Synthase of Chlamydomonas Reinhardtii: Import and Cleavage of the Precursor Protein (Chloroplast Coupling Factor 1/Nuclear Encoded/Transcription/Translation) LLOYD M

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 factors 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. reinhardtii strain CC 124 as described and of the anti-CF1 serum used for immunoprecipitations. (30). The Coomassie-stained polyacrylamide gel shows purified Hybridizations. After electrophoresis, nucleic acids were CF1 and whole cell protein (lanes 1 and 2). Identical samples transferred to GeneScreen filters by capillary action and were electrophoresed from the gel to nitrocellulose filter fixed by UV irradiation (31). Nick-translated probes (Be- papers and the (blocked) filters were probed with either thesda Research Laboratories, Gaithersburg, MD; instruc- anti-y-subunit serum or anti-CF1 serum. Antigen-antibody tions for catalog 8162SA) containing [a-32P]deoxycytidine complexes were detected as described. This anti-y-subunit were hybridized to the filters at 65°C-68°C (31) and were serum is highly specific for the y subunit found either in visualized by autoradiography after washing. purified CF1 or in a background of cellular protein (lanes 3 Transcription/Translation. Plasmid templates were linear- and 4). Anti-CF1 serum is specific for the 4-subunit CF1 ized with HindIII (28) and the runoff in vitro transcription A B C reaction was catalyzed by T7 RNA polymerase (Promega 2 3 4 5 6 Biotec, Madison, WI; Technical Bulletin 002). In vitro translation reactions were carried out as suggested by the manufacturer using rabbit reticulocyte lysates (Bethesda Research Laboratories, Gaithersburg, MD) at 88.5 mM K+, 1.17 mM Mg2", and 20 ,uCi of [35S]methionine (1 Ci = 37 .ON GBq) in 30-,l reaction volumes. Polyadenylylated RNA from C. reinhardtii at 1 ,g per reaction mixture gave 20-30 times more acid-precipitable radioactivity than did a reaction mixture with no added RNA. Immunoprecipitations. The immunoprecipitation of Na23sSO4-labeled cell extracts and in vitro translation reaction mixtures was performed essentially as described (30). Crude C. reinhardtii CF1 was prepared from 35S-labeled thylakoid membranes by chloroform extraction; the high- speed aqueous supernatant, brought to 200 mM NaCl, was FIG. 1. Specificity of the anti-v-subunit serum and anti-CF1 immunoprecipitated with anti-CF1 serum without the addi- serum. (A) Coomassie brilliant blue-stained polyacrylamide gel; tion of detergents. In all cases, the pellets of IgGsorb (The identical samples were transferred to nitrocellulose and probed with Enzyme Center, Boston, MA), which contained the antigen- preadsorbed anti-v-subunit serum (B) or with anti-CF1 serum (C). Lanes: 1, 3, and 5, purified 4-subunit C. reinhardtii CF1 (5 ,g); 2, 4, antibody complexes, were washed with 50 mM Tris-HCl (pH and 6, whole cells of C. reinhardtii (=12 ,ug of chlorophyll and 115 8.0), 200 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P40. ,ug of protein). Antigen-antibody complexes were visualized by first Prior to gel electrophoresis, the pellets were resuspended binding an enzyme-conjugated second antibody and then adding a and heated in sample buffer containing 3% NaDodSO4, 2.5% substrate for color development. The subunits of CF1 are labeled on 2-mercaptoethanol, and 8 M urea to solubilize proteins. the far left. Downloaded by guest on September 28, 2021 Biochemistry: Yu et al. Proc. NaMl. Acad. Sci. USA 85 (1988) 1371 isolated from C. reinhardtii, either as the purified protein or 2137 CC 124 --l in a background of cellular protein (lanes 5 and 6). The y N1-1 Q~~~~~~~~~( subunit is recognized by the anti-y-subunit serum as well as (i 0 by the anti-CF1 serum. ~~'~~~~ ~ ~ c cDNA Isolation. The first screening of the Agtll library, containing 106 phage, yielded 25 positive signals. These 14.8W regions were excised from the agar plates, and after three additional screenings, 14 purified phage remained. Of these, 9 were arbitrarily chosen. A reverse immunoblot (23) dem- _W 4.8 onstrated that these cDNAs, when expressed, were able to specifically remove the anti-y-subunit antibodies from the - 2.3 anti-CF1 serum (data not shown). DNA was isolated from - - these phage (36) and digested with EcoRl prior to electro- 6m0 1.4 phoresis. The released inserts ranged in size from about 1300 to 1900 base pairs. The three largest inserts (approximately 1900, 1760, and 1360 base pairs) were excised from the 0.4 agarose gel and were ligated into the EcoRI site in the plasmid pTZ18R (pTZ18R-yCFj-1A, pTZ18R-yCF,-4A, and pTZ18R-yCF,-9A, respectively); these three plasmids were 0.2 used to transform E. coli strain JM101. RNA Blot Analysis. The plasmids pTZ18R-yCF,-lA, FIG. 3. Autoradiograph of Southern blot hybridizations of pTZ18R-yCF,-4A, and pTZ18R-yCF,-9A and the host plas- genomic DNA from C. reinhardtii strains 2137 (Left) and CC 124 total (Right) with a cDNA probe of the