Plant Molecular Biology 20: 569-574, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

569

Update section

Short communication

Import of the precursor of the chloroplast Rieske iron-sulphur protein by pea chloroplasts A. Hugh Salter 1, Barbara J. Newman 2, Johnathan A. Napier 3 and John C. Gray* Department of Plant Sciences and Cambridge Centrefor Molecular Recognition, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK (* authorfor correspondence),"present addresses: 1Department of Biochemistry, Arrhenius Laboratoryfor Natural Sciences, University of Stockholm, S-10691 Stockholm, Sweden,"2AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4A T, UK; 3AFR C Institute of Arable Crops Research, Long Ashton Research Station, Long Ashton, Bristol BSI8 9AF, UK Received 16 March 1992; accepted 2 June 1992

Key words:Rieske FeS protein, cytochrome bf complex, chloroplast import, thylakoid membrane, presequence

Abstract cDNA clones encoding the precursor of the Rieske FeS protein of the chloroplast thylakoid membrane have been isolated from a pea leaf cDNA library in 2gt 11, following screening with antibodies to purified pea chloroplast Rieske FeS protein. The longest cDNA insert of 880 bp encodes a polypeptide of 230 amino acid residues, of which 50 residues constitute an N-terminal cleavable presequence and 180 residues make up the mature protein. Transcription and translation of the cDNA in vitro produced a polypeptide of 26 kDa which was efficiently imported by isolated pea chloroplasts and processed to the mature 20 kDa protein. Southern hybridisation to pea genomic DNA indicated the presence of a single gene encoding the Rieske FeS protein in the haploid genome.

The Rieske FeS protein is the only nuclearencoded subunit of the cytochrome bf complex of the chloroplast thylakoid membrane [21]. The other components, including cytochrome f, cytochrome b-563 and subunits IV and V, are all encoded by the chloroplast genome [6, 8, 25]. The Rieske FeS protein is synthesised in precursor

form on cytosolic poly(A)-rich RNA [1] and imported into the chloroplasts where it is processed to the correct size [2, 21]. Analysis of cDNA clones encoding the precursors of the spinach and tobacco Rieske FeS proteins indicated that the proteins are synthesized with N-terminal cleavable presequences of 52-68 amino acid residues

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X63605.

570 attached to the mature polypeptide of 179 amino acid residues [13, 16, 20]. The mature polypeptides contain two short highly conserved sequences containing the putative ligands for the [2Fe2S] centre, but otherwise show little primary sequence similarity to mitochondrial or bacterial Rieske FeS proteins [20]. The aim of the present work was to isolate cDNA clones encoding the pea chloroplast Rieske FeS protein for studies on the assembly of the cytochrome bf complex in pea. A pea leaf cDNA library in 2gt 11 [5] was screened with rabbit antibodies to the purified pea Rieske FeS protein, isolated as described by Hurt et al. [9]. Positive signals were obtained from 40 plaques out of a total of 150000, and five of these were purified by two more rounds of screening at lower density but none encoded a full-length precursor protein. The longest insert, corresponding to nucleotides 45-789 in Fig. 1, was 32p-labelled by the random hexanucleotide primer method and used to screen the same c D N A library in 2gt 11 by

hybridisation. Approximately 80 plaques gave a positive hybridisation signal out of a total of 100000 plaques. Ten plaques were purified by two more rounds of hybridisation screening and their c D N A inserts were released with Eco RI and characterised. The longest insert of 880 bp contained an open reading frame of 230 codons with homology to the cDNAs encoding the spinach and tobacco Rieske FeS proteins [13, 16, 20]. The nucleotide sequence and deduced amino acid sequence are shown in Fig. 1. The open reading frame starts with ATG at position 44-46 and consists of 50 codons encoding an N-terminal presequence and 180 codons encoding the mature Rieske FeS protein. There are no stop codons or initiation codons upstream of the putative initiation codon at position 44-46. The sequence around the putative initiation codon (5'-CCACCATGTC) matches the consensus for eukaryotic translation start sites (5'-ACCATGG) originally determined by Kozak [10], although more recent compilations have detected differences in

