Plant Molecular Biology 20: 549-554, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

549

Update section

Short communication

Import and processing of the precursor of the delta subunit of tobacco chloroplast ATP synthase J.A. Napier

1, K.H.

Larsson 2, F. Maduefio and J.C. Gray*

Department of Plant Sciences and Cambridge Centrefor Molecular Recognition, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK (*author for correspondence)," Present addresses: 1AFR C Institute of Arable Crops Research, Long Ashton Research Station, Long Ashton, Bristol BS18 9AF, UK; 2Department of Cell Research, Swedish University of Agricultural Sciences, Box 7055, S-75007, Uppsala, Sweden Received 16 March 1992; accepted in revised form 8 June 1992

Key words: ATP synthase, CF1CFo, coupling factor, chloroplast import, presequence

Abstract A cDNA clone encoding the complete precursor of the b subunit of chloroplast ATP synthase has been isolated from a tobacco (Nicotiana tabacum) leaf cDNA library in 2gt11. The 880 bp insert encodes a polypeptide of 248 amino acid residues, of which 61 residues constitute an N-terminal presequence and 187 residues make up the mature b subunit. Transcription and translation of the cDNA in vitro produced a protein of 29 kDa which was imported by isolated pea chloroplasts and processed to the mature 20 kDa subunit. The b subunit precursor was processed to the mature size by a processing peptidase present in pea stromal extracts. Hybridisation of the cDNA to Southern blots of tobacco genomic DNA suggests the presence of two genes in the haploid genome.

The b subunit is one of three nuclear-encoded subunits of the chloroplast ATP synthase in higher plants [23]. The protein is synthesised on cytosolic ribosomes [5, 16], in a larger precursor form [ 17, 24, 25] and is subsequently imported into chloroplasts before assembly into the CF1 complex of the ATP synthase [22]. In the absence of chloroplast-encoded subunits of the ATP synthase, in heat-treated rye leaves, the newly im-

ported b subunits were degraded with a half-life of approximately 4 h [4]. The isolation of cDNA clones encoding the spinach ~ subunit revealed that the protein was synthesised with an Nterminal presequence of 70 amino acid residues, and that the mature subunit showed only limited sequence similarity to b subunits from bacterial and mitochondrial ATP synthases [ 10]. The aim of the present research was to isolate cDNA

The DNA sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X63607.

550 clones encoding the complete precursor of the subunit from tobacco to enable further studies on the synthesis, assembly and function of the chloroplast ATP synthase. We have previously described the isolation of tobacco c D N A clones encoding the precursor of the 7 subunit and have demonstrated the import and processing of the 7 precursor by chloroplasts [ 11 ]. In this paper, we describe the characterisation of tobacco c D N A clones encoding the precursor of the ~ subunit of the chloroplast ATP synthase. A tobacco leaf c D N A library in 2gtll was screened with a spinach c D N A encoding the subunit of chloroplast ATP synthase [10], and 6 plaques out of a total of 400 000 gave a positive hybridisation signal. These plaques were rescreened at lower densities and three plaques remained positive through two further rounds of screening, c D N A inserts of 0.7-0.9 kb were released from these clones by digestion with Eco RI, and the longest insert was completely sequenced (Fig. 1). The nucleotide sequence of 880 bp is slightly smaller than the transcript size of 1.1 kb, determined by northern hybridisation (data not shown), but the c D N A does not contain a poly(A) tract and this probably accounts for the difference in size. The c D N A encodes a protein of 248 amino

