Proc. Nati. Acad. Sci. USA Vol. 85, pp. 787-791, February 1988 Botany Structure and expression of spinach leaf cDNA encoding ribulosebisphosphate carboxylase/oxygenase activase (photosynthesis/Arabidopsis thaliana/nucleotide binding site/mRNA processing) JEFFREY M. WERNEKE*, RAYMOND E. ZIELINSKI*, AND WILLIAM L. OGRENtt *Department of Plant Biology, University of Illinois, Urbana, IL 61801; and tAgricultural Research Service, U.S. Department of Agriculture, 1102 South Goodwin Avenue, Urbana, IL 61801 Contributed by William L. Ogren, October 12, 1987

ABSTRACT Ribulosebisphosphate carboxylase/oxygen- gene, expressed the cDNAs in Escherichia coli, and used the ase activase is a recently discovered enzyme that catalyzes the clones as hybridization probes to address the specific nature activation of ribulose-1,5-bisphosphate carboxylase/oxygenase of the rca mutation.§ ["rubisco"; ribulose-bisphosphate carboxylase; 3-phospho-D- glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] in vivo. MATERIALS AND METHODS Clones of rubisco activase cDNA were isolated immunologi- Purification of Rubisco Activase. Intact spinach chloro- cally from spinach (Spinacea oleracea L.) and Arabidopsis plasts were lysed by 1:10 dilution into 20 mM Tris HCI, pH thaliana libraries. Sequence analysis of the spinach and Ara- 8/4 mM 2-mercaptoethanol (8). After centrifugation at bidopsis cDNAs identified consensus nucleotide binding sites, 10,000 x g for 10 min, the supernatant was passed through consistent with an ATP requirement for rubisco activase a 22-ptm Milex filter. Forty milligrams of soluble protein was activity. A derived amino acid sequence common to chloro- then loaded onto a Mono Q column (Pharmacia) equilibrated plast transit peptides was also identified. After synthesis of in the same buffer. Protein was eluted from the column with rubisco activase in vitro, the transit peptide was cleaved and a KCI gradient, and the fractions at 170-210 mM KCI were the protein was transported into isolated chloroplasts. Analy- combined. Solid ammonium sulfate was added to 35% satu- sis of spinach and Arabidopsis nuclear DNA by hybridization ration and the precipitated protein was collected by centrif- indicated a single rubisco activase gene in each species. Leaves ugation. After resuspension in a minimal vol of 100 mM of spinach and Arabidopsis wild type contained a single 1.9- Tris HCI, pH 8/4 mM 2-mercaptoethanol, rubisco activase kilobase rubisco activase mRNA. In an Arabidopsis mutant protein was further fractionated by gel filtration on a Super- lacking rubisco activase protein, mRNA species of 1.7 and 2.1 ose 12 column (Pharmacia) equilibrated with the same kilobases were observed under conditions of high-stringency buffer. A portion of each fraction was removed, precipitated hybridization with a wild-type cDNA probe. This observation with acetone, resuspended in NaDodSO4 sample buffer, and indicates that the lesion in the mutant arises from an error in run on polyacrylamide gels to determine purity. Fractions mRNA processing. containing rubisco activase eluted in the 100- to 200-kDa size range and consisted of two polypeptides, with apparent Ribulose-1,5-bisphosphate carboxylase/oxygenase ["ru- molecular masses of 45 and 41 kDa. bisco"; ribulose-bisphosphate carboxylase; 3-phospho-D- Antibody Preparation. Antibodies against spinach activase glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], the ini- were produced in BALB/c mice (9), with two immunizations tial enzyme in photosynthetic carbon dioxide reduction and 3 weeks apart. The antigen was prepared by suspending 15 photorespiratory carbon oxidation, must be converted to an ,ug of acetone-precipitated protein in 100 ,ul of 10 mM activated state for catalytic competency (1). The activation Tris HCI (pH 8.0) and emulsifying in an equal volume of process in vivo had been considered to occur by the spon- Freund's complete adjuvant. Ascites fluid was collected and taneous addition of CO2 and Mg2+ to a lysine residue of the centrifuged briefly to remove clotted material, divided into enzyme (1, 2), but it was recently shown that activation