C. reinhardtii y subunit. Genomic mid (pTZ18R) were nick-translated and used to probe DNA (1 ,ug) was restricted overnight with various restriction endo- RNA from C. reinhardtii for hybridizing sequences (Fig. 2). nucleases, separated by gel electrophoresis, and then transferred to The plasmids that contained cDNA inserts hybridized pri- a nylon filter. The blot was probed with the nick-translated plasmid marily to a single RNA species from C. reinhardtii (Fig. 2, pTZ18R-yCF1-1A. This cDNA probe was derived from strain 2137 lane 1 vs. lanes 2, 3, and 4). Using a calibration curve polyadenylylated RNA. Within this cDNA, HindIII and Sst I/Sac I constructed with ethidium bromide-stained RNA molecular have no recognition sites, Hinfl and Pst I each have two recognition weight standards (Bethesda Research Laboratories), this sites, and Pvu II has one recognition site. Size markers in kilobases RNA was -1900 bases long and was about the same size as are indicated in the center lane. the largest cDNA. Genomic Southern Analysis. Nick-translated cDNA ments are generated for closely linked gene copies. A similar (pTZ18R-yCF,-lA) was used to probe restricted and elec- analysis of the banding patterns from the CC 124 digestions trophoresed genomic DNA from two strains of C. reinhard- yields the same conclusion: the y subunit is probably en- tii. Strain 2137 (Fig. 3 Left) was the source of our cDNA coded by an intron-containing single-copy gene that, in both library, and strain CC 124 (also known as 137c; Fig. 3 Right), strains, contains one Pst I and one Sst I/Sac I restriction site when mated with strain 21 gr, produced strain 2137. The within the intron. number and sizes of the bands in Fig. 3 were considered Interestingly these Southern analyses also reveal restric- along with the ethidium bromide-stained patterns of the tion fragment length polymorphisms. They are attributable cDNA probe after restriction with the same enzymes fol- to two differences between the strains. An insertion present lowed by electrophoresis (the cDNA restriction data are in strain 2137 (and absent in strain CC 124) suffices to recorded in Fig. 3). reconcile the differences in the lengths of the hybridizing The HindIll, Pvu II, and Sst I/Sac I banding patterns fragments derived from the HindIII, Hinfl, and Pvu II generated with strain 2137 DNA (Fig. 3) are consistent with digestions. Flanking the y-subunit gene in strain 2137, this the 'y subunit being encoded by either a single-copy gene insert must contain at least one HindIII and one HindI containing an intron or, alternatively, two closely linked restriction site. The other difference can be ascribed to a gene copies. However, the Hinfl- and Pst I-derived banding second Pst I restriction site within the y-subunit gene in patterns for strain 2137 DNA (Fig. 3) are only consistent strain 2137. Thus, the larger hybridizing fragment derived with the former possibility since too few hybridizing frag- from the Pst I digestion of strain CC 124 DNA is apparently split into two smaller hybridizing fragments in the Pst I 2 3 4 digestion of strain 2137 DNA. -9.5 -7.5 In Vitro Translation, In Vivo Labeling, and Immunoprecip- -4.4 itation. Three linearized plasmids were transcribed in vitro " " -2.4 and the transcription products were isolated and then trans- i.4 lated in vitro in the presence of [35S]methionine. The sam- :.:s- ples were electrophoresed in a 15% polyacrylamide gel before and after immunoprecipitation and 35S labeling was -0.3 visualized by fluorography (Fig. 4). The vector (pTZ18R) yielded translation products identical to the minus RNA control (lanes 7 and 5); in neither case did treatment with FIG. 2. Hybridization of nick-translated plasmids to total RNA anti-y-subunit serum yield an immunoprecipitable product samples from C. reinhardtii. Total RNA samples were denatured (lanes 15 and 13). The plasmids pTZ18R-yCF1-lA and and electrophoresed in an agarose gel containing formaldehyde, pTZ18R-,yCF1-lC contained the same cDNA insert in oppo- which was then blotted and fixed to a nylon filter. The filter was site orientations. The translation product from pTZ18R- divided and incubated with 32P-labeled plasmid DNA produced by of low molecular nick-translation. Lanes: 1, host plasmid DNA (pTZ18R); 2, cDNA- yCF1-1C (lane 6) was indiscrete, weight, containing plasmid pTZ18R-yCF,-lA; 3, cDNA-containing plasmid and was not immunoprecipitated by anti-y-subunit serum pTZ18R-yCF,-4A; 4, cDNA-containing plasmid pTZ18R-yCF1-9A. (lane 14). However, in the other orientation, the transcribed Markers on right denote the approximate number of kilobases cDNA was translated to give essentially one product (lane gauged from RNA molecular weight standards. 2), which was readily immunoprecipitated with anti-)y- Downloaded by guest on September 28, 2021 1372 Biochemistry: Yu et al. Proc. Natl. Acad. Sci. USA 85 (1988)

2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 b _ _

_ _

_ y p.-~~__.