acid residues, which may be identified as the precursor of the 6 subunit of chloroplast ATP synthase by comparison with the sequence of the spinach protein [10] and with the determined N-terminal amino acid sequence of the isolated tobacco b subunit. The CF1CF 0 complex was isolated from tobacco thylakoid membranes by the method of Pick [20], as described previously [ 11 ]. The purified protein from the sucrose gradient was subjected to electrophoresis in a 13~o polyacrylamide gel in the presence of SDS [12] and the polypeptides electroblotted onto polyvinylidene difluoride (PVDF) membrane [14]. The first 24 residues of the mature ~ subunit were determined to be Val-Ala-Ser-Ala-Ala-Gly-SerTyr-Ala-Asn-Ala-Leu-Ala-Asp-(Val/Ile)-AlaLys-Ser-Asn-Gly-Thr-Leu-Glu-Gln. Some indication of heterogeneity was obtained at position 15 where both valine and isoleucine were detected. The N-terminal sequence corresponds to residues 62-85 of the predicted precursor protein. This indicates that the mature tobacco subunit of 187 residues is synthesised with an N-terminal presequence of 61 amino acid residues. The tobacco presequence is 9 amino acid residues shorter than reported for spinach [10]. A comparison of the amino acid sequences of the

i

AGCGAGATTAATGGCGGCACTACAACAAACTCCGATAACTTTCCAGTCCAGGTCACCGCCGCCGACTCAAATCATCAGCGGACCGACGGCAAAGCTCTCCTTTTCCGGCGGCCTCAAGCT MetA•aA•aLeuGlnG•nThrPr•I•eThrPheG•nSerArgSerPr•Pr•Pr•ThrG•nI•eI•eSerG•yPr•ThrA•aLysLeuSerPheserG•yG•yLeuLysLe

120

121

CCCGAAACTAACCATCAAGCTCCGCTCTAATCGCACCTCCCGCCGAGGCGGTGGTGCTGCCGGATCAAAAATGGTGGCTTCCGCTGCCGGAAGTTACGCGAACGCACTCGCGGACATAGC

240

241

CAAGTCGAACGGAACCCTAGAACAAACCACCGCCGACCTCGAAAAAATCGAAAAAATCTCCGACGACGAAGCAGTGTTCAACTTCTTCGTGAGCCCTATTGTcGGCGAAGAGAAGAAACG aLysSerAsnG•yThrLeuG•uGlnThrThrA•aAspLeuG•uLysI•eGluLysI•eSerAspAspG•uA•aVa•PheAsnPhePheVa••erPr•IleVa•G•yG•uG•uLysLysAr

360

361

CGAACTCGTGGACGAGATCGTTTCATCGTCGAGCATCCAGCCGCACGTGGCGAATTTCCTGAACATTTTAGTAGACATGAAGCGCGTGGAGCTAATCAAAGAAATTGTAAAGGAGTTCGA gG•uLeuVa•AspG•uI•eva••er•erSerSerI•eG•nPr•HisValA•aAsnPheLeuAsnI•eLeuVa•AspMetLysArgVa•G•uLeuIleLysG•uI•eVa•LysG•uPheG•

480

481

GAAAGTGTACAACACGCTTACGGACACGGAACTTGCTGTGGTCACTTCTGTTGTGAAATTGGAATCGCAGCATTTGGCTCAGATCGCGAAAGGTGTACAGCGATTGACAGGTTCGAAAAA uLysValTyrAsnThrLeuThrAspThrG•uLeuA•aVa•Va•Thr•erVa•Va•LysLeuGluSerG•nHisLeuA•aG•nI•eAlaLysG•yValG•nArgLeuThrG•ySerLysAs

600

601

CGTGAGGATTAAAACGGTGATTGATGAATCGCTAGTAGCTGGATTTACAATAAGGTACGGAAATTCAGGATCAAAGTTGATTGATATGAGTGTcAAGAAACAACTTGAGGATATTGCTGC nVa•ArgI•eLysThrVa•I•eAspGluSerLeuValA•aG•yPheThrI•eAr•TyrGlyAsnSerG•ySerLysLeuI•eAspMet•erVa•LysLysG•nLeuG•uAspI•eA•aA•

