Aspects of Subunit Interactions in the Chloroplast ATP Synthase'

Plant Physiol. (1993) 102: 241-249

Aspects of Subunit Interactions in the Chloroplast ATP Synthase’

1. lsolation of a Chloroplast Coupling Factor 1-Subunit 111 Complex from Spinach Thylakoids Downloaded from https://academic.oup.com/plphys/article/102/1/241/6066835 by guest on 23 September 2021

Carolyn M. Wetzel’ and Richard E. McCarty* Field of Botany, Cornell University, Ithaca, New York 14853 (C.M.W.); and Department of Biology, The Johns Hopkins University, Baltimore, Maryland 2121 8 (R.E.M.)

decreasing molecular mass. CFl has a molecular mas of A chloroplast ATP synthase complex (CFl [chloroplast-coupling 400,000 D (Moroney et al., 1983) and a subunit stoichiometry factor l]-CFo[membrane-spanning portion of chloroplast ATP syn- of a3P3yk(Süss and Schmidt, 1982; McCarty and Moroney, thase]) depleted of all CF, subunits except subunit 111 (also known 1985). CFo contains four different subunits, with a probable as the proteolipid subunit) was purified to study the interaction stoichiometry of 111121116-121VIand molecular mass (for 11112) between CFl and subunit 111. Subunit 111 has a putative role in of 171,000 D (Süss and Schmidt, 1982; Fromme et al., 1987a, proton translocation across the thylakoid membrane during pho- 1987b; Grotjohann and Graber, 1990). Based on hydropathy tophosphorylation; therefore, an accurate model of subunit interactions involving subunit 111 will be valuable for elucidating the plot analysis of the amino acid sequences of CFo subunits I, mechanism and regulation of energy coupling. Purification of the 111, and IV, each appears to contain at least one membrane- complex from a crude CFl-CFo preparation from spinach (Spinacia spanning region in addition to hydrophilic regions (Hudson oleracea) thylakoids was accomplished by detergent treatment et al., 1987). A clear understanding of the association of the during anion-exchange chromatography. Subunit 111 in the complex two components of the ATP synthase will be invaluable for was positively identified by amino acid analysis and N-terminal determining the role of proton flow in enzyme function. sequencing. lhe association of subunit 111 with CF, was verified by Subunit interactions between CFl and CFo have been stud- linear sucrose gradient centrifugation, immunoprecipitation, and ied by a variety of methods, but the connections are still not incorporation of the complex into asolectin liposomes. After incor- clearly understood. It has been suggested that the a subunit poration into liposomes, CF, was removed from the CFl-III complex of CFl is associated with CFo, because the a subunit of CFl by ethylenediaminetetracetate treatment. lhe subunit Ill-proteoliposomes were competent to rebind purified CFl. lhese results in thylakoids is much more resistant to proteolysis than that indicate that subunit 111 directly interacts with CFl in spinach of the solubilized enzyme (Moroney and McCarty, 1982a, thylakoids. 1982b). The proximity of the.a subunit of thermophilic bacterial F1 to the membrane has been indicated by labeling of a with a photoreactive phospholipid (Gao and Bauerlein, Membrane-associated ATP synthesis in chloroplasts, many 1987). 6 and E are not required for attachment of CFl to CFo, bacteria, and mitochondria is accomplished by Fl-Fo ATP but membranes reconstituted with the enzyme lacking either synthases. The chloroplast ATP synthase is composed of two subunit are proton leaky and unable to synthesize ATP (Patrie distinct components, CFl and CFo. CFl is an exhinsic mem- and McCarty, 1984; Richter et al., 1984). Chemical cross- brane protein that contains the sites of nucleotide binding, linking of Vicia fuba CFl-CFo subunits revealed possible as- catalytic activity, and regulation of the enzyme. CFo is an sociations among a-11, p-I, p-11, 7-11, 6-1, and 6-111 (Süss, integral membrane protein complex with the function of 1986). Rott and Nelson (1983) also carried out cross-linking translocating protons across the thylakoid to a specifically studies of spinach CFl-CFo. Both of these studies were carried attached CFl moiety (for reviews, see McCarty and Carmeli, out before the full complement of CFo subunits was identi- 1982; Hammes, 1983; Strotmann and Bickel-Sandkotter, fied; consequently, their results are difficult to interpret. 1984; Futai et al., 1989; Junge, 1989). CF, contains five Analysis of Chlamydomonas reinhardtii photophosphorylation mutants lacking various Fo subunits suggests that, in different subunits, denoted a, p, 7, 6, and 6 in order of C. reinhardtii, subunit 111 is required for binding of CFl to CFo This work was funded by National Science Foundation grants and that subunits I and IV may function to stabilize the DMB 88-03608 and DMB 91-04742 to R.E.M. Additionally, this association (Lemaire and Wollman, 1989a, 1989b). In the material is based on work partially supported by a National Science Foundation Graduate Fellowship to C.M.W. Abbreviations: CFo, membrane-spanning portion of the chloro- Present address: Department of Botany, Bessey Hall, Iowa State plast ATP synthase; CF,, chloroplast-coupling factor 1; CF1-CFo, University, Ames, IA 5001 1. chloroplast ATP synthase, holoenzyme; CF1-III, chloroplast-coupling * Corresponding author; fax 1-410-516-5213. factor 1 associated with subunit I11 of CF,; IgG, immunoglobulin G. 241 242 Wetzel and McCarty Plant Physiol. Vol. 102, 1993 structurally similar Escherichia coli FI-Fo, incubation with the same buffer as used for equilibration; most of the pig- antibodies against subunit c (equivalent to subunit 111) pre- mented impurities eluted. The flow rate was 1 mL min-' vented binding of F1 to Fo in membrane vesicles. The anti- during protein application and until the pigmented front bodies were also able to displace F1 from FOwhen added to reached the bottom of the column, and then the flow was nonstripped membranes (Deckers-Hebestreit and Altendorf, adjusted to 2 mL min-' for the rest of the procedure. 