i0 20 30 40 50 60 70 80 90 i00 ii0 CATTCTCAACCCAACAGAATCTTCTTCATCAGGAGCTTCCACCATGTCTTCCAcCACTCTATCTCCCACAACCCCTTCTCAGCTATGTTCTGGAAAGAGTGGGATTTCATGTCCCTCAAT MetSerSerThrThrLeuSerProThrThrProSerGlnLeuCysSerGlyLysSerGlyIleSerCysProSerll

120

130 140 150 160 170 ~80 190 200 210 220 AGCTCTTCTTGTGAAACCAA•AAGGAcGCAGATGACTGGGAGGGGTAATAAGGGAATGAAAATTACTTGCcAAGCTAcCAGCATTCCTGCTGATCGTGTACCTGATATGAGTAAGAGGAA eA•aLeuLeuVa•LysPr•ThrArgThrG•nMetThrGlyArgG•yAsnLysGlyMetLysIleThrCysG•nA•aThrSerI•ePr•A•aA••ArgVa•Pr•AspMegSerLysArgLy

230

240

350

360

A 250 260 270 280 290 300 310 320 330 340 GACCTTGAATTTGCTTCTTCTTGGTGCTCTTTCACTTCCTACTGCTGGAATGCTTGTTCCATATGGTTCCTTCCTTGTACCTCCAGGCTCAGGTTCTTCAACTGGTGGTACCGTTGCCAA

sThrLeuAsnLeuLeuLeuLeuG•yA•aLeuSerLeuPr•ThrAlaG•yMetLeuVa•Pr•TyrG•ySerPheLeuVa•Pr•Pr•GlySerG•ySerSerThrG•yGlyThrVa•AlaLy 370 380 390 400 410 420 430 440 450 460 GGATGCCGTTGGCAATGATGTAGTTGCAACGGAATGGCTCAAGACTCATGCACCTGGCGACCGTACTCTTACACAAGGATTAAAGGGTGATCCTACCTACCTTGTGGTAGAGAAAGACAG sAspA•aVa•G•yAsnAspVa•Va•A•aThrG•uTrpLeuLysThrHisA•aPr•G•yAspArgThrLeuThrG•nGlyLeuLysG•yAspPr•ThrTyrLeuVa•Va•G•uLysAs•Ar

470

480

490 500 510 520 530 540 550 560 570 580 AACACTTGCAACATTTGCGATTAACGCCGTATGCACT•ATCTCGGGTGTGTCGTGCCGTTTAATCAAGCTGAGAACAAGTTCATCTGCCCCTGCCATGGATCTCAGTACAATGACCAAGG

590

600

gThrLeuA•aThr•heA•aI•eAsnA•aVa••y•ThrHisLeuG•yCysValVa•Pr••heAsnGlnA•aG•uAsnLys•heI•eCysPr•CysHisG•ySerG•nTyrAsnAspGlnG• 610

620

630

640

650

660

670

680

690

700

710

720

AAGAGTTGTGAGAGGACCTGCACCTTTGTCTCTAGcATTGGCACATTGTGATGTTGGTGTGGAGGATGGGAAGGTTGTGTTTGTTcCTTGGGTTGAAACAGATTTCAGAACAGGTGA•GC yArgVa•ValArgG•yPr•AlaPr•LeuSerLeuAlaLeuAlaHisCysAspValG•yVa•G•uAspG•yLysVa•Va•PheValPr•TrpVa•G•uThrAspPheArgThrG•yAspAl

730 740 750 760 770 780 790 800 810 820 TCCATGGTGGTCTTAGATTTCTTAAACTTGTCCTATATGTAATATATATTCAAATCTGAAATTCAAACATTTATGTTTTGTATTAATGCATAAACACTTTTTGTAATCAAATTAATATTA aProTrpTrpSer***