720

721

TCAACTTGAAATTGGGGATATTCAACTAGCTGTATAATTGATCTGCTTTTAAAATTATATTTTTTTTCGGTGATTAATTTTGTAACTTAATTTGCTTCAAAGGCAAATGGAAGATACTGT aGlnLeuGluIleGlyAspIleGlnLeuAlaValEnd

840

841

AT TGTTCTAAATTTTTGGATGAATATATATTAGTAGCTAC

uPr~LysLeuThrI~eLysLeuArg~erAsnArgThr~erArgArgG~yG~yG~yAlaAlaGlySerLysMet~a~AlaSerA~aAlaG~ySerTyrA~aAsnA~aLeuA~aAspI~eAl

880

Fig. 1. Nncleotide sequence and deduced amino acid sequence of the precursor of the tobacco ~ subunit. Nucleotide sequencing was carried out by the dideoxynucleotide chain-termination method on plasmid DNA, using a USB Sequenase kit. The single Acc I site (nucleotides 431-436) was used to generate subclones in pUC18. Confirmation of sequence and the presence of the single Acc I site was obtained using specific oligonucleotides as primers. The processing site in the precursor protein is marked with an arrowhead.

551 /5subunits from other oxygenic photosynthetic organisms is shown in Fig. 2. Only the tobacco and spinach proteins have a long N-terminal presequence for targeting the protein to the chloroplasts. The mature tobacco and spinach /5 subunits share identical residues at 64~o of comparable positions, whereas only 36~o of the residues of the presequences are identical. Within the mature /5 subunit, sequence conservation is higher in the C-proximal half of the protein; identical residues are present at only 46 ~o of positions in the N-proximal region (residues 62-154 in Fig. 2) and at 79~o of positions in the C-proximal region (residues 155-248). Considerable variation in the sequence of the N-terminal region between species has been noted previously following sequencing of the spinach and maize /5

subunits [2]. The higher level of conservation in the C-proximal region is also found in comparisons with /5 subunits from other organisms (Fig. 2), although overall levels of sequence conservation are poor. The tobacco /5 subunit contains identical residues at 26-27 ~o of comparable positions of the cyanobacterial 6 subunits [6, 15 ], and only 23 ~o identical residues compared to the chloroplast-encoded /5 subunit from Odontella sinensis [ 19]. The c D N A for the tobacco /5 subunit precursor, as a 0.9 kb Eco RI fragment, was inserted into the transcription vector pGEM-9Zf(-) and transcribed with SP6 polymerase, as described by Newman and Gray [18 ], with the plasmid in the supercoiled state. Translation of the transcripts in a wheat germ extract in the presence of [35S]-

Tobacco

MAALQQTPITFQSRSPPPTQIIS

Spinach

MAALQN-PVALQSRTTTAVAALSTSSTTSPPKPFSLSFSSSTATFNPLRLKILTASKLTA

..... GPTAK--LSFS---GGLKLPKLTIKLRSNRTS

50 59

Tobacco

RRGGGAAGSK~ASAAGS~N~LADIAKSNGTLEQTTA~LEKIEKISDDEAVFNFFVSPI

ii0

Spinach

KPRGGALGTRMVDSTAS S LADVADVTGTLEATNS VEKLIRIFSEEPVYYFFANPV MSINPLASKIAAPIYAIR~LFDFSVDQNLMHQITA~FQNLEVFLNKTPDLTEYLSNP MTSTSQLFDPIYAIE~LMAIAREQGLEDRFGE~AALFRSTLAASADLRHLLENP MTSKVANTEVAQPIYAIQ~LLSIAKSKSLTEEFGT~ARTLLNLLTENQQLRNFIDNP MSEFITVARPY~_~K~AFDFAVEHQSVERWQ-~MLAFAAEVTKNEQMAELLSGA

