Synthase of Chlamydomonas Reinhardtii: Import and Cleavage of the Precursor Protein (Chloroplast Coupling Factor 1/Nuclear Encoded/Transcription/Translation) LLOYD M
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Proc. Nail. Acad. Sci. USA Vol. 85, pp. 1369-1373, March 5, 1988 Biochemistry Isolation of a cDNA clone for the y subunit of the chloroplast ATP synthase of Chlamydomonas reinhardtii: Import and cleavage of the precursor protein (chloroplast coupling factor 1/nuclear encoded/transcription/translation) LLOYD M. YU*t, SABEEHA MERCHANT*§, STEVEN M. THEG*, AND BRUCE R. SELMAN* *Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706; and tThe Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138 Communicated by Henry Lardy, October 19, 1987 ABSTRACT A cDNA library from Chlamydomonas rein- enzyme (11), and the mechanism of protein import into the hardti, constructed in the phage expression vector Agtll, was chloroplast. probed with antiserum directed against the nuclear-encoded y Chlamydomonas reinhardtii is a genetically malleable subunit of the chloroplast H+-transporting ATP synthase green alga (12) that contains one large chloroplast per cell. It [ATP phosphohydrolase (H+-transporting) or chloroplast is possible to isolate intact chloroplasts (13-15) that can coupling factors 0 and 1, EC 3.6.1.34] of C. reinhardtii. A import precursor proteins (16). Precursors of the nuclear- cDNA was isolated and transcribed in vitro. The transcript was encoded subunits are difficult to detect in vivo since their translated in vitro and immunoprecipitated with anti-y- half-lives are very short (9, 10). The study of the import of subunit serum to yield a product that coelectrophoresed with these subunits and their assembly into the complex is most the immunoprecipitated product from in vitro-translated poly- easily accomplished by using isolated chloroplasts; thus, it adenylylated RNA. These proteins were larger than the ma- becomes essential to synthesize these proteins in vitro. ture y subunit, either immunoprecipitated as chloroplast Toward this end, we have constructed a cDNA library coupling factor 1 or as the individual subunit. Thus, the y from C. reinhardtii in the chimeric protein expression vector subunit is synthesized as a precursor of greater molecular Agtll and cloned a cDNA for the precursor to the y subunit weight in C. reinhardtii. Furthermore, the precursor protein of CFo and CF1. While this work was in progress, Tittgen et encoded by the cDNA is imported into pea chloroplasts and al. (17) published work describing the isolation of a cDNA processed to a lower molecular weight polypeptide that coe- for the y subunit of the spinach ATP synthase. The two lectrophoreses with mature C. reinhardtii y subunit. The subunits probably fulfill similar roles in their respective largest cDNA isolated is about the same length as the corre- complexes. Regardless of the degree of amino acid sequence sponding mRNA (--1900 bases long) and probably contains the homology, their nucleotide sequences should be markedly entire coding region. Southern blot analyses revealed restric- different since the nuclear DNA of C. reinhardtii uses tion fragment length polymorphisms and that the y subunit is codons biased toward a guanine or cytosine in the second probably encoded by an intron-containing single-copy gene. and third positions (18-21). Proton-transporting ATP synthases [ATPases; ATP phos- MATERIALS AND METHODS phohydrolase (H + -transporting) or chloroplast coupling fac- tors 0 and 1, EC 3.6.1.34] are a class of multisubunit Library Construction. A cDNA library of C. reinhardtii enzymes found in energy-transducing membranes. The com- strain 2137 polyadenylylated RNA was constructed in the position of these coupling factors 0 and 1 (F0-F1) type expression vector Agtll as described (22). enzymes varies. For instance, the number of subunit types Screening. The Agtll library was screened with antibodies found in the Escherichia coli enzyme is less than the number directed against the y subunit of C. reinhardtii CF1 after of subunit types found in the chloroplast ATP synthase, removal of E. coli cross-reactive antibodies (23). Positive which in turn contains fewer types than the mitochondrial plaques were purified by rescreening three times. enzyme (1-3). Nevertheless, the mechanism of ATP synthe- Preparation of Antigens and Antisera. C. reinhardtii CF1 sis/hydrolysis, although unknown, appears to be conserved was purified as described (24) with slight modifications. The and, indeed, the catalytic subunits (a, I, and y) share washed membranes, at 3.0 mg of chlorophyll per ml, were conserved stoichiometries (4, 5); the f3 subunits, in particu- diluted 1:2 with a buffer containing 500 mM sucrose, 50 mM lar, share conserved amino acid sequences (6). Tris SO4 (pH 8.0), 4 mM dithiothreitol, 4 mM ATP, and 2 The chloroplast coupling factors 0 and 1 (CFo and CF1) mM EDTA (25) before the CF1 was extracted by swirling comprise the thylakoid-bound