1992). The binding of subunit 111 to CFl has not been posi- Next, the column was washed with 1 bed volume of 50 tively demonstrated in higher plant chloroplasts. Chloroplast m NaH2P04-NaOH, 2 mM Zwittergent 3-12, and 0.02% subunit 111, isolated by organic extraction, is unable to bind asolectin and then by a gradient of 2 bed volumes of this CF1 when incorporated into liposomes, although it is capable same buffer to 2 bed volumes of 5 mM NaH2P04-NaOH, 20 of slow proton translocation across the membrane (Nelson et mM Zwittergent 3-12, and 0.02% asolectin, followed by 1 Downloaded from https://academic.oup.com/plphys/article/102/1/241/6066835 by guest on 23 September 2021 al., 1977; Sigrist-Nelson and Azzi, 1980). bed volume of the gradient end-point buffer. This removes The aim of this study was to isolate an enzyme complex subunits I and IV. To remove subunit I1 and most of the that was depleted of a11 CFo subunits except 111, for the Rubisco, the column was washed with 3 bed volumes of 5 purpose of examining interactions between subunit 111 and mM NaH2P04-NaOH, 0.5% (w/v) Na-cholate, and 0.02% CFl. Subunit 111, also historically known as the proteolipid, is asolectin, followed by a gradient (2 bed volumes of each a very hydrophobic membrane-spanning polypeptide (Hud- buffer) of this same buffer to 190 mM NaH2P04-NaOH,0.5% son et al., 1987) that is present in six to 12 copies per enzyme Na-cholate, and 0.02% asolectin. (Süss and Schmidt, 1982; Fromme et al., 1987a, 1987b; The CFI-I11 complex was then eluted from the column by Grotjohann and Graber, 1990). It is part of the putative 2 bed volumes of 100 mM NaH2P04-NaOH, 0.05% Na- proton channel (Nelson et al., 1977; Sigrist-Nelson and Azzi, cholate, 0.01% asolectin, and 0.5 M (NH4)$504.Peak protein 1980) and can be blocked by covalent modification with fractions were combined, and ATP and asolectin were added N,N''-dicyclohexylcarbodiimide (Sigrist-Nelson et al., 1978). to final concentrations of 0.1 mM and 0.1% (w/v), respec- Its role in proton translocation indicates a close association tively, before the purified CFl-III was stored in small aliquots with CFl. However, past evidence for direct interactions is under liquid nitrogen. Purity of the complex was determined ambiguous. Feng and McCarty (1990a, 1990b) introduced a by SDS-PAGE. The yield and purification of CFl-111 from chromatographic method for purifying CFi-CFo and for ob- crude CFl-CFo were calculated based on total and specific taining the complex lacking either subunit IV or subunits I sulfite-stimulated Mg2+-ATPase activities of the complexes and IV. Their work suggested that it might be possible to (Richter et al., 1987; Wetzel and McCarty, 1993). purify a CF1-subunit 111 complex. In this paper, we describe a chromatographic method for purification of a CF1-subunit ldentification of Subunit 111 I11 complex, positive identification of subunit 111, and verifi- cation of the association between CF1 and subunit 111. Finally, The identity of subunit 111 was verified by amino acid we give evidence that CFl can rebind to isolated subunit I11 analysis and N-terminal amino acid sequencing, based on the in liposomes. In the accompanying paper (Wetzel and Mc- reported sequence for spinach subunit 111 (Deno et al., 1984). Carty, 1993), we describe the characterization of enzymic A sample of CFl-I11 was electrophoretically separated into activity of the purified complex. subunits using SDS-PAGE and then electroblotted onto a polyvinylidene fluoride membrane (Problot, Applied Biosys- tems) according to the manufacturer's instructions. The pro- MATERIALS AND METHODS tein was visualized by Coomassie blue staining, and the band Preparation of CF,, Crude CFl-CF,, Purified CFl-CF,, and corresponding to subunit 111 (apparent molecular mass 8 kD) CF1-III from Spinacia oleracea was excised from the surrounding material and dried. The sample was divided, with one-half used for amino acid CF1 was purified according to the method of Shapiro and analysis (Cohen et al., 1984) and the other half subjected to McCarty (1990). Crude CFI-CFo was isolated by cholate/ gas-phase N-terminal sequencing (Shively, 1986). Both anal- octylglucoside extraction of thylakoids (Pick and Racker, yses included blanks from nonprotein-staining regions of the 1979; Feng and McCarty, 1990a). Purified CFl-CFo was pre- electroblot. pared according to the method of Feng and McCarty (1990a). CF1-111 was prepared as follows. A11 steps were carried out Association of CFl-III at 4OC. Crude CFl-CFo(100-120 mg of protein) was dialyzed for 8 to 12 h against 1 L of 50 mM NaH2P04-NaOH(pH 7.0), The association of subunit I11 with CFI was analyzed using 10% (v/v) glycerol, 1 mM MgC12, and 1 mM DTT. Triton X- Suc density gradient ultracentrifugation, by immunoprecipi- 100 was added to the dialyzed protein from a 10% stock to tation of the complex with anti-0 antibodies, and by incor- a final concentration of 0.5%. Note: A11 of the following poration into asolectin liposomes. buffers contained 10% (v/v) glycerol, 1 m MgC12, and 1 mM For Suc density gradient ultracentrifugation, solid DTT with additional components as described for each step (NH4@04 was added to approximately 3 mg of CFl-I11 in and were titrated to pH 7.0 with either NaOH or HC1. The 10% glycerol, 100 mM NaH2P04-NaOH (pH 7.0), 1 mM cyde CFI-CFowas applied to a 2.5- X 9-cm DEAE-Trisacryl- MgC12, 1 mM DTT, and 0.5 M (NH&S04 (adjusted to pH 7.0) M column that was equilibrated with a buffer containing 50 to a final leve1 of 45% saturation at OOC, and the mixture mM NaH2P04-NaOH, 0.5% Triton X-100, and 0.01% (w/v) was allowed to sit at 4OC for 1 h. The protein precipitate was asolectin. The column was washed with 4 bed volumes of pelleted, resuspended in 0.4 mL of 30 mM Tris-succinate (pH lsolation of a Chloroplast Coupling Factor 1-Subunit III Complex 243