I19 55 52 55 51

Tobacco

VGEEKKRELVDEIVSSSSIQPHVANFLNILVDMK~VELIKEIVKEFEKVYNTLTDTELAV

170

Spinach Odontella sinensis Synechococcus 6301 Anabaena 7120 E. coil

ISIDNKRSVLDEIITTSGLQPHTANFINILIDSENINLVKEILNEFEDVFNKITGTEVAV LISAKSKEEVLNKTL-KS-QINKETFKFLIVLVN~SRINLLEPIIASYLNLVYNAASVKM TLFSSQKKAVLNQVFGSSVHPLVLNFLNLLVDRN~IAFLDGIADRYQALLRKLRNVVRAD FIAAENKKALIKQILSEAS-PYLRNFLLLLVDKR~IFFLPEILQQYLALLRQLNQTVLAE LAPETLAESFIA-VCGEQLDENGQNLIRVMAENG~LNALPDVLEQFIHLRAVSEATAEVD

179 113 112 114 ii0

Tobacco

VT-~VVKLESQHLAQIAKGVQRLTGSKNVR-IKTVI--~ESLVA~FT~RYGNSGSKLI~M

226

Spinach

VT- VVKLENDHLAQIAKGVQKITGAKNVR-IKTVI-PSLVA FT R Y G N E G S K L V IEV~TAYAFT-NLQKNPLIKKLKELT-NAREIRLVITV~SSLIG~FL~KT-N--SKVL~F VS-~AVPLTEAQVQVITEKVKQLTGAAGVEIESQV---~ADLLG~VI~K---VGSQVL~A VT-~AVALTEDQQQAVTEKVLALTKARQVELATKV---~SDLIG~VI~K---VGSQVI~S VI-~AAALSEQQLAKISAAMEKRL-SRKVKLNCKI---~KSVMA~VI~R---AGDMVI~G

Odontella sinensis Synechococcus 6301 Anabaena 7120 E. coli

Odontella sinensis Synechococcus 6301 Anabaena 7120 E. coli

M

235 168 165 167 162

4,

Tobacco

S VKKQ~ED

Spinach Odontella sinensis Synechococcus 6301 Anabaena 7120 E. coli

IAAQ~E

I GD I Q L A V

248

SVKKQ

EEIAAQ .T .I.KN . .Q]L[Q . .K .LA . .K HILID H E. M .S .VD.LD.EV IT L A V

257

S LRGQILIKRi S I SILIAA S I RGQILIRRL S L RILISN S S V R G R ~ E RLADV~JQ S

187 180 183 177

Fig. 2. Comparison of amino acid sequences of 6 subunits of ATP synthases. Sequences are derived from nucleotide sequences presented as follows: spinach [ 10], Odontella sinensis [ 19], Synechocoecus 6301 [6], Anabaena 7t20 [ 15], Escherichia coli [8 ]. Amino

acid residues present in all six sequences are boxed. Amino acid residues identical in the tobacco and spinach sequences are marked with an asterisk (*). The processing site of the tobacco precursor protein is marked with an arrowhead. The numbering refers to amino acid residues.

552 methionine produced a labelled polypeptide of 29 kDa (Fig. 3). This polypeptide was imported by isolated pea chloroplasts and processed to the mature subunit of 20 kDa (Fig. 3). Treatment of the reisolated chloroplasts with thermolysin demonstrated that the 20 kDa protein was protected from degradation indicating that it had been translocated across the chloroplast envelope. The mature 20 kDa polypeptide was also generated from the 29 kDa precursor by incubation with a pea stromal extract (data not shown). The processing by the stromal extract was inhibited in the presence of 2 mM 1,10-phenanthroline, as has been shown for processing of other nuclearencoded chloroplast precursor proteins [1, 21]. Hybridisation of the c D N A insert to a Southern blot of tobacco genomic D N A digested with Eco RI, Hind III or Sty I gave a relatively simple pattern of hybridisation, with two main hybridising bands in each of the restriction enzyme digests (Fig. 4). Hybridisation was obtained with