FIG. 5. Polyacrylamide gel electrophoretic and fluorographic FIG. 4. Polyacrylamide gel electrophoretic and fluorographic analysis of the import of the pre-'y subunit from C. reinhardtii into analysis of in vitro translations of transcribed cDNAs and poly- pea chloroplasts. pTZ18R-yCF1-1A was transcribed in vitro and the adenylylated RNA and of in vivo labeled cells. Lanes: 1-7, total transcription products were translated in vitro in a rabbit reticulo- translates or cell extracts prior to immunoprecipitation; 8-15, im- cyte lysate in the presence of [35S]methionine. Prior to starting the munoprecipitates of total translates or cell extracts; 1, 5, 9, and 13, import assay, the lysate was incubated for 10 min at room temper- labeled products from in vitro translations containing [35S]methi- ature either with buffer alone or with glucose and hexokinase to onine with either no RNA (lane 5) or polyadenylylated RNA (lane 1) deplete the lysate of nucleoside triphosphates. The lysates were and the 35S-labeled products immunoprecipitated from these in vitro then incubated with intact pea chloroplasts at 25TC for 20 min in the translations with anti-y-subunit serum (lanes 13 and 9, respectively); presence (lanes 3 and 4) or absence (lanes 5-8) of light. The dark 3 and 11, 3sS products from whole cells, labeled in vivo with incubations were either supplemented with 5 mM ATP (lanes 5 and Na235SO4, after detergent solubilization (lane 3) and immunoprecip- 6) or contained the glucose- and hexokinase-treated lysate (lanes 7 itation with anti-y-subunit serum (lane 11); 4, 8, and 12, a crude and 8). After the incubation period, each sample was divided in half chloroform extract of C. reinhardtii membranes (lane 4) derived and one portion was treated with thermolysin (lanes 4, 6, and 8) or from cells labeled in vivo with Na235SO4 and the "S-labeled prod- with buffer alone (lanes 3, 5, and 7) for 10 min at 250C. Intact ucts immunoprecipitated with anti-CF1 serum (lanes 8 and 12); 6 and chloroplasts were then isolated on Percoll gradients, solubilized in 14, 7 and 15, and 2 and 10, [35S]methionine-labeled in vitro transla- sample buffer, and electrophoresed. Labeled products were re- tion reactions using transcription products from linearized plasmids vealed by fluorography. Lane 2, immunoprecipitated (35S-labeled) pTZ18R-yCF1-lC (lane 6), pTZ18R (lane 7), and pTZ18R-yCF1-lA C. reinhardtii CF1 standard; lanes 1 and 9, sample ofthe reticulocyte (lane 2), and the 35S-labeled products immunoprecipitated from lysate prior to incubation with the pea chloroplasts pretreated with these in vitro translations with anti-v-subunit serum (lanes 14, 15, (lane 9) or without (lane 1) glucose and hexokinase. and 10, respectively). The subunits of CF1 are labeled on the far right. polypeptides appeared that were resistant to thermolysin and presumably imported (lanes 3 and 5 vs. lanes 4 and 6). subunit serum (lane 10). Several smaller products, which When the reticulocyte lysate was first depleted of nucleoside appeared upon prolonged exposure, may represent miscued triphosphates (by the addition of glucose and hexokinase), translation initiation/termination events (lanes 2 and 10). chloroplasts incubated in the dark bound the precursor but The major translation product derived from pTZ18R-yCF1- did not process it (lane 7). The largest of the exogenous 1A comigrated with the product, immunoprecipitated with protease-resistant (imported) polypeptides comigrated with anti-y-subunit serum, from in vitro translated polyadenyly- the mature y subunit found in the C. reinhardtii CF1 (lane 1). lated RNA from C. reinhardtii (lanes 1 and 9). Both of these bands migrated above the mature y subunit immunoprecipitated either from detergent extracted cells with anti-y- DISCUSSION subunit serum (lanes 3 and 11) or from a crude CF1- In this article, we show that we have isolated a cDNA for the containing extract with anti-CF1 serum (lanes 4, 8, and 12). pre-y subunit of the thylakoid ATP synthase from C. rein- This presumed precursor of the y subunit has an approxi- hardtii. Our largest cDNA (pTZ18R-yCFj-1A) probably con- mate molecular weight of 42,000. Since the mature subunit tains the entire coding region for this nuclear-encoded sub- has a molecular weight of =38,000, the precursor to the y unit and does contain sufficient information for the in subunit probably carries an amino-terminal transit sequence vitro-synthesized polypeptide to be transported into and of -30 amino acids. processed by energetically competent pea chloroplasts. Import into Pea Chloroplasts. Plasmid pTZ18R--yCF1-lA We screened our cDNA library, constructed in the expres- was transcribed in vitro and the transcription products were sion vector Agtll, with a highly specific antiserum directed translated in vitro in the presence of [35S]methionine. The against the y subunit (Fig. 1). After selecting and rescreening reticulocyte lysates were incubated with pea chloroplasts in promising phage to homogeneity, cDNAs were cloned into vitro and, after 20 min at 25°C, the suspensions were treated the transcription/sequencing plasmid pTZ18R. The hybrid- with protease or with buffer alone. The chloroplasts were ization of nick-translated cDNA to a sample of total RNA then reisolated from a Percoll gradient, dissolved in sample from C. reinhardtii shows that our largest cDNA was ap- buffer, and electrophoresed in a polyacrylamide gel. Radio- proximately the same size as the corresponding mRNA (Fig. labeled products were visualized by fluorography (Fig. 5). 2). The reticulocyte lysate, containing the pre-y subunit (lane 1), Southern blot analysis of strain CC 124 and its F1 progeny also labeled an indigenous protein that migrated just below strain 2137, from which the cDNA library was constructed, the ,8 subunit ofthe C. reinhardtii CF1 (lane 2). In the light or suggests that the pre-y subunit is encoded by an intron- in the dark, the precursor bound to chloroplasts (lanes 3, 5, containing single-copy gene (Fig. 3). Furthermore, the and 7) in a protease-sensitive manner (lanes 4, 6, and 8). In Southern blot analysis ofthese two strains reveals restriction the presence of light or exogenous ATP, faster-migrating fragment length polymorphisms consistent with a flanking Downloaded by guest on September 28, 2021 Biochemistry: Yu et al. Proc. Natl. Acad. Sci. USA 85 (1988) 1373 insertion and the acquisition of a restriction site in strain 5. Merchant, S., Shaner, S. L. & Selman, B. R. (1983) J. Biol. 2137. With an array of phenotypes, these polymorphisms Chem. 258, 1026-1031. could be used as markers in the mapping ofthe 6. Runswick, M. J. & Walker, J. E. (1983) J. Biol. Chem. 258, pre-y-subunit 3081-3089. gene. 7. Nelson, N. (1981) Curr. Top. Bioenerg. 11, 1-33. The electrophoretic mobilities of immunoprecipitated 8. Nelson, N., Nelson, H. & Schatz, G. (1980) Proc. Natl. Acad. polypeptides, translated in vitro or in vivo, show that the y Sci. USA 77, 1361-1364. subunit is synthesized as a larger precursor and that our 9. Chua, N.-H. & Schmidt, G. W. (1979) J. Cell Biol. 81, 461- cDNA probably encodes the entire length ofthis polypeptide 483. (Fig. 4). Our gel system cannot resolve proteins of only 10. Schmidt, G. W. & Mishkind, M. L. (1986) Annu. Rev. Bio- chem. 55, 879-912. slightly different molecular weights; it remains a possibility 11. Merchant, S. & Selman, B. R. (1984) Plant Physiol. 75, that our cDNA does not contain the entire coding region for 781-787. the pre-y subunit. Confirmation of the length and informa- 12. Sager, R. (1972) in Cytoplasmic Genes And Organelles (Aca- tion content of our cDNA must await its sequence determi- demic, New York), pp. 49-104. nation. 