in aliquots, and stored at - 80°C. vivo is catalyzed by a soluble chloroplast protein (3, 4). First Affinity Purification of Monospecific Polyclonal Antibodies. evidence that rubisco activation in vivo is not a spontaneous Approximately 2 mg of soluble spinach leaf protein was process came from the isolation of an Arabidopsis thaliana fractionated on a preparative-scale NaDodSO4/polyacrylam- nuclear gene mutant (rca) deficient in the ability to activate ide gel, transferred electrophoretically to nitrocellulose, and rubisco (5). Subsequently, it was demonstrated that activa- incubated with rubisco activase antiserum. Antibodies tion could be restored in reconstituted mutant chloroplasts bound to each polypeptide were eluted separately by a 30-s by adding chloroplast extracts from spinach or Arabidopsis incubation of nitrocellulose strips in a minimal vol of 250 mM wild type (3, 4). The protein that restored activation, rubisco glycine hydrochloride (pH 2.4). This solution was then activase, has been purified and found to occur in all higher removed from the nitrocellulose and neutralized with an plant species examined (6). equal vol of 1 M Tris-HCI (pH 8.2). The eluted antibodies Little is known about the reaction mechanism of rubisco were diluted 1:50 into buffer containing 0.5% gelatin and activase other than requirements for ribulosebisphosphate 0.02% sodium azide and stored at 4°C. (4) and ATP (7). Likewise, there is little information on the RNA Extraction and RNA Blot Analysis. Cytoplasmic RNA nature of the lesion in the Arabidopsis rubisco activation was prepared from fresh or frozen (- 80°C) leaf tissue (10). mutant except that the pattern of inheritance is Mendelian Poly(A)+-enriched RNA fractions were prepared by affinity (3, 5), indicating that the protein is nuclear encoded. In the Pharmacia P-L experiments reported here, we have isolated cDNA clones chromatography on poly(U)-agarose (type 6, to determine the primary structure of the rubisco activase tTo whom reprint requests should be addressed. §The sequence reported in this paper is being deposited in the The publication costs of this article were defrayed in part by page charge EMBL/GenBank data base (Bolt, Beranek, and Newman Labora- payment. This article must therefore be hereby marked "advertisement" tories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg) in accordance with 18 U.S.C. §1734 solely to indicate this fact. (accession no. J03610). 787 Downloaded by guest on October 1, 2021 788 Botany: Werneke et al. Proc. Natl. Acad. Sci. USA 85 (1988) Biochemicals), dissolved in sterile water, and stored at - 80'C. RNA was fractionated in agarose-formaldehyde gels 1 2 (11) and transferred to nitrocellulose (12). Blotted and baked filters were rehydrated (12) and prehybridized in 0.675 M NaCl/50 mM NaHPO4/Na2HPO4/5 mM Na2EDTA, pH 7.4/5 x Denhardt's solution (1 x Denhardt's solution = 0.02% bovine serum albumin/0.02% Ficoll/0.02% polyvinyl- pyrrolidone)/0.1% NaDodSO4/100 1Lg of denatured salmon sperm DNA per ml at 420C for 6-16 hr. Hybridization was carried out for 6-24 hr at 420C in a similar solution except that formamide was added to 40%o, salmon sperm DNA was omitted, and 32P-labeled probe was included at 106 cpm/ml. cDNA Cloning and Immunological Screening. Double- stranded cDNA was synthesized by the following modifica- tion of a published method (13): first strand synthesis was FIG. 1. Cross-reactivity of the 45- and 41 kDa rubisco activase carried out with 2-5 ug of poly(A) + RNA in a vol of 50 Al, polypeptides. Anti-rubisco, activase antibodies were bound to pre- using 200 units of Molony murine leukemia virus reverse parative immunoblots of spinach leaf polypeptides. Antibodies transcriptase (Bethesda Research Laboratories). Reverse reacting with the 45- and 41-kDa polypeptides were eluted sepa- transcription was performed at 370C for 60 min rately and used to probe a second blot. Antibodies eluted from the and was 45-kDa band reacted with both the 45- and the 41-kDa polypeptides terminated by the addition of EDTA to 20 mM. The reaction on a second blot (lane 1). Likewise, antibodies eluted from