ATP synthase and together with 0.5 vol of redistilled chloroform. The phases were contain nine of separated by centrifugation at 39,000 x g for 15 min and the subunits (2, 3) which three (y, 6, and II) are aqueous phase was recentrifuged once before application to synthesized in the cytosol and encoded by nuclear DNA (7). a DEAE Sephadex A-50 column. The eluted CF1 was The initial translation products for these subunits presum- concentrated and stored as an ammonium sulfate precipitate ably bear N-terminal transit sequences (8-10), which are before use as an antigen. The subunits of CF1 were separated cleaved during import into the chloroplast. The ability to by preparative gel electrophoresis and the y subunit was synthesize the nuclear-encoded subunits of the CFo and CF1 electroeluted (26) from the appropriately excised portion of in vitro would aid in the study of their assembly into the the gel after visualizing with KCI (27). Antisera against the holoenzyme, the possible turnover of subunits in the mature Abbreviation: CF1, chloroplast coupling factor 1. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §Present address: Department of Chemistry and Biochemistry, in accordance with 18 U.S.C. §1734 solely to indicate this fact. University of California, Los Angeles, CA 90024. 1369 Downloaded by guest on September 28, 2021 1370 Biochemistry: Yu et al. Proc. Natl. Acad. Sci. USA 85 (1988) purified CF1 or the 'y subunit of CF1 were collected from Gel Electrophoresis. NaDodSO4 denatured proteins were immunized rabbits. separated on discontinuous polyacrylamide gels (32) and Immunoblots. Samples of purified CF1 and whole C. 35S-labeled proteins were visualized by fluorography (33). reinhardtii cells were denatured in sample buffer and elec- Denatured RNA was separated on 1.2% agarose gels con- trophoresed in a polyacrylamide gel. Two-thirds of the gel taining formaldehyde, and DNA was separated on 0.8% was transferred to nitrocellulose filter paper (4 hr, 60 V, at agarose gels containing a Tris-borate buffer (28). When 40C) in a buffer containing 25 mM Tris HCI, 192 mM glycine, appropriate, nucleic acids were visualized by ethidium bro- and 20% (vol/vol) MeOH; the remaining one-third was mide staining. stained with Coomassie brilliant blue. The filter was blocked Cells: Growth, Labeling and Sample Preparation. Strain with 5% fetal calf serum in 50 mM Tris (pH 8.0) and 200 mM CC 124 of C. reinhardtii was grown photoheterotrophically NaCl and then cut in half. Each portion was incubated to late logarithmic or early stationary phase on Tris- overnight at room temperature with 7.5 ml of a 1:500 dilution acetate/phosphate- medium (34) containing 200 MuM S042- of either anti-CF1 serum or anti-y-subunit serum. The anti- and 3 mCi of Na235SO4 in 125 ml of culture. The cells were gen-antibody complexes were detected by incubating the pelleted at 40C at 3000 x g for 3 min and washed once in 10 filters with horseradish peroxidase conjugated to goat (anti- mM NaPi buffer (pH 7.1). The final pellet was resuspended rabbit) antibodies followed by the addition of hydrogen in phosphate buffer. A 30-,p1 sample, containing 50 ug of peroxide and 4-chloro-1-naphthol to develop color. This chlorophyll, was frozen at - 80'C and thawed at 250C two detection procedure was carried out according to the manu- times before detergent solubilization and immunoprecipita- facturer's instructions [Bio-Rad Laboratories, Richmond, tion (30). The remainder of the cells were used to prepare CA; the Bio-Rad Immun-Blot (GAR-HRP) assay kit instruc- crude thylakoid membranes by expulsion through the orifice tions] except that three washes of the filters occurred prior of a cold (00C-40C) nitrogen cavitation bomb at 1000-750 psi to adding a reagent, and 0.05% (vol/vol) Nonidet P-40 was (1 psi = 6.89 kPa) followed by differential centrifugation at included only in the first of these three washes. 3000 x g for 45 sec. Three cycles through the bomb gave Plasmids. The cDNA inserts were prepared by EcoRI almost complete breakage; crude washed thylakoids and a digestion of Agtll DNA, agarose gel electrophoresis (28), CF1-containing extract were then prepared as described and freeze/thaw rupture and centrifugation of excised gel above. fragments (29). The inserts were ligated into the EcoRI site In Vitro Import into Pea Chloroplasts. The preparation of of the polylinker in the plasmid pTZ18R (United States pea chloroplasts and the in vitro import assays were per- Biochemical, Cleveland, OH; Genescribe-Z technical man- formed as described (35). ual) before transformation of the host cell JM101. Plasmids were isolated by alkaline detergent lysis of the host cells RESULTS followed by polyethylene glycol precipitation (Promega Bio- tec, Madison, WI; technical manual on sequencing). Immunoblots. Fig. 1 shows the specificity of the anti-y- RNA Preparation. Total RNA and polyadenylylated RNA subunit serum used during the screening of the Agtll library were isolated from C.