13. Klein, U., Chen, C., Gibbs, M. & Platt-Aloia, K. A. (1983) Nevertheless, the precursor to the C. reinhardtii y subunit Plant Physiol. 72, 481-487. is imported by energetically competent pea chloroplasts- 14. Belknap, W. R. (1983) Plant Physiol. 72, 1130-1132. 15. Mendiola-Morgenthaler, L., Leu, S. & Boschetti, A. (1985) i.e., it is removed to a protease-protected space in the dark, Plant Sci. 38, 33-39. provided that (Mg)ATP is present, or in the light (Fig. 5). 16. Boschetti, A., Breidenbach, E., Clemetson-Nussbaum, J., Furthermore, this import is associated with the processing of Leu, S. & Michel, H. P. (1986) Progress in Photosynthesis the precursor to a product that comigrates with the mature y Research, ed. Biggins, J. (Nijhoff, Boston), Vol. 4, 585-588. subunit from C. reinhardtii. Thus it appears, although we are 17. Tittgen, J., Hermans, J., Steppuhn, J., Jansen, T., Jansson, C., not certain, that the amino-terminal transit peptide is faith- Andersson, B., Nechushtai, R., Nelson, N. & Herrmann, fully removed by the pea processing enzyme. Within the R. G. (1986) Mol. Gen. Genet. 204, 258-265. chloroplast, the locations ofthe imported mature-sized poly- 18. Chiang, K.-S. & Sueoka, N. (1967) Proc. Natl. Acad. Sci. USA 57, 1506-1513. peptide and the smaller degradation products are unknown. 19. Youngblom, J., Schloss, J. A. & Silflow, C. D. (1984) Mol. In C. reinhardtii, the rate of subunit turnover in CF1 is low Cell. Biol. 4, 2686-2696. and the pool sizes of unassembled CF1 subunits are <1% of 20. Silflow, C. D., Chisholm, R. L., Conner, T. W. & Ranum, the total pool sizes (11). If pea chloroplasts can synthesize L. P. W. (1985) Mol. Cell. Biol. 5, 2389-2398. significant amounts of the ATP synthase subunits encoded 21. Goldschmidt-Clermont, M. & Rahire, M. (1986) J. Mol. Biol. therein during a 20-min import assay, the lack of the other 191, 421-432. nuclear-encoded subunits, 6 and II, would probably limit the 22. Merchant, S. & Bogorad, L. (1987) J. Biol. Chem. 262, assembly of complete CFO-CF1 complexes. Schmidt and 9062-9067. Mishkind (37) have shown that newly synthesized and im- 23. Young, R. A. & Davis, R. W. (1985) in Genetic Engineering Principles and Methods, eds. Setlow, J. K. & Hollander, A. ported small subunits of ribulose-1,5-bisphosphate carbox- (Plenum, New York), Vol. 7, pp. 29-41. ylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), 24. Selman-Reimer, S., Merchant, S. & Selman, B. R. (1981) EC 4.1.1.39] are degraded in cells of C. reinhardtii treated Biochemistry 20, 5476-5482. with chloramphenicol. Presumably, a lack of free large 25. Younis, H. M., Winget, G. D. & Racker, E. (1977) J. Biol. subunit, which precludes assembly of the holoenzyme, trig- Chem. 252, 1814-1818. gers the selective proteolysis of the unassembled small 26. Allington, W. B., Cordry, A. L., McCullough, G. A., Mit- subunit. Thus, even with a homologous import system one chell, D. E. & Nelson, J. W. (1978) Anal. Biochem. 85, 188- might expect the degradation of the y subunit once it has 196. been into chloroplasts. In our 27. Nelles, L. P. & Bamburg, J. R. (1976) Anal. Biochem. 73, imported heterologous import 522-531. system, it would not be surprising if the C. reinhardtii pre-y 28. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular subunit is not assembled into the holoenzyme, but rather is Cloning: A Laboratory Manual (Cold Spring Harbor Labora- degraded after import into pea chloroplasts. tory, Cold Spring Harbor, NY), pp. 55-186. 29. Tautz, D. & Renz, M. (1983) Anal. Biochem. 132, 14-19. This work was supported in part by grants from the College of 30. Merchant, S. & Bogorad, L. 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