6.5), 0.5 mM EDTA (pH %O), 0.1 mM ATP, 0.1% asolectin, modifications of the procedures of Admon et al. (1985) and and either 0.4% (w/v) Na-cholate or 0.2% (v/v) Triton Krupinski and Hammes (1986) were used as follows: the X-100, and then layered onto a 4.4-mL SUCgradient. The enzyme complexes (0.14 mg of protein) were precipitated gradients contained the same components as the resuspen- with solid (NH,),SO, (to 50% saturation at OOC) and resus- sion buffer, with a linear SUCconcentration gradient of 7 to pended in a buffer containing 0.9% (w/v) Na-cholate, 0.8% 40% for the Na-cholate buffer samples and 7 to 30% for the (w/v) sonicated asolectin (final aso1ectin:protein weight ratio Triton X-1OC buffer samples. Two Na-cholate gradients were of 40:1), 2 mM MgC12, 150 mM KC1, 0.5 mM EDTA (PH 8.0), run, one containing 100 m~ Na2S03and one lacking it. The and 10 mM Tricine-NaOH (pH 8.0) and incubated on ice for Suc gradients were centrifuged at 164,00Og, 4OC, for 16 to 10 min. To remove cholate and thus cause liposome forma- 18 h. Fractions (0.3-0.4 mL) were collected by puncturing tion, each sample was passed by centrifugation through 3 the bottom of the tube, and aliquots of each fraction were mL of Sephadex G-50 (fine) in a disposable filter column Downloaded from https://academic.oup.com/plphys/article/102/1/241/6066835 by guest on 23 September 2021 added to double-strength gel sample buffer for analysis by (Penefsky, 1977) that had been equilibrated with 2 mM SDS-PAGE. MgC12, 150 mM KCl, 0.5 mM EDTA (pH 8.0), and 10 mM CFl-I11 was precipitated by anti-P antibodies as another Tricine-NaOH (pH 8.0). Excess buffer was removed by an means of verifying the association of the complex. IgGs were initial centrifugation step before the columns were used. isolated from rabbit anti-P antiserum according to the pro- The second method used for incorporation of enzyme cedure of Livingston (1974). Antibody titer was determined complexes into asolectin vesicles was that of Schmidt and by the ELISA, with the activity of peroxidase conjugated to Graber (1985). Protein (0.4 mg) was mixed with 35 mg goat anti-rabbit antibody used for detection. Specificity of of sonicated asolectin in 2% (w/v) Na-cholate pH @.O), 1% the IgGs was verified by immunoblot analysis (Towbin et al., (w/v) Na-deoxycholate, 10 mM Tricine-NaOH (pH 8.0), 0.1 1979), using the same second antibody as described above in mM EDTA (pH 8.0), and 0.5 mM DTT in 4 mL of total volume. conjunction with the Amersham ECL Western Detection The mixture was dialyzed in 6000 to 8000 mo1 wt cutoff System. dialysis tubing against 1 L of 10 mM Tricine-NaOH (pH 8.0), Immunoprecipitation of CFl, purified CFl-CFo, and CFl-111 2.5 mM MgC12, 0.2 mM EDTA (pH %O), and 0.5 mM DTT at by anti-P IgGs was carried out by the following procedure. 3OoC for 12 h. As suggested by Grotjohann and Graber Each of the complexes (700 pg) was precipitated with solid (1990), the dialyzed proteoliposomes were centrifuged (NH4)2S04,pelleted, and resuspended in 300 pL of buffer (11,290g for 6 min) to remove large vesicles and denatured containing 10% glycerol, 50 mM NaHJ’04-NaOH (pH 7.0), material. and 1 mM MgC12. Each sample was dialyzed in 6000 to 8000 To determine the extent of complex incorporation, each mo1 wt cutoff dialysis tubing against 1 L of the same buffer, sample was subjected to centrifugation at 96,6OOg,,,,, for 15 with one change, for 15 h at 4OC. Na-cholate was added to min, the supernatants were removed and saved, and the the dialyzed material from a 20% stock to a final concentra- pelleted liposomes were resuspended to the same volume in tion of 0.5% to solubilize the protein. The protein concentra- the buffer used for column equilibration and centrifuged tion of each sample was measured by the Pierce BCA protein again. Aliquots of the original liposome samples, the first assay, and cholate buffer (dialysis buffer supplemented with supernatants and pellets, and second supernatants and pellets 0.5% Na-cholate) was added as needed to equalize each were each used to measure sulfite-stimulated Mg2+-ATPase sample to 1 mg of protein mL-’. Aliquots of each sample activity (Richter et al., 1987; Wetzel and McCarty, 1993). The were removed and saved as pretreatment protein samples. percentage of total activity partitioning with the pellet was Anti-P IgG was added to 300 pL of each precipitation sample, assumed to be the percentage of the whole functional com- plus a zero-protein control, and the samples were mixed end- plex incorporated into liposomes, with the CFl values as a over-end for 1 h at room temperature and overnight at 4OC. control for nonspecific binding. Protein A covalently coupled to Sepharose CL-4B (4.5 mg), swollen in cholate buffer, was added to each sample and Reconstitution of CF, with CF1-Depleted mixed end-over-end for 2.5 h at room temperature. The Subunit III Proteoliposomes samples were centrifuged to pellet the Sepharose beads and bound protein. The supernatants, containing unprecipitated The gel filtration method described above was used to protein, were carefully removed. The pretreatment samples prepare CFl-I11 and CFl-CFo proteoliposomes and control and the unprecipitated protein samples were each prepared (zero protein) liposomes with the following modifications: for SDS-PAGE by adding an equal volume of double-strength approximately 200 pg of each complex type was precipitated gel sample buffer and heating to 37OC for 1 h. The protein and then incorporated into liposomes, in a final buffer (600 A-Sepharose beads were washed four times with cholate pL) containing 30 mM KCl, 10 mM Tricine-NaOH (pH 8.0), buffer, resuspended in 90 pL of gel sample buffer, heated to 0.5 mM EDTA (pH &O), and 2 mM MgC12 (buffer A). 37OC for 1 h, boiled for 1.5 min, and then centrifuged to Each sample was divided into three (200 pL) aliquots and pellet the Sepharose beads. The supernatants, containing centrifuged (lOO,OOOg, 10 min) to pellet the (pro- precipitated protein, were carefully removed and, along with teo)liposomes. The supernatants were discarded. One aliquot the prepared gel samples of pretreatment and unprecipitated of each sample (“pretreatment”) was resuspended in 150 pL protein, were electrophoresed in a 15% SDS-polyacrylamide of buffer B (buffer A plus 50 mM DTT) and set aside on ice gel. until assayed for enzyme activity. The second aliquot of each Purified CFl-CFo, CFl-111, and CFl were each reconstituted sample (“nonstripped”)was resuspended in 200 pL of buffer into asolectin liposomes by two different methods. First, A. The final aliquot of each sample (“EDTA stripped”) was 244 Wetzel and McCarty Plant Physiol. Vol. 102, 1993