the mixture was then extracted with phenol/chloroform and 41-kDa polypeptide on the preparative blot reacted with both precipitated twice with ethanol. Second strand reactions polypeptides (lane 2). contained 1 ,ug of cDNA-mRNA hybrids, 20 mM TrisHCI, pH 7.5/5 mM MgCl2/100 mM KCl/100 ,uM each dNTP/ pletely absent in the rca mutant (6). To determine whether bovine serum albumin (50 1kg/ml)/0.75 unit of E. coli RNAse these two p'olypeptides are related,im polyclonal antibodies H (Bethesda Research Laboratories)/25 units of DNA poly- reacting with the 45- or the 41-kDa polypeptides were eluted merase I (Bethesda Research Laboratories), in a final vol of separately from a preparative immunoblot and used to probe 100,ul and were incubated at 12°C for 2 hr. The reaction was duplicate preparations of spinach leaf soluble proteins im- terminated by phenol/chloroform extraction, adjusted to 200 mobilized on nitrocellulose. Antibodies eluted from either mM NaCl, and precipitated with ethanol. EcoRI sites on the the 45- or 41-kDa bands react with both polypeptides on a cDNAs were protected by methylation with 10 units of second immunoblot (Fig. 1). This result indicates that the EcoRI methylase (New England Biolabs) and 0.4 mM S- two polypeptides are derived from the same, or very similar, adenosylmethionine in 10 Al. After phenol/chloroform ex- molecules. The two polypeptides may arise from processing traction and ethanol precipitation from 200 mM NaCl, EcoRI upon, or after, import into the chloroplast, or may result linkers were ligated onto the cDNAs. After cleavage with from a protease-sensitive site on the protein. Genomic EcoRI, excess linkers were removed by gel filtration on a Southern blot analysis indicates that there is only one activase column of Bio-Gel A1.5m. The cDNAs were then cloned gene per genome in both spinach and Arabidopsis (Fig. 2), s0 into the EcoRI site of Agtll (14) and packaged in vitro it is unlikely that the two polypeptides are separate subunits according to the manufacturer's directions (Promega Biotec, transcribed from different, but related, genes. Madison, WI) to yield 106 recombinant phage per ug of Isolation of Spinach Activase cDNA Clones. Ascites fluid mRNA. The cDNA libraries were screened with mouse containing anti-rubisco activase antibodies was used to anti-rubisco activase, and immune complexes were detected screen spinach and Arabidopsis leaf cDNA libraries cloned with an alkaline phosphatase-conjugated secondary antibody. in Agtll. Of 2.5 x i01 spinach clones screened, 2 gave In Vitro Translation and Immune Precipitation. For detec- tion of rubisco activase mRNAs, aliquots of poly(A)+ mRNA were translated in vitro in a wheat germ cell-free 1 23 4 56 system (15) using labeled [35S]methionine (>600 Ci/mmol; 1 Ci = 37 GBq). Immune complexes were precipitated from translation mixtures using Protein A-Sepharose (16). Immu- noprecipitated polypeptides were analyzed by NaDodSO4/ 5-0 PAGE and fluorography. Transport into Chloroplasts. A 1.9-kilobase (kb) cDNA coding for the entire rubisco activase polypeptide was sub- qw cloned into plasmid pTZ18U at the EcoRI site. The plasmid was linearized downstream of the rubisco activase coding ..:.4..:.:. region with BamHI and then added to a reaction mixture containing T7 RNA polymerase and NTPs to produce large amounts of rubisco activase mRNA. Transcription was terminated by phenol/chloroform extraction and the mRNA was recovered by ethanol precipitation. Wheat germ cell- free translations were performed as described above, and FIG. 2. Southern blot analysis of the rubisco activase gene. Ten contained 1,ug ofT7-generated activase mRNA per 40 /l of micrograms of genomic DNA from spinach, Arabidopsis wild type, reaction mixture. Transport of rubisco activase into chloro- and the Arabidopsis rca mutant was digested with EcoRI or HindIII, plasts was determined essentially as described (17). electrophoresed on a 1% agarose gel, and transferred to nitrocellu- lose. The spinach blot was probed with a 1.6-kb spinach rubisco RESULTS activase cDNA, and the Arabidopsis blot was probed with a 1.2-kb Arabidopsis