Fig. 3. Chloroplast import and processing of the tobacco 6 subunit precursor. Isolated pea chloroplasts (25/~g chlorophyll) were incubated with 3sS-labelled precursor in the presence of 1 mM methionine in a final volume of 150/~1 of import buffer (50 mM Hepes-KOH pH 8.0, 330 mM sorbitol) for 30 min at 20 °C in the light. Thermolysin (100 #g/ml final conc) was added and incubated for 30 min on ice before intact chloroplasts were reisolated through 40~o Percoll. The chloroplasts were washed in import buffer, resuspended in SD S-sample buffer [ 12] and analysed on 15 ~ polyacrylamide gels in the presence of SDS [12]. 35S-labelled polypeptides were visualised by fluorography. Lane 1, 35S-labelled precursor of ~ subunit; lane2, reisolated chloroplasts; lane 3, thermolysin-treated chloroplasts. Marker proteins were serum albumin (68 kDa), ovalbumin (46 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12 kDa).

Fig. 4. Hybridisation of b subunit c D N A to Southern blots. Tobacco leaf D N A (10 #g) was digested for 16 h with 100 units ofEco RI (lane 1), Hind III (lane 2) and Sty I (lane 3) in a volume of 400/~1, ethanol precipitated and suspended in 10/, 1 10 mM Tris-HC1 pH 8.0, 1 mM Na2EDTA. These samples were fractionated by electrophoresis in 0.7% agarose, capillary blotted to GeneScreen Plus (NEN Research Products, DuPont) and hybridised at 65 °C with 32P-labelled cDNA, labelled by the random hexanucleotide primer method [7]. The filter was washed twice with 2 x SSC at room temperature, twice with 2 x SSC, 0.1% SDS at 65 °C and twice with 0.1 x SSC at room temperature, as described in the GeneScreen Plus manual. Markers were 2 D N A cut with Hind III.

4.7 kb and 9.4 kb Eco RI fragments, 7.0 kb and 23 kb Hind III fragments and 6.9 kb and 8 kb Sty I fragments. This suggests the presence of two genes, the minimum number expected in the haploid genome of Nicotiana tabacum, an amphidiploid species which arose following hybridisation between the ancestors of N. sylvestris and N. tomentosiformis [ 9]. Each of these progenitor plants would have provided one copy of the 6 subunit gene, and both copies would have been retained in the haploid genome of N. tabacum. Two genes encoding the 7 subunit of the chloroplast ATP synthase are also present in the tobacco haploid genome [ 11 ]. Single-copy genes encoding the 7 and 6 subunits of the chloroplast ATP synthase are present in the spinach genome [3, 13]. The presence of two genes encoding the b subunit in tobacco could lead to heterogeneity in the 6 sub-

553 u n i t p r e s e n t in t h e A T P s y n t h a s e c o m p l e x . E v i d e n c e for this is p r o v i d e d b y t h e h e t e r o g e n e i t y at p o s i t i o n 15 in t h e d e t e r m i n e d N - t e r m i n a l seq u e n c e . B o t h V a l a n d Ile w e r e d e t e c t e d at this p o s i t i o n , a l t h o u g h t h e s e q u e n c e o f t h e c D N A ind i c a t e d Ile (Fig. 1). T h i s s u g g e s t s t h a t c h a r a c t e r isation of additional cDNA clones may provide t h e s e q u e n c e o f t h e p r o t e i n c o n t a i n i n g Val-15.

Acknowledgements W e w o u l d like to t h a n k P r o f e s s o r D r R . G . H e r r m a n n for t h e gift o f t h e s p i n a c h 5 s u b u n i t c D N A , P r o f e s s o r D r R . J . B e r z b o r n for gifts o f a n t i s e r a , D r L. P a c k m a n for c a r r y i n g o u t N - t e r m i n a l seq u e n c i n g a n d C a t h e r i n e S m a r t f o r t h e gift o f t h e tobacco leaf cDNA library. K.H.L. was supported by a grant from The Swedish Institute, Stockholm and F.M. was supported by an EMBO Fellowship and by a Fellowship from the Spanish Ministry of Education. This work was s u p p o r t e d b y a r e s e a r c h g r a n t f r o m t h e U . K . Science and Engineering Research Council.