resuspende ^0 f EDT0.7Lo M 20 M 8.0)H 5m m n A i (p d1 , RESULTS Tricine-NaOH (pH 8.0). Both of these treatments proceeded Preparatiof no t rooa n m mi temperature fo0 3 r the d samplee an ,nth s were centrifuged to pellet the (proteo)liposomes. procedure Th e describe purificatioe th r dfo CFj-IIf no I from Each pelle resuspendes twa fiLbuffef 0 o 20 n rdi containing crude CFi-CFo is a modification of that used by Feng and Tricine-NaOM KC1HI M m 0 ,1 EDT 0 M 3 m 8.0)H AH5 (p 0. , McCarty (1990b) to prepare other subunit-deficient forms of 1 (pH 8.0), and 1 mg mL" of BSA. Each sample was then CFrCF0 .e impuritie Mosth e crudf th o t en i s mixture ar e divided into two, 100-jtL aliquots (12 total). One of each set eluted from the DEAE-Trisacryl-M column by the initial 0.5% of aliquots ("minus-CFr) was mixed with 100 jiL of buffer B, Triton X-100, 50 mM sodium phosphate buffer wash, while and the others ("plus-CFi") were mixed with 100 juL of (1 mg the CFi-CFo complex remains bound to the matrix. Subse- Downloaded from https://academic.oup.com/plphys/article/102/1/241/6066835 by guest on 23 September 2021 mL"1) DTT-reduced CFi suspended in buffer B. Finally, quent washes with staggered, inverse gradient f Zwittero s - gent sodiu3-1d an 2 m phosphate remove, subunitIV d an sI MgCl2 e (0. stockTh . 5 M s addel aliquotal )mM wa o 5 dt o st reconstitution was allowed to proceed for 20 min at room d the e potentiallan nth y destabilized subuni I (FenI t d gan temperature before centrifugation to pellet the (pro- McCarty, 1990b) is eluted by a sodium phosphate gradient teo)liposomes. Each f pelle o s resuspendeL wa ju t 0 20 n i d presence th n i f 0.5eo % Na-cholate. buffer B, centrifuged again, and resuspended in 150 pL of Results from the purification procedure are displayed in buffer B. All samples were then placed on ice until assayed the SDS-PAG f Figuro l . LanEge 1 e show1 e subunie th s t composition of the crude CF^CFo, and lanes 2 through 4 for sulfite-stimulated Mg2+-ATPase activity (Richter et al., show purified CFi-CF , CFj-III, and CFi, respectively. The 1987; Wetzel and McCarty, 1993). 0 most persistent contaminants are the small and large subunits