rubisco activase cDNA. Lanes: 1, spinach (HindIII); 2, Antisera Specificity. Anti-rubisco activase antibodies re- spinach (EcoRI); 3, Arabidopsis wild type (HindIII); 4, Arabidopsis acted specifically with two polypeptides present in leaf wild type (EcoRI); 5, Arabidopsis rca mutant (HindIII); 6, Arabi- extracts of spinach, wild-type Arabidopsis, and all other dopsis rca mutant (EcoRI). Approximate sizes in kb are indicated on higher plants examined (6). These polypeptides were com- the left. Downloaded by guest on October 1, 2021 Botany: Werneke et al. Proc. Natl. Acad. Sci. USA 85 (1988) 789 1 2 3 4 56 7 8910 1 2 3 4 5 6 78910 66-66 45 . _ 45--a- 31 ~ e31-

21--- 21------IND.-

FIG. 3. Expression of rubisco activase in E. coli. (Left) Lanes: 1, molecular mass standards (in kDa); 2-6 and 8-10, E. coli extracts; 7, spinach leaf extract. (Right) Anti-rubisco activase immunoblot of the same gel. positive signals. One of these recombinants contained a A series of BAL-31-deleted clones were produced and 1.6-kb cDNA insert, which was subcloned into the EcoRI site subcloned into M13 vectors for sequence analysis. Both ofpUC8 for bacterial expression. E. coli clones harboring this strands of the cDNA were sequenced for >90% of the length plasmid, designated pRCA1.6, were then assayed for expres- of the gene, using the dideoxy chain-termination method (18) sion of activase fusion proteins by immunoblot analysis of E. (Fig. 4). To confirm the identity of the cDNA, a sample of coli protein extracts. One-half of the recombinant clones purified rubisco activase (6) was subjected to N-terminal produced immunoreactive rubisco activase fusion proteins sequence analysis. The predicted amino acid sequence from with apparent molecular masses nearly identical to rubisco the cDNA clone (residues 59-77 of the precursor polypep- activase isolated from spinach leaves (Fig. 3). Further analy- tide) and the sequence of the first 19 amino acids from the sis by digestion with HindIII showed that the clones not N-terminal end of the purified protein, determined by Ed- producing immunoreactive protein contained the cDNA in the man degradation, were identical. Also, the fusion protein opposite orientation relative to the lacZ promoter. produced in E. coli from the 1.6-kb cDNA possessed rubisco

TTTTAGGAATGGAGACCTACAACAAATTAAATTMACCMCAAAACTTTGAGACTATTTATTCATTATTTACAGAGTAAACAGCTCGCTATAACACAAAACGCATAT TAAAATTACAAAAACAGAACAACTACTACTTCTCACTTCTCAGGGCTTTCTTCTAC CCAAAAAAAGTAACACTCTTTG CTMATCTTTTTAGTGTTCTACC CATCCTCC CCATCGGATCTCGTCGCCCGTTGGATTGATATCGGCTACTGCTGTCTCGACCGTTGGAGCTGCCACCAGGGCACCTTTGAACTTGAATGGGTCAAGCGCAGGGGCATCA M A T A V S T V C AA T R A P L N LN C SS A G A S 26 GTCCCAACATCAGGTTTCTTGGGGAGCCAGCTTAAAGAAGCATACAAATGTTAGATTCCCAAGCCAGCTCCAGGACAACCTCAATGACCGTCAAGGCCGCCGCACAATGAG V P T S C F L C S S L KK H T N V R F P SSS R TT S MT V K A A E N E 62 MT M I T N S F F F L GAGAAGAACACCGACAAATCGGGCTCATTTGGCTMGGCACTTTTCTGATGACCAACTTGACATCCGTACGGGGTAAGGGTATGGTTGACAGTCTCTTCCAAGCTCCTGCT E K NT D K WA H L A K D F S D D Q L D I R R G K G M V D S L F Q A P A 98 GATGCCCGGTACCCACGTTCCCATTCAGAGTTCCTTTGMATACGAGAGCCCAAGGTCTTCGAMAGTACGACATTGACAACATGTTGGGTGATCTCTACATTG CCCCTGCC D A C T H V P I Q SS F E Y E S Q C L R KY D I D N M L C DL Y I A P A 134 TTTATGGACMAGCTTGTTGTTCACATCACCAAGAACTTCTTGAACTTG CCCAACATCAAGATACCACTCATCTTGGGTGTTTGGGGAGGCAAGGGTCAAGGTAAATCC F M D K L VV H I T K N F L NL P N I K I P L I L C V W C C K C Q G K S 170 TTCCAATGTGAGCTTGTGTTCGCCAAGTTAGGAATAAACCCCATCATGATGAGTGCCCGGAGAATTGGAAAGTGGAAATGCAGGAGAGCCCAGCTAAGTTGATCAGGCAA F Q CE L V F A KL C I N P I M M S A C E LE S C N A G E P A K L I R Q 206 AGGTACCGTGAGGCAGCAGACTTGATTG CTAAGGGTAAGATGTGTGCTCTATTCATCAACGATCTGGAACCCGGTGCTGGACGTATGGGAGGCACCACCCAATACACC R Y R E A A DL I A K G K M C A L F I N D L E P G A C R M G G TT Q Y T 242 GTAAACAACCAGATGGCTTAACGCCACACTCCTGAACATTGCTGACAACCCAACCAATGCTCCAACTCCCTGGTATGTACAACAAGCCACGACAATGCCCGTGTCCCCATC V N N Q M V N A T L L N I A D N P T N V Q LP G M Y N K Q DN A RV P I 278 ATTGTTACTGGTAACGATTTCTCCACCTTGTACGCTCCCCTTATCCGTGATG GTCGTATGCGAGAAGTTCTACTCGGGCTCCCACCCGTGAGGACCGTATTGGTGTCTGT I VT G N D F S T LY A P L I R D G R M E K F Y W A P TR E D R I G V C 314 ACCGGTATTTTCAAGACTGACAAAGTTC CTGCAGAACACGTTGTTAAG CTCGTTGACGCCTTCCCTGGACAATCTATCCACTTTTTCGGAGCCGTTGAGCGCCTCGTGTA T G I F K T DK V P A E H V V K LV D A F P C Q S I D FF C A L R A R V 350 TACGACGATGMAGTMAGGMGTGGGTTAATAGTGTAGGAGTGGACAATGTAGGAAAGAAG CTGGCTGAACTCAAAGGATGGACCACCAGTGTTTGCAGCAACCACAAATC Y DD E V R K W V N S V C V D N V G K K L V N S K D C P P V F E Q PE M 386 ACCTTACAAAAGTTGATGGAGTACGGAAACATGCTTGTGCAAGAGCAAGAGAATGTCAAGAGAGTC CAACTTGCTGACCAGTACATGAGCTCCCGCTCCACTTCGTGAT T L Q KL M E Y C N M L V Q E Q E N V K R V Q L A D Q Y M SS AA 1 C D 422