850 860 870 TGTCAATTCAAGTTTGTATAAGGTGAAAAGGAAAAAAAAA

830

840

880

Fig. 1. Nucleotide sequence of a cDNA encoding the precursor of the pea chloroplast Rieske FeS protein. Nucleotide sequencing was carried out by the dideoxynucleotide chain-termination method on single-stranded DNA produced by cloning restriction fragments in M13 rap18 and mpl9 [19]. Both strands were sequenced and all restriction sites used for cloning were confirmed by sequencing. A translation of the longest open reading frame is shown below the nucleotide sequence. The putative cleavage site between the presequence and the mature polypeptide is marked with an arrowhead.

571 the consensus sequences for plants and animals [3, 11]. The sequence around the putative initiation c o d o n of the pea c D N A is more similar to the vertebrate consensus ( 5 ' - C C A C C A T G G C ) than to the dicotyledonous plant consensus ( 5 ' - A A A A A A T G G C ) . A short poly(A) tail is present at the end of the insert (nucleotides 872-880). The c D N A is similar in size to the m R N A present in total R N A extracted from pea leaves, as determined by northern hybridisation using the ~ P labelled c D N A insert as a probe. A single hybridising b a n d o f 950 nucleotides was obtained (data not shown). The deduced amino acid sequence of the precursor of the pea Rieske FeS protein is c o m p a r e d to the sequences of the spinach [17], tobacco [13, 16] and cyanobacterial [9, 12, 24] proteins in

Pea Tobacco Spinach

Pea Tobacco Spinach Nostoc 7906 Synechocystis 6803 Synechococcus 7002

P~a Tobacco Spinach Nostoc 7906 Synechocystis 6803 Synechococcus 7002

Pea

Tobacco

Spinach Noatoc 7906 Synechocystis 6803 Synechococcus 7002 Pea Tobacco Spinach

Nostoc 7906 Synechocystis 6803 Synechococcus 7002

Fig. 2. The pea presequence (50 amino acid residues) is similar in size to the tobacco presequence (49 residues) and is strikingly shorter than the spinach presequence (68 residues). The putative cleavage site, m a r k e d with an arrowhead, was identified from the determined N-terminal sequence of the pea Rieske FeS protein. The sequence A l a - T h r - S e r - I l e - P r o - A l a - A s p - A r g - V a l was obtained from 40 pmol protein electroblotted onto Immobilon m e m b r a n e and is in complete agreement with the sequence predicted from the c D N A inserts. The spinach presequence contains an additional 17 amino acid residues at the N-terminus which are not present in the pea and tobacco presequences. There is no similarity in nucleotide sequence or deduced amino acid sequence between pea and spinach in the region

MSSTTLSPTTPSQLCSGKSGISCPSIALLVKPTRT MASSTLSPVT--QLCSSKSGLSSVSQCLLVKPMKI MIISIFNQLHLTENSSLMASFTLSSATPSQLCSSKNGMFAPSLAL-AKAGRV

Q - M T G R G N - K G M K I T C Q~A T - ~ I P A ~ - R ~ M

~-s~n~a~wc~T-N~N~pNw~l~

35 33 51

S~

ETLINL~I~5~A~S~T~Y~

NVLISKERiRGMKLTCQAT-~IP~DI-NIVPDIMQ~

QFMINLLITFNTVTGVALGA MTQLNGSSN--IVPDILGN

34

QFLNL~_~JWVNTAAGTALGG

34

~NVV~ZpPI~SN~a~I~ITTaI~OI~ YNAVKYLIIPPISSNGSGIGGIVTAIKDIAL

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R T LA T F A~V[C

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T H L G CVV PIF~QA~IIC

P C H G S Q YIND Q ~ R

135 135 153 86 86 86

187

DP TYILlVVEINDGTZ.ATYGFNAlV[CTHLGCVVP[FINIAAINNKFII]CP CNGSQYINNQIGIR 187 DPTYIT]VWlSDKTLATFGIZNAIVFT~LGCVVPFNAAIENKFtZlCPCSGSQYINNQ[Sla205 D P T NIIVVEINKQ A I KD X sire NAIIIC T H L ~ CVV PIWNV~.IE N K FIKICP C H G S Q YID E T[G]K