of Rubisco and subunits of the Cyt b6// complex. Cyt-con- Miscellaneous Procedures taining protein was identified spectrophotometrically from the difference spectrum of reduced versus oxidized protein SDS-PAGE was performed according to the method of (Hurt and Hauska, 1981), and the Rubisco subunits were Flin Gregersod gan n (1986), wit l samplhge e buffer modified identifie l relativ ge y thei b a dS o ret SD position e th n o s from tha f Grotjohano t Grabed nan r (1990 contaio t ) % n10 standard. Greater purity can be obtained by repeating the glycerol, 4% (w/v) SDS, 10% (w/v) /3-mercaptoethanol, and DEAE-Trisacryl-M chromatography step and/or passing the Tris-HCM m 6.8)5 H 12 l(p , wit finae adjustehth H p l 1 7. do t protein complex through a Rubisco immunoaffinity column, with NaOH. Gels were stained either with Coomassie bril- overalle th t bu yiel specifid dan c activit enzyme th f yo e will e silvelianth y r b t staininblur o eR g metho f . Wrado al t ye decrease e purpose th r investigation r ou Fo f . o s e chroon , - (1981). matography stejudges pwa resulacceptablo n dt a n i t e level Protein was determined either by the method of Lowry et of purification. al. (1951), modification e Lowrth f so y method (Bensadoun Table I shows typical values for recovery of CFi-III during purification from crude CFi-CF0. Activity was measured as and Weinstein, 1976; Markwell et al., 1978), or the Pierce 2+ BCA protein assay system, depending on the presence of sulfite-stimulated Mg -ATPase after reduction with DTT. interfering compounds. Apparent protein concentration val- yiel e degred Th dan purificatiof eo n were somewhat variable. s werue e adjuste accouno t de presenc th r fo tf asolectin o e , base standarn do d curves. Asolectin interfereA s BC wit e hth protein assay system in an inconsistent manner (Kessler and 1234 Fanesril, 1986). With the method of Lowry et al. (1951), asolectin (final sample concentrations up to 0.05%) results in an increase of the intercept (zero protein) of the standard 0.0o t curve uni2p t A doe u t chang bu f t sso no slope eth f eo the standard curves. BSA was used for all of the standard curves. -IV -I Chemicals e -II S DEAE-Trisacryl-M (IBF Biotechnics s obtainewa ) d from Serva Biochemicals (Paramus, NJ). Zwittergent s 3-1wa 2 III purchased from Calbiochem Corp. (San Diego, CA). Asolec- tin was obtained from Associated Concentrates (Woodside, NY). Cholat purifies ewa treatmeny db t with activated char- coal, followed by repeated recrystallization (Kagawa and Figur . SDS-PAG1 e f crudEo e CF,-CF0, purified CF,-CF0, CF,-III, and CF,. Protein (35 Mg per lane) was prepared as described in Racker, 1971). Protei covalentlA n y couple Sepharoso t d e "Material SDS-polyacrylamid% 15 Methods, d a san n o n ru " e gel, CL-4B was purchased from Sigma Chemical Co. Other re- and stained with Coomassie blue. Lane 1, Crude CFi-CF0; lane 2, agents were of normal laboratory grade and were purchased purified CF,-CF0; lane 3, CFi-III; lane 4, CFi. S indicates location of from routine sources. the small subunit of Rubisco. lsolation of a Chloroplast Coupling Factor I-Subunit III Complex 245

McCarty, 1993). When in the presence of Triton X-100, Table 1. Recovery of CFl-/ll after purification from crude CF,-CFo conversely, subunit I11 dissociates from CFI and sediments at Sulfite-stimulated MgZ+-ATPaseactivity (5-1 5 pg of protein) was much lighter densities of 14 to 16% Suc (Fig. 2B). The CFl measured in 4 mM ATP, 2 mM MgCI2, 10 mM Tris-HCI (pH 8.0), and 100 mM Na2S03at 37°C for 10 min. continues to sediment to 22 to 24% SUC.The purified CF1- CFo holoenzyme remains associated during Triton X-100 SUC Crude CF,-111 density centrifugation (Feng and McCarty, 1990b), which CF,-CFo suggests that subunits I, 11, and/or IV are necessary for Total protein (mg) 1 O0 6.7 stabilizing the complex in the presence of the detergent. Total activity (pmol of Pi 283 60 CFI-111 complex association was further tested by immu- min-') noprecipitation with anti-/3 IgGs. Figure 3 shows the SDS-