GCCAACMAAGATGCCATTGACAGAGGMACTTTCTTCGG CAMAGCAGCTCAGCcMGTMAGTTTGCCAGTTGCTCAAG GTTGTACAGACC CTCAGGCC AAAAACTATGAT A N K D A I D R C T F F G K A AQ Q V S L P V AQ C C T D P E A K N Y D 458

CCAACTGCAAGGAGTGATGATGGGAG CTGCACGTACAATTTGTAGGTCTTACTCAATTTGTTGC AACTGGATATCAGAAAAAGGGGAACAATTTTAGTTAATTTGGC P T A R S DD G S C T Y N L Z 472 TCTCTTAATTAGAGGGATTATTATTCCACTTCCTATACTTTGCCTATrTTTTTTCTTTTTTAATTTTTGTCTCGTTGC1 GAGTTGTTTCTCCTCTAATTTTCTCTT FIG. 4. Nucleotide sequence of the 1.9-kb spinach rubisco activase cDNA. The preprotein is cleaved between amino acids 58 and 59 upon import into the chloroplast (indicated by the first arrow). Consensus nucleotide binding sites (19) are located at positions 163-170 and 218-227 (underlined). The amino acid sequence of the rubisco activase 1.6-kb cDNA (pRCA1.6) expressed in E. coli begins at position 30. The protein contains substituted amino acids in positions 30-40 as indicated in the lower line. The pRCA1.6 cDNA terminates translation after amino acid 435 (indicated by the second arrow). Amino acids are identified by the single-letter code. Downloaded by guest on October 1, 2021 790 Botany: Werneke et al. Proc. Natl. Acad. Sci. USA 85 (1988) activase activity (J.M.W., J. M. Chatfield, and W.L.O., tively and was found to harbor a 0.5-kb Arabidopsis cDNA unpublished data). Inspection of the derived amino acid insert. Sequence analysis of the Arabidopsis cDNA showed sequence of the spinach rubisco activase gene revealed two considerable similarity to the spinach cDNA at the amino regions (amino acid residues 163-170 and 218-227) that acid level, while the third positions of the codons varied share homology with the nucleotide binding domains identi- considerably between the two species (data not shown). fied in a variety of polypeptides from both animals and Using this 0.5-kb cDNA as a hybridization probe, we recov- bacteria (19, 20). The amino acid sequences of these regions ered a 1.9-kb Arabidopsis wild-type cDNA and a 1.4-kb were identical in the spinach and Arabidopsis cDNAs. These cDNA from a AgtlO cDNA library made from rca mutant observations are consistent with an ATP requirement for mRNA. rubisco activase-mediated activation of rubisco in vitro (7). The spinach and Arabidopsis activase cDNA clones were The 1.6-kb cDNA was used as a hybridization probe to used as hybridization probes in RNA blot experiments to rescreen the spinach Agtll library for larger activase determine the size of the activase transcripts from a variety cDNAs. A 1.9-kb cDNA was recovered and common se- of plants and to explore the nature of the rca mutation in quences were found to be identical with the 1.6-kb cDNA. Arabidopsis (5). Poly(A)+ mRNA isolated from spinach, The 1.9-kb cDNA also encoded the entire transit peptide and barley, pea, and Arabidopsis contained a single species of a portion of the 5' untranslated region of the activase mRNA, -1.9 kb long, which hybridized to both the spinach mRNA. The nucleotide and derived amino acid sequences or Arabidopsis cDNA clones. However, poly(A) + mRNA are given in Fig. 4. fractions isolated from the rca mutant of Arabidopsis con- Presence of a Transit Polypeptide. Many chloroplast poly- tained two rubisco activase mRNA species, one 0.2 kb peptides are encoded by the plant nuclear genome, synthe- smaller and the other 0.2 kb larger than the authentic rubisco sized in the cytoplasm, and then imported into the chloro- activase mRNA. This observation is consistent with the plast posttranslationally (21). These proteins are character- possibility