpH, on the P. Itttherii chloroplast chromosome indicates that there are obvious differences in the chloroplast genuine organisation and structure of this alga when compared with chloroplast genomes from other plant phyla. In all cases, thus far reported, secYforms part of or is located

Table I Pairwise comparison for predicted SecY proteins from P, httherii, C, puradoxa, Crvptomonas. E. col~ and B. .s'ubtilis

P. h~llteHi C. paradoxtt Cr),ptomonas

P. httherii

C, parudoxa

28/50 32/55

28150 29/48

Cr),ptomono.s' 32/55 29/48 -

Numbers refer to percent identity/similarity.

120

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B, subtiHs

31/49 25/46 34/56

26/53 25/'46 33/57

Volume 304, number ! 3

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June 1992 •

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Fig. 1. Nucleotide sequence o~ the P. ~ h e r i i sec Y gent and surrounding s e q u e n t . Hindll I r~tfiction sites and the SecY pr~icted protein ~ q u e n ~ arc shown. T h e nueleotide ~equcnces u s ~ to construct PCR primers (A and B) are ovcrlined.

within the rp gene cluster from which the analogous gene cluster in higher plants appears to be derived [10].

However, the sec Y gene on the P. lutherii chloroplast genome is independent of the rp gene cluster and is 121

Volume 304, number 2,3 1 2 3

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genome of P. lutherii and its possible role within the chloroplast of this alga is of considerable importance. In E. coB, sec Y is one of six genes that code for protein transloeation components. Protein transloeation across the prokaryotic membranes involves an array of components (SeeA, B, D, E, F and Y), termed 'export apparatus' which include soluble and membrane-associated proteins as well as signal peptides in the translocated protein. These features are common to the translocation process across the eukaryotic rough eitdoplasmic reticulure [24]. Suh et al. (1990) [8] suggest that signal peptide recognition proteins [25,26] and the soluble cytoplasmic SecA proteins [27,28] are likely candidates for interaction with SecY in order to facilitate bacterial protein translocation. A chloroplast-encoded gene which predicts a protein related to bacterial SecA [27,28] is also present in P. lutherii (Scaramuzzi, PhD Thesis, 1991). The amino terminus of P. lutherU SecA is 52% identical to the equivalent region o f the E. coli and B. subtiBs SecA proteins. The amino terminus o f SecA is essential lbr its function [27] by coupling ATP hydrolysis to precursor protein transport [29-31]. To date, secA has not been reported from any chloroplast genome but its presence, together with secY on the chloroplast genome of P. lutherii strongly implies that this organism has an alternative mechanism for protein translocation into and within the chloroplast, and which may be similar to bacterial protein translocation. Further support for this is the discovery o f a chloroplast-encoded Hsp70 in P. lutherii [4]. The chloroplasts of chromophytic algae are surrounded by two extra membranes, the chloroplast endoplasmic reticulum (CER) which bear eukaryotic ribosomes [32]. These features have been implicated in the import of nuclear-encoded, chloroplast light-harvesting proteins in the diatom, Phaeodactylum tricor, utum [33]. That the chromophytic algae have additional mechanisms accommodating chloroplast protein translocation has also been proposed for another diatom O. sinensus [20]. An O R F located upstream of the azpl gene predicts a protein that is approximately 20% identical to a prokaryotic periplasmic ATP-binding transport

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Fig. 2, Comparison of the derived amino acid sequencesof P. haherii S¢¢Y (I) with Sexy proteins o f Cr)ptomonas(2) and C, paradoxa (3), The predictedprotein sequenceis shown for all SecYproteins. The ten putative MSR are overllng~d.Identicalamino acid residuesare indicated ~ith an asterisk (*);conservativesubstitutionsare indicatedwith a dot (e). Alignmentswerecarried out using the CLUSTALprogram [34], Substitutionsare designatedconservativeif both amino acids fall within one of the followingexchangegroups: T, S, A. G, P: g, K, H: F, W, Y: D, E. Q. N: I. L, M, V: and C [35], linked to atpI/H. Both these findings support the hypothesis that the chloroplast genome of P. lutherii differs from those of higher plants, where the atplHFA and the Iio gene clusters are separated by approximately 50 kb [11] and secY is not present. It also differs from the cryptophytic alga and C paradoxa, where sec Y is present and linked to the spc operon, of the rp gene cluster [6,9]. The presence of the secY gene on the chloroplast Hind~II

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Fig. 3. Nucleotid¢ sequence of the P. ~therli, a ~ H 8¢ne and the 3' regionof the prE~ding a~! gcnu. NuclEotid¢sequen~s us~ to construct P C R primers (C and D) are overlln~. ThE prEdictEd amino acid sequences of AtpH and the carboay tE~inus of Atpl are shown. 122

Volume 304. number 2,3

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II I~tl i!asll ~ho,I 4..- did CD

11.okp I Fig. 4. Map of pMAQg03 which contains an 8.0.kb fragment of P. lutherilchlorophtst DNA showing the positions of sec Y, atpl and atpH. The sec Y gene is located on a 2.3-kb EcoRl fragment and the atp genes arc located on a 1,6-kb EcoRl/Xholl fragment. The two DNA fragments containing the genes are contiguous. The bold bars represent the regions of DNA sequenced. The length and position of the top arrows correspond to the location of the respective genes, The direction of presumed transcription is indicated. Bottom arrows indicate the position and dire=tion of the PCR primers (A. B, C, D),

protein b u t does not display any similarity to the SeeY protein r e p o r t e d here. T h e a u t h o r s [20] conclude t h a t the "peculiar o r g a n i s a t i o n o f the c h r o m o p h y t i c plastids including four a d d i t i o n a l m e m b r a n e s requires additional t r a n s p o r t systems'. M o v e m e n t o f proteins within the chloroplast across thylakoid m e m b r a n e s m a y also b e facilitated via these proteins. The discovery o f c h l o r o p l a s t - e n c o d e d sec Y, .~'ecA a n d hsp70 genes and the linkage ofsec Yto the atpl a n d atpH genes reveal that the c h l o r o p l a s t genov.,,e o f P. lutherii differs substantially f r o m that o f other plant phyla. It m a y also be n o t e d t h a t the percent idemi~:y/similarity o f P. lutherii SecY with Cryptomonas cp SecY is similar to t h a t with C. paradoxa cyanelle SecY a n d bacterial SecY a n d that there is only a low percent identity/similarity in the C - t e r m i n a l region o f c h l o r o p l a s t - e n c o d e d H s p 7 0 proteins (27% identity/48% similarity in 128 C - t e r m i n a l residues) f r o m P. haherii and the red algae Porphyra umbilica/is [5]. We tentatively suggest t h a t the c h r o m o phytic algae f o r m a lineage distinct f r o m the C h l o r o phyta, C r y p t o p h y t a , R h o d o p h y t a and the cyanelle c o n taining g r o u p o f algae.

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Characterisation of a chloroplast-encoded secY homologue and atpH from a chromophytic alga. Evidence for a novel chloroplast genome organisation.

secY is a prokaryotic gene that encodes the SecY protein, an integral membrane component of the prokaryotic protein translocation apparatus. A chlorop...
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