D P TYIIIVV~ISDD WZ ANY ~lI NAIVlCT H L GCVVPlWNA SlE NK~IMICP CHG S QY NANNK

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D P T Y]VV~_V_~GD N T I S S Y GII NAIIIC T H L G C V V PIW~T A N~_N_K~MIC E P C H G S Q Y D g T~GIK

138

~V R G P A P L S L A L ~ C D v GV E~G~VV F V P ~ V ~ G D A ~ S ~ V R G P A P L S L A LIAIHIAD I - - DIDIGIKIVV F V PIWIVlET D F R TIG E DIP W WIA V V R G P A P L S L A LIAIHICD V - - DIDIGIKIVV F V PIWITIET D F R TIG E AIP W WIS A

230 228 247

~ V R G P AP ~ s ~,ALIAInIAN T-VDpI-IKII I L S PiwFI~ T n~ RTIGI~AIP~WIA

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~ V R G P A P L S L A LIAIHIAT V - T DpIDIKILV L S TIWITIET D F R T p E DIP WWIA V V R G P A P L S L A L]VlH~AE V - T E~DIKII S F T DIWITIET D F R TIDE PA~_W_~A

180 180

Fig. 2. Comparison of amino acid sequences of the Rieske FeS proteins from pea, spinach and cyanobacteria. The spinach sequence was obtained from Steppuhn etal. [20], tobacco from Palomares etaI. [16] as corrected by Maduefio etal. [13], Nostoc PCC 7906 from Kallas et al. [ 10], Synechocystis PCC 6803 from Mayas and Barber [ 14] and Synechococcus PCC 7002 from Widger

[24]. Residues which are identical in all six species are boxed. The putative cleavage site between the presequence and the mature polypeptides of pea, tobacco and spinach is marked with an arrowhead. The pea sequence presented above differs at several positions from the preliminary amino acid sequence presented previously [14, 15].

572 upstream of the proposed initiation codon for pea (nucleotides 44-46 in Fig. 1). The spinach presequence contains a methionine residue (Met-18) which corresponds to the initiating methionine residue in pea and tobacco (Fig. 2), and thereafter there is a high level of similarity between the presequences with identical amino acid residues at 46-55 ~ of comparable positions. The pea presequence has many of the features of a typical chloroplast transit peptide [22]. It contains a high proportion ( 3 2 ~ ) of serine and threonine residues, and no acidic glutamate and aspartate residues. The first 10 residues, which may be important in envelope binding, contain 3 serine and 4 threonine residues and no basic residues. The basic residues, 4 lysine and 2 arginine residues, are found mainly in the C-terminal region of the presequence where they may be important for processing. There appear to be no reasons to believe that the presequence is atypical, as has been suggested for the presequence of the spinach Rieske FeS protein [20]. The presequence does not contain any extended hydrophobic regions which might act as thylakoid targeting signals. The information for targeting the Rieske polypeptide to the thylakoid membrane has been shown to reside within the mature polypeptide [2]. It seems probable that the extended hydrophobic region near the N-terminus of the mature polypeptide acts as a thylakoid targeting signal. The amino acid sequence of the mature pea Rieske FeS protein is very similar to the spinach and tobacco proteins [13, 16, 20]. Identical residues are present at 85-88 To of comparable positions. The spinach protein contains an additional amino acid residue at the C-terminus, and the pea protein contains two additional amino acid residues, Gly-Val at positions 155 and 156 of the mature protein. Similarity to the Rieske FeS protein of cyanobacteria [ 10, 14, 24] is much less, with identical residues present at only 55-61 ~o of comparable positions. The most highly conserved regions are in the C-proximal half of the protein and include the two cysteine-containing motifs, Cys-Pro-Cys-His-Ser-Gly and Cys-Thr-His-CysPro-Gly, which provide the ligands for the [2Fe2S] cluster. These residues are conserved in

all other Rieske FeS proteins, of bacterial or mitochondrial origins [7], and two cysteine and two histidine residues have been implicated as ligands