that the rca mutation alters normal processing of ized by the presence of a semiconserved N-terminal transit the rubisco activase mRNA precursor. peptide sequence that is cleaved upon import into the plastid (22, 23). The presence of a rubisco activase transit peptide DISCUSSION was demonstrated by translating spinach and pea poly(A)+ Purified spinach rubisco activase preparations contain two mRNA in a wheat germ extract and then using the anti- immunologically related polypeptides of -41 and -45 kDa. rubisco activase ascites fluid to immunoprecipitate rubisco The enzyme is initially synthesized as a 51-kDa precursor, activase precursor polypeptides. The size of the immunopre- and the 45-kDa polypeptide is the primary product after cipitated rubisco activase precursor was =51 kDa on chloroplast import. A second processing event appears to NaDodSO4/polyacrylamide gels for spinach (Fig. 5) and pea occur after uptake, generating the 41-kDa species. The (data not shown). presence of two polypeptides may also be the result of To confirm that the 51-kDa polypeptide contained a func- susceptibility to proteolytic cleavage during extraction, but tional transit sequence, the 1.9-kb rubisco activase cDNA this explanation is unlikely since activity in vitro was asso- was subcloned into a vector containing a T7 promoter and ciated with the smaller polypeptide (J.M.W., J. M. Chat- used to produce rubisco activase mRNA in vitro. This field, W.L.O., unpublished data). It has been observed that mRNA was then used to produce precursor polypeptides in the relative ratios of the 41- and 45-kDa polypeptides vary a wheat germ translation system. The polypeptides produced greatly between plant species (6). The 41-kDa polypeptide is from the cloned cDNA were identical in molecular mass to always prominent, but the amount of the 45-kDa polypeptide the polypeptides that were immunoprecipitated (Fig. 5). In may approximately equal the 41-kDa polypeptide (spinach), addition, when added to isolated chloroplasts, the 51-kDa be much reduced (barley), or be completely absent (maize). polypeptide was imported and cleaved to yield a major Within each species examined, the ratio of the polypeptides polypeptide of 45 kDa and a minor polypeptide of 41 kDa. was always the same. No differences have been observed in N-terminal sequence analysis of purified rubisco activase the relative amounts of the two polypeptides during devel- indicates the cleavage during transport occurs between the opment or in response to light. two alanine residues at positions 58 and 59 (Fig. 4). Several lines of evidence indicate that there is a single Arabidopsis cDNA Clones and the rca Mutation. Rubisco rubisco activase gene in spinach, Arabidopsis, and perhaps activase cDNA clones were isolated from an Arabidopsis other plants. In Arabidopsis, the rca phenotype is inherited wild-type cDNA library, constructed and screened in the as a simple Mendelian trait (3, 5). In both spinach and same fashion as the spinach cDNA library. Of 1.25 x iOs Arabidopsis, genomic Southern blot analysis revealed sim- plaques screened with spinach antisera, one reacted posi- ple hybridization patterns (Fig. 2). DNA sequencing of the 3' untranslated regions of several cDNA clones isolated from 1 2 34 5 spinach and Arabidopsis revealed an invariant nucleotide sequence within each species (data not shown). Finally, both 66 the 45- and 41-kDa rubisco activase polypeptides can be js derived from a single molecular species of 51-kDa rubisco 45 ! activase precursor (Fig. 4). Although these observations do not preclude the existence of multiple structural genes FIG. 5. Immunoprecipitation and transport of spinach rubisco encoding rubisco activase, they are consistent with a single activase into isolated chloroplasts. A O.5-,ug aliquot of poly(A)+ gene hypothesis. spinach leaf mRNA was translated in a wheat germ extract (lane 1). Comparing the derived amino acid sequence of rubisco Anti-rubisco activase antisera was then used to precipitate a 51-kDa activase (Fig. 4) with N-terminal amino acid sequences of polypeptide from the translation (lane 2). The