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Structure and Expression of Spinach Leaf Cdna Encoding

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Structure and Expression of Spinach Leaf Cdna Encoding 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.
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