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restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 (1983). 8. Gay NJ, Walker JE: The atp operon: nucleotide sequence of the promoter and the genes for the membrane proteins, and the 5 subunit of Escherichia coli ATP-synthase. Nucl Acids Res 9:3919-3926 (1981). 9. Gray JC, Kung SD, Wildman SG, Sheen SJ: Origin of Nicotiana tabacum L detected by polypeptide composition of Fraction I protein. Nature 252:226-227 (1974). 10. Hermans J, Rother C, Bichler J, Steppuhn J, Herrmann RG: Nucleotide sequence of cDNA clones encoding the complete precursor for subunit delta of thylakoid-located ATP synthase from spinach. Plant Mol Biol 10:323-330 (1988). 11. Larsson KH, Napier JA, Gray JC: Import and processing of the precursor form of the gamma subunit of the chloroplast ATP synthase from tobacco. Plant Mol Biol 19:343-349 (1992). 12. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 (1970). 13. Mason, J, Whitfeld, PR: The ~-subunit of spinach chloroplast ATP synthase: isolation and characterisation of cDNA and genomic clones. Plant Mol Biol 14: 10071018 (1990). 14. Matsudaira P: Sequence from picomole quantities of protein electroblotted onto polyvinylidine difluoride membranes. J Biol Chem 262:10035-10038 (1987). 15. McCarn DF, Whitaker RA, Alam J, Vrba JM, Curtis SE: Genes encoding the alpha, gamma, delta and four F 0 subunits of ATP synthase constitute an operon in the cyanobacterium Anabaena sp. strain PCC 7120. J Bact 170:3448-3458 (1988). 16. Nechushtai R, Nelson N, Mattoo AK, Edelman M: Site of synthesis of subunits to photosystem I reaction center and the proton-ATPase in Spirodela. FEBS Lett 125: 115-119 (1981). 17. Nelson N, Nelson H, Schatz G: Biosynthesis and assembly of the proton-translocating adenosine triphosphatase complex from chloroplasts. Proc Natl Acad Sci USA 77: 1361-1364 (1980). 18. Newman BJ, Gray JC: Characterisation of a full-length cDNA clone for pea ferredoxin-NADP ÷ reductase. Plant Mol Biol 10:511-520 (1988). 19. Pancic PG, Strotmann H, Kowallik KV: The 8 subunit of the chloroplast ATPase is plastid-encoded in the diatom Odontella sinensis. FEBS Lett 280:387-392 (1991). 20. Pick U: Isolation of the ATPase complex (CFo-CF1). In Edelman M, Hallick RB, Chua N-H (eds) Methods in Chloroplast Molecular Biology, pp. 873-880. Elsevier, Amsterdam, (1982). 21. Robinson C, Hlis RJ: Transport of proteins into chloroplasts. Partial purification of a chloroplast protease involved in the processing of imported precursor polypeptides. Eur J Biochem 142:337-342 (1984). 22. Shinohara K, Minami E-I, Watanabe A: Synthesis and

554 assembly of H +ATPase complex by isolated 'rough' thylakoids. Arch Biochem Biophys 260:452-460 (1988). 23. 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).

24. Watanabe A, Price CA: Translation of mRNAs for subunits of chloroplast coupling factor 1 in spinach. Proc Natl Acad Sci USA 79:6304-6308 (1982). 25. Westhoff P, Nelson N, Btinemann H, Herrmann RG: Localization of genes for coupling factor subunits on the spinach plastid chromosome. Curr Genet 4:109-120 (1981).