Specific activity (pmol of 2.8 9.0 Downloaded from https://academic.oup.com/plphys/article/102/1/241/6066835 by guest on 23 September 2021 Pi mg-' min-') PAGE-separated subunit profiles of CFI, CFl-111, purified Purification (fold) 1 3.2 CFI-CFo, and a zero-protein control. Pretreatment profiles Yield (% by activity) 1O0 21 (left panel, lanes 1-4) show the complete subunit composi- tions of the complexes. Precipitated profiles (center panel, lanes 1-4) show the subunits that fractionated with the pellet after binding of anti-p IgG and protein A-Sepharose beads. The purity of the crude CFI-CFostarting material was a major CFI is precipitated by the antibodies (center panel, lane 3). source of the variation because preparations from a common The CFl-111 lane (center panel, lane 4) clearly shows that crude CF1-CFo stock showed similar yield and purity (not subunit 111 remains associated with CFI throughout the pre- shown). Specific activity of CFI-111 is considerably higher cipitation procedure. Purified CFl-CFo was not effectively than for the crude material, with a calculated 3.2-fold puri- precipitated under these conditions (center panel, lane 2); it fication, whereas a comparison of total activity values indi- is possible that CFo subunits I, 11, and/or IV partially occlude cates a yield of 21%. The range of yields was 14 to 21%. antigenic sites on /3. The right panel (lanes 1-4) shows the Because of the uncertainty about the subunit stoichiometry unprecipitated material from each complex. Lane 1 of each and mo1 wt of CFo, and of the subunit 111 complex, specific panel contains the IgG and buffer control. Results of the activity values must be viewed with caution. Some CFl elutes precipitation experiments support the hypothesis of CFl- from the column during the final sodium phosphate gradient, subunit I11 association. contributing to a decrease in recovery. The partitioning of sulfite-stimulated Mg2+-ATPaseactivity of DTT-reduced enzyme complexes upon incorporation into ldentification of Subunit 111 The putative subunit I11 band electroblotted onto a polyvinylidene fluoride membrane from a 15% SDS-PAGE gel was subjected to amino acid analysis and gas-phase N- Table II. Comparison of published (expected)mino acid terminal sequencing. The observed amino acid composition composition (molar percent) and N-terminal sequence" for spinach subunit 111 (Deno et a/., 7984) with those observed for subunit 111 (molar percent) and N-terminal sequence closely agree with isolated from the purified CF1-///complex predicted values based on published sequence information See "Materiais and Methods" for procedural details. (Table 11). This agreement indicates positive identification of subunit 111 in the purified complex. It was reported by Howe Amino Acid Analysis Amino Acid et al. (1982) that sequencing required removal of a putative Expected Observed blocked N terminus in subunit 111, but this was not necessary in our analysis. M % Ala 21.0 21.7 Leu 14.8 15.8 Association of CF1-III G lY 13.6 13.8 The presence of subunit-111 in the same fractions as CFI Glu + Gln 8.6 9.0 Val does not evince their physical association; therefore, the 8.6 8.6 Ile 7.4 7.3 association of the complex was tested in severa1 ways. Figure Pro 4.9 5.5 2 shows SUCdensity centrifugation fraction profiles of CF1- Ser 3.7 3.1 111 in the presence of Na-cholate plus Na2S03 and Triton X- Thr 3.7 3.4 100, respectively. In Na-cholate plus Na2S03, subunit 111 Phe 3.7 3.4 sediments with CFI to a leve1 of 27% SUC,indicating an Asp + Asn 2.5 2.3 association that remains stable during the centrifugation. Arg 2.5 2.7 Similar results were obtained in a gradient containing Na- Met 2.5 1.3 cholate without NazSOa but the equilibrated protein band TY r 1.2 1.3 was more diffuse (not shown). It appears that most of the LYS 1.2 0.5 subunit I11 sedimented with only half of the CFl (Fig. 2A, His O O O 0.6 fraction 4 versus 5); this is probably an artifact due to the CY5 prolonged centrifugation and should not be taken as an a N-terminal sequence: Expected, (N terminal) Met-Asn-Pro-Leu- indication of a large population of CFl lacking subunit 111 in Ile-Ala-Ala-Ala-Ser-Val; observed, (N terminal) Met-Asn-Pro-Leu-lle- the CFI-111 complex (see accompanying paper, Wetzel and Ala-Ala-Ala-Clu-VallGlv. 246 Wetze McCartd an l y Plant Physiol. Vol. 102, 1993