immunoprecipitate is the two mature polypeptides indicates that the molecule the same molecular mass as the polypeptide produced from mRNA contains a transit peptide 58 amino acids long. After import generated in vitro from the 1.9-kb spinach activase cDNA (lane 3). and cleavage, the apparent size of the polypeptide is reduced The 51-kDa precursor polypeptide, generated from a spinach cDNA by -6 clone, was imported into isolated chloroplasts and led to the kDa (Fig. 5). There are several nuclear-encoded appearance of both the 45- and 41-kDa polypeptides within the chloroplast proteins for which transit peptide sequences are stroma (lane 4). The imported polypeptides are the same size as known. Within these precursors there appears to be a high those observed in immunoblots of spinach leaf polypeptides (lane 5). degree of amino acid sequence conservation at the N termi- Numbers on left are kDa. nus (22). Less sequence similarity is evident at the sites of Downloaded by guest on October 1, 2021 Botany: Werneke et al. Proc. Natl. Acad. Sci. USA 85 (1988) 791

cleavage that form the mature polypeptides. The proteolytic well characterized for the human f-globin genes (25). An processing site of the spinach rubisco activase transit pep- insertion or deletion in the rubisco activase gene might also tide appears similar to the site of plastocyanin cleavage, in cause transcription abnormalities leading to larger or smaller that both are cleaved between two alanine residues. How- mRNAs. This has probably not occurred in the Arabidopsis ever, the processing site in plastocyanin, a polypeptide asso- rca mutant, since the size of the wild-type and mutant ciated with the inner thylakoid membrane, is directly pre- rubisco activase genomic DNA restriction fragments were ceded by a group of uncharged amino acids. These residues identical (Fig. 2). may be necessary to span the thylakoid membrane prior to The data presented here are consistent with previous translocation (23). The rubisco activase precursor polypep- observations and conclusions drawn from genetic and bio- tide sequence bears no resemblance to the plastocyanin chemical experiments with rubisco activase. The Arabi- precursor sequence in this region, as might be expected for a dopsis rca mutation followed a single Mendelian inheritance protein whose destination is the stromal compartment of the pattern (3, 5), and there is likely only one gene encoding this plastid. The site of cleavage of the rubisco activase transit protein (Fig. 2). The derived amino acid sequence from peptide is preceded by a serine- and threonine-rich region, analysis of rubisco activase cDNA (Fig. 4), together with much like the rubisco small-subunit transit peptides (22, 23). partial N-terminal amino acid sequence of the mature poly- Rubisco activase requires ATP for activity (7), and the peptides, has identified a 58-amino acid transit peptide. deduced amino acid sequence of rubisco activase contains Thus, the protein is nuclear encoded, synthesized in the two regions with sequences similar to those suggested to be cytoplasm, and transported into the chloroplast as a 45-kDa involved in nucleotide binding (19). These regions are lo- polypeptide. Subsequent processing in the chloroplast yields cated at amino acids 163-170 and 218-227 of the precursor a 41-kDa polypeptide (Fig. 5), and it appears that only the polypeptide (Fig. 4). In chloroplasts of illuminated leaves, 41-kDa polypeptide possesses activity. Little is presently the ribulosebisphosphate concentration is in the range of3-6 known about the nature of the activity other than a require- mM. At this concentration, ribulosebisphosphate is a potent ment for ATP (7). The deduced amino acid sequence (Fig. 4) inhibitor of rubisco activity (24) and prevents spontaneous identified two regions, which are suggested (19) to represent rubisco activation (J.M.W., J. M. Chatfield, and W.L.O., nucleotide-binding sites in ATP-utilizing enzymes. The re- unpublished data). However, when ATP and rubisco acti- action mechanism of rubisco activase and the role of ATP in vase were added to a reaction mixture containing 3 mM this mechanism remain to be determined. ribulosebisphosphate at atmospheric C02, rubisco became activated. Thus, ATP appears to provide the energy needed We thank J. Mark Chatfield for providing the N-terminal amino to activate the acid sequence ofthe two rubisco activase polypeptides. J.M.W. was rubisco-ribulosebisphosphate complex. supported by a grant from the McKnight Foundation. Rubisco activase is encoded in several species by a single mRNA of =1.9 kb (Fig. 6). The rca mutant of Arabidopsis, 1. Lorimer, G. H., Badger, M. R. & Andrews, T. J. (1976) Biochem- which lacks detectable rubisco activase activity and poly- istry 15, 529-536. peptides (3, 6), contains two mRNA species. These mRNAs 2. Perchorowicz, J. T., Raynes, D. A. & Jensen, R. G. (1981) Proc. are =200 nucleotides and smaller than the Nati. Acad. Sci. USA 78, 2985-2989. larger wild-type 3. Salvucci, M. E., Portis, A. R., Jr., & Ogren, W. L. (1985) Photo- rubisco activase mRNA. Mutations disrupting normal tran- synthesis Res. 7, 193-201. scription initiation or termination, or pre-mRNA splicing, 4. Portis, A. R., Jr., Salvucci, M. E. & Ogren, W. L. (1986) Plant might be expected to alter the number or size of individual Physiol. 82, %7-971. mRNA transcripts. Of these possibilities, an alteration in 5. Somerville, C. R., Portis, A. R., Jr., & Ogren, W. L. (1982) Plant pre-mRNA splicing is most likely to produce multiple Physiol. 70, 381-387. mRNA without 6. Salvucci, M. E., Werneke, J. M., Ogren, W. L. & Portis, A. R., transcripts corresponding protein accumula- Jr. (1987) Plant Physiol. 84, 930-936. tion. If the rca mutation altered a nucleotide bordering either 7. Streusand, V. J. & Portis, A. R., Jr. (1987) Plant Physiol. 85, the 3' or 5' splicing sites, the rubisco activase mRNA 152-154. precursor might be spliced at normally unused "cryptic" 8. Salvucci, M. E., Portis, A. R., Jr., & Ogren, W. L. (1986) Anal. splice sites within an intron or adjacent exon. As a conse- Biochem. 153, 97-101. quence, two aberrant mRNAs would be produced, one 9. Lacy, M. & Voss, E. (1986) J. Immunol. Methods 87, 169-177. and one smaller than the 10. Cashmore, A. R. (1982) in Methods in Chloroplast Molecular larger wild-type transcript. Such Biology, eds. Edelman, M., Hallick, R. B. & Chua, N.-H. (Elsevier mutations and subsequent selection ofcryptic splice sites are Biomedical, Amsterdam), pp. 387-392. 11. Lizardi, P. M., Williamson, R. & Brown, D. D. (1974) Cell 4, 199-205. 1 2345 1 2 3 12. Thomas, P. S. (1983) Methods Enzymol. 100, 255-266. A1I B 13. Gubler, U. & Hoffman, B. J. (1983) Gene 25, 263-269. 14. Huynh, T. V., Young, R. A. & Davis, R. W. (1985) in DNA Cloning, ed. Glover, D. M. (IRL, Oxford), Vol. 1, pp. 49-78. -0- 15. Erickson, A. H. & Blobel, G. (1983) Methods Enzymol. 96, 38-50. -0- 16. Anderson, D. J. & Blobel, G. (1983) Methods Enzymol. 96, 111- 120. 's 17. Pain, D. & Blobel, G. (1987) Proc. Natl. Acad. Sci. USA 84, 3288-3292. 18. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 19. Duncan, T. M., Parsonage, D. & Senior, A. E. (1986) FEBS Lett. 208, 1-6. 20. Higgins, C. F., Hiles, I. D., Salmond, G. P. C., Gill, D. R., Dow- FIG. 6. RNA blot identification of rubisco activase nie, J. A., Evans, I. J., Holland, I. B., Gray, L., Bell, A. W. & mRNAs. Hermodson, M. A. Nature 448-450. Poly(A)+ mRNAs were fractionated on formaldehyde gels, trans- (1986) (London) 323, ferred to 21. Ellis, R. J. (1981) Annu. Rev. Plant Physiol. 32, 111-137. nitrocellulose, and hybridized with either the 1.6-kb 22. Karlin-Neumann, G. A. & Tobin, E. M. (1986) EMBO J. 5, 9-13. spinach rubisco activase cDNA (A) or a 0.5-kb Arabidopsis rubisco 23. Schmidt, G. W. & Mishkind, M. L. (1986) Annu. Rev. Biochem. activase cDNA (B). The sources of the mRNAs are as follows. (A) 55, 879-912. Lanes: 1, spinach; 2, barley; 3, pea; 4, Arabidopsis wild type; 5, 24. Jordan, D. B. & Chollet, R. (1983) J. Biol. Chem. 258, 13752-13758. Arabidopsis rca mutant. (B) Lanes: 1, spinach; 2, Arabidopsis wild 25. Treisman, R., Orkin, S. H. & Maniatis, T. (1983) Nature (London) type; 3, Arabidopsis rca mutant. 302, 591-596. Downloaded by guest on October 1, 2021