[3]. To establish that the c D N A insert encodes a complete, functional precursor for the Rieske FeS protein, the 880 bp Eco RI fragment was inserted into pSP64, the hybrid plasmid was transcribed with SP6 polymerase and the transcripts translated in a wheat germ extract in the presence of [35S ]-methionine. An 35S-labelled polypeptide of 26 kDa was the major product of the incubation (Fig. 3). A polypeptide of 26 k D a was also immunoprecipitated from the translation products of pea leaf poly(A)-rich R N A in the wheat germ extract (data not shown), suggesting that the pea leafmRNA encodes an identical polypeptide. The 26 kDa polypeptide produced by transcription and translation in vitro was efficiently imported into isolated pea chloroplasts and processed to a polypeptide of 2 0 k D a (Fig. 3). The 2 0 k D a polypeptide was not accessible to added thermolysin indicating that the processed form of the

Fig. 3. Import and processing of the precursor of the Rieske

FeS protein by isolated pea chloroplasts. 35S-labelled precursor was synthesised in vitro by transcription of the cDNA in pSP64 and by translation of the transcripts in a wheat germ extract [15]. Translation products (4 x 10 4 dpm) were incubated with intact pea chloroplasts containing 100 #g chlorophyll in a volume of 150 #1 [ 15]. Chloroplasts were reisolated, treated with thermolysin to assess the location of the polypeptides and analysed by SDS-PAGE. Lane 1, markers; lane 2, total translation products; lane 3, chloroplasts incubated with translation products; lane4, chloroplasts incubated with translation products, then treated with thermolysin.

573 Rieske FeS protein was located behind the chloroplast envelope. This indicates that the 26 kDa precursor protein contains the sequences necessary for chloroplast import. Further experiments have indicated that the imported Rieske FeS protein is correctly inserted into the thylakoid membrane and integrated into the cytochrome bf complex in vitro (J.A. Napier, F. Maduefio & J.C. Gray, unpublished). Comparisons of all the c D N A inserts encoding parts of the pea Rieske FeS protein suggested that they were all derived from a single m R N A species. Nucleotide sequence and restriction enzyme sites were completely conserved, suggesting the presence of a single functional gene for the Rieske FeS protein in the pea haploid genome. Southern hybridisation of the 32P-labelled c D N A insert to pea genomic D N A digested with Barn HI (not shown), Hind III and Bam HI plus Hind III gave single bands of hybridisation in each track (Fig. 4). Hybridising bands were obtained at 12 kbp with Bam HI, a 9 kb with Hind III and at

Fig. 4. Southern hybridisation of pea genomic DNA. Pea nuclear DNA (10/~g) was digested with restriction enzymes [ 15] and separated by 0.75 % agarose gel electrophoresis. The DNA was transferred to a GeneScreen nylon mebrane (NEN Research Products) and probed with 3ap-labelled cDNA encoding the Rieske FeS protein at 65 °C overnight as described by Wahl et al. [23]. Hybridising bands were detected by autoradiography. Lane 1, Pea nuclear DNA digested with Barn HI and Hind III; lane 2, pea nuclear DNA digested with Hind III. Sizes in kbp were determined in relation to 32p-end-labelled Hind III fragments of )~DNA.

7 kb in a double digest with Bam HI and Hind III. This suggests the presence of a single gene encoding the precursor of the Rieske FeS protein in the pea haploid genome, as has been suggested for the spinach protein [21].

Acknowledgements We wish to thank Dr L. Packman for protein sequencing, and Dr D.L. Willey and Dr D.S. Bendall for their help and advice. This work was supported by grants from the Science and Engineering Research Council. A.H.S. was supported by an SERC Research Studentship.

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574 9. Hurt E, Hauska G, Malkin R: Isolation of the Rieske iron-sulfur protein from the cytochrome b6-f complex of spinach chloroplasts. FEBS Lett 134:1-5 (1981). 10. Kallas T, Spiller S, Malkin R: Primary structure of cotranscribed genes encoding the Rieske FeS and cytochrome fproteins from the cyanobacterium Nostoc PCC 7906. Proc Natl Acad Sci USA 85:5794-5798 (1988). 11. Kozak M: Compilation and analysis of sequence upstream from the translational start site in eukaryotic mRNAs. Nucl Acids Res 12:857-872 (1984). 12. L/itcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA: Selection of AUG initiation codons differs in plants and animals. EMBO J 6:43-48 (1987). 13. Maduefio F, Napier JA, Cejudo FJ, Gray JC: Import and processing of the precursor of the Rieske FeS protein of tobacco chloroplasts. Plant Mol Biol, in press. 14. Mayes SR, Barber J: Primary structure of the psbN-psbHpet@etA gene cluster of the cyanobacterium Synechocystis PCC 6803. Plant Mol Biol 17:289-293 (1991). 15. Newman BJ, Gray JC: Characterisation of a full-length cDNA clone for pea ferredoxin-NADP ÷ reductase. Plant Mol Biol 10:511-520 (1988). 16. Palomares R, Herrmann RG, Oelmtiller R: Different blue-light requirement for the accumulation of transcripts from nuclear genes for thylakoid proteins in Nicotiana tabacum and Lycopersicon esculentum. J Photochem Photobiol B Biol 11:151-162 (1991). i7. Salter AH, Newman BJ, Gray JC: Characterization of cDNA clones encoding the pea chloroplast Rieske Fe-S protein. In: Barber J, Malkin R (eds) Techniques and New Developments in Photosynthesis Research, pp. 473-476. Plenum, New York (1989).

18. Salter AH, Gray JC: Characterisation of a full-length cDNA clone encoding the pea Rieske Fe-S protein: import and processing by isolated chloroplasts. In: Baitscheffsky M (ed) Current Research in Photosynthesis, vol 3, pp. 267-270. Kluwer, Dordrecht (1990). 19. Sanger F, Coulson AR, Barrell BG, Smith AJH, Roe BA: Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol 143:161-178 (1980). 20. Steppuhn J, Rother C, Hermans J, Jansen T, Sainikow J, Hanska G, Herrmann RG: The complete amino-acid sequence of the Rieske FeS-precursor protein from spinach chloroplasts deduced from cDNA analysis. Mol Gen Genet 210:171-177 (1987). 21. Tittgen J, Hermans J, Steppuhn J, Jansen T, Jansson C, Andersson B, Nechushtai R, Nelson N, Herrmann RG: Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins. Mol Gen Genet 204: 258265 (1986). 22. von Heijne G, Steppuhn J, Herrmann RG: Domain structure of mitochondrial mad chloroplast targeting peptides. Eur J Biochem 180:535-545 (1989). 23. Wahl GM, Stern M, Stark GR: Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl paper and rapid hybridisation by using dextran sulphate. Proc Natl Acad Sci USA 76:3683-3687 (1979). 24. Widger W: The cloning and sequencing of Synechococcus sp. PCC 7002 petCA operon: implications for the cytochrome c-553 binding domain of cytochrome f Photosynth Res 30:71-84 (1991). 25. Willey DL, Auffret A, Gray JC: Structure and topography of cytochrome f in pea chloroplast membranes. Cell 36:555-562 (1984).