A: Na-cholate asolectin liposome describes i s Tabln di e IIIactivite .Th y that partitioned with the pelleted liposomes was assumed to rep- resent enzyme complexes incorporated in liposomes. The CFl Fractions 1-12 results from the gel filtration method of liposome incorporation are displayed in Table III. For CFi-III, there was 18% incorporation r purifiefo d an d, CFi-CF- in % 025 ther s ewa corporation. The CFi sample showed 4% of total activity in a the pelleted liposomes, which is a measure of nonspecific P liposomese th o t bindin i CF . f Washingo pelletee gth d liposomes resulte somn i d e change e totath ln i sactivit y ratios,

but these changes difficule ar asseso t t s because thers wa e Downloaded from https://academic.oup.com/plphys/article/102/1/241/6066835 by guest on 23 September 2021 6 overall loss or gain of total activity for some of the samples. nonadditivite Th fractione attributee th b f yo n ca s experdo t - e imental error: volumes and total amounts of protein were typically very small, making handling and quantitation dif- III ficult. However, the pellets of CFi-CF0 and CFi-III proteoliposomes retained an appreciably greater percentage of total activity than that of the CFi liposomes even after washing. Similar results were obtained each time this experimens wa t p to bottom repeated (dat t shown)ano . 40% sucros% 7 e CFi-CF CFi-IId oan I were also incorporated into liposomes by a cholate/deoxycholate dialysis procedure (Table III). CFi- B: Triton X-100 CF0 showe incorporation% d23 , wit los% h1 t after washing, d CFj-IIan I e incorporatetotath f l o activit e % th 19 n di y liposome fraction with a loss of 2% due to washing. There CFl Fractions 1-12 i controthin i CF s n o experimentru n l s wa e abilitTh . o yt become incorporated into liposomes indicates i s i thaCF t associated with a lipophilic moiety, either CFo or subunit III. a P Pre-treiitment Precipitated Unnrecinitatcd 1234 1234 1234 8

E a III P

bottom top 30% sucrose 7%

densite FigurSu . ye2 gradient centrifugatio f CF,-!!npreso e th -n i ! ence of Na-cholate or Triton X-100. CF,-III was centrifugally equilibrated in a 4.4-mL linear Sue gradient containing 30 mM Tris- succinat ATP EDTAM M m ,m 6.5) H 1 0.15 e,0. (p 0. %, asolectind an ,

either 0.4% Na-cholate, 100 mM Na2SO3, and 7 to 40% Sue (A) or 0.2% Triton X-100 and 7 to 30% Sue (B). Fractions were collected, aliquotd an SDS-polyacrylamids% wer15 n eo preparen ru d dan e III gels. The gels were stained with Coomassie blue. A shows subunit I remaininII g associated wit presence h th CF n i tf Na-cholateo e , Figure 3. Immunoprecipitation of CFi, purified CFrCF0, and CF,- and B shows that Triton X-100 causes dissociation of subunit III. III with anti-|8 IgCs. Protein complexes were precipitated with The first lane of each gel is a CF) standard. antibody plus protei covalentlnA y boun Sepharoseo dt . Unprecip- itated materia separates wa l d fro precipitatee mth centrifugay b d - tion. Sample f pretreatmento s , precipitated d unprecipitatean , d material were prepared and subjected to SDS-PACE. Lane 1, IgC,

zero-enzyme control; lane 2, IgC plus purified CFi-CF0; lane 3, IgC plus CF!, staines pluC - Ig lanwa s l , dCFe4 ge wit e rlllhTh . Coo- massie blue. lsolation of a Chloroplast Coupling Factor 1-Subunit III Complex 247

Association of the complex was confirmed by severa1 meth- Table 111. Partitioning of ATPase activity upon incorporation of enzyme complexes in asolectin liposomes ods. Cosedimentation of subunit I11 with CF, during SUC CF,, purified CFl-CFo, and CF,-III were each incorporated in density centrifugation in the presence of cholate indicates asolectin liposomes as described in "Materiais and Methods." Cho- association. Immunoprecipitation of the CF1 by an antibody late removal was either by Sephadex column or by dialysis. The against the P subunit results in concomitant precipitation of liposome preparations were pelleted by centrifugation, and the subunit 111, again indicating association. Finally, the associa- pellets were washed and resuspended. Samples from precentrifu- tion of subunit 111 with CF1 confers to CFl the ability to be gation, first pellet and supernatant, and washed pellet and super- incorporated into asolectin vesicles. Specificity of the binding natant were each assayed for sulfite-stimulated MgZ+-ATPaseactiv- of subunit I11 to CFI is explored in the accompanying paper ity (37"C, 10 min). N.D., Not detectable. The initial total activity (Wetzel and McCarty, 1993). values (nmol of Pi min-') were CFl-CFo = 59, CFl-III = 22, and CF, The incorporation of both CFl-I11 and CFl-CFo into lipo- Downloaded from https://academic.oup.com/plphys/article/102/1/241/6066835 by guest on 23 September 2021 = 55 when cholate was removed by Sephadex column and CF1- somes could be improved. Reconstitution of protein com- CFo = 153 and CF,-III = 73 when cholate was removed by dialysis. plexes into artificial membranes is still as much art as science. Percentage of lnitial Total Although the activity that partitioned with the vesicles is an Fraction Activity indication of successfully incorporated complexes, the activity CF.-CF, CF.-III CF. that remained in the soluble fraction does not necessarily Cholate removal by indicate that the complexes were unable to become reconsti- Sephadex column tuted. With further experimentation, especially with pro- Pellet 25 18 4 tein:lipid:detergent ratios in the liposome formation mixture, Supernatant 83 81 87 a much higher leve1 of enzyme incorporation may be achieved. Washed pellet 16 6 1 Evidence supporting (C)F1-proteolipid interaction has been Supernatant 10 7 3 obtained for bacteria and for lower plant chloroplasts but is ambiguous in the case of higher plant chloroplasts. The Cholate removal by primary argument against the association is based on the dialysis observation that isolated subunit 111 will not bind purified Pellet 23 102 Supernatant 78 102 CFl (Nelson et al., 1977; Sigrist-Nelson and Azzi, 1980). In these experiments, organic solvent extraction was used to Washed pellet 22 17 prepare subunit I11 before it was incorporated into liposomes. Supernatant 6 N.D. Our results clearly demonstrate that subunit 111 is competent to bind CF, in the presence of MgC12. The asolectin membrane environment may stabilize a subunit 111 complex struc- Reconstitution of CF, with CFI-Depleted ture that is potentially disrupted during organic solvent Subunit 111 Proteoliposomes extraction. The stoichiometry of subunit 111 remains an open question. CF, will bind to isolated subunit 111 or CFo in proteolipo- Lacking a clean antibody against subunit 111, immunological somes but not to zero-protein liposomes. Table IV shows the results of sulfite-stimulated Mg*+-ATPase activity measure- ments of EDTA-stripped, nonstripped, CF1-reconstituted, and -unreconstituted liposomes. The amount of total activity re- Table IV. Reconstitution of CF, with subunit 111 or CF, in liposomes flects the amount of CF, bound to the proteoliposomes. CF1-III and CF1-CFowere each incorporated into asolectin lipo- Clearly, treatment with EDTA under low salt conditions somes. The preparations were either treated with EDTA under low- removes CF1 from both CFl-CFo and CFI-111 proteoliposomes. salt conditions to strip CF, from the membranes (Stripped) or left in buffer (Nonstripped).Following centrifugation to remove liber- Reconstitution of CF, with the stripped vesicles results in ated CF, from the proteoliposomes, fresh DTT-reduced CF, was restoration of activity. CFl will bind to nonstripped proteoli- added for reconstitution. Buffer containing an equivalent amount posomes, indicating that some CFo or subunit I11 sites are of DTT was added to control samples. Unbound CF, was removed, vacant in these samples. The zero-protein liposomes bound and all samples were assayed for sulfite-stimulated Mg2+-ATPase an undetectable amount of CFI, supporting the hypothesis activity (990 pL of 4 mM ATP, 2 mM MgC12, 10 mM Tricine-NaOH that the binding of CF, to CFo or subunit 111 in proteolipo- [pH 8.01 100 mM Na2S0, and 10 pL of proteoliposomes, 37"C, 10 somes is specific. min). Liposomes prepared in the absence of the protein complexes did not bind a significant amount of CF, either before or after EDTA treatment. Total activity is given as nmol of Pi formed per min. DISCUSSION Total ATPase Activity Evidence presented in this paper supports a direct associ- Liposomes Reconstitution Bound ation between CFl and subunit 111. The enzyme purified as described is clearly deficient in a11 CFo subunits except subunit NonstriDDed StriDDed 111, although it may be slightly contaminated with Rubisco CFi-III Buffer 0.21 <0.02 and Cyt b6/f complex subunits. The presence of non-ATPase CFi 0.54 0.16 complex proteins does not affect the conclusions reached CFi-CFo Buffer 0.33 10.02 from the presented data. CFi 0.64 0.44 248 Wetzel and McCarty Plant Physiol. Vol. 102, 1993

techniques could not be used. Dye binding and silver staining Owens, and Karl-Heinz Siiss for their helpful discussions. We are of subunit I11 are inconsistent from gel to gel, precluding especially grateful to Dr. Jane Gibson for use of her laboratory space densitometry as a quantitative approach. Fromme et al. at Come11 University. (1987a) suggest a model of a complex of 12 subunits of subunit 111, based on the presence of a high molecular mas Received January 12, 1993; accepted Febmary 15, 1993. Copyright Clearance Center: 0032-0889/93/102/0241/09. (100 kD) protein band that correlates with the absence of the typical 8-kD band for subunit I11 under certain SDS-PAGE LITERATURE ClTED conditions. The same phenomenon was observed by us in some of the gels run for this study (unpublished observation). Admon A, Pick U, Avron M (1985) ATP-induced ApH formation in chloroplast ATP synthase proteoliposomes. J Membr Biol 86: Solubilization by cholate versus Triton X-100 and the re-

45-50 Downloaded from https://academic.oup.com/plphys/article/102/1/241/6066835 by guest on 23 September 2021 sultant association or dissociation of subunit 111, respectively, Apley EC, Wagner R (1990) Fast rotation of EITC-labelled proteo- provides a useful method for obtaining isolated subunit I11 lipid after reconstitution of chloroplast ATP synthase in bilayer for further study. One obstacle faced in using subunit 111 membranes. Biochim Biophys Acta 1015: 29-40 isolated by this method is remova1 of Triton X-100 from the Bensadoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70: 241-250 preparation, because the low critica1 micellar concentration Cohen SA, Tarvin TL, Bidlingmeyer BA, Tarr GE (1984) Analysis of Triton X-100 makes it difficult to extract. Previously pub- of amino acids: using precolumn derivatization with phenylisothi- lished methods for purifying subunit I11 require harsh or ocyanate. Am Lab 16: 48-59 denaturing treatments, including organic solvents (Fillin- Deckers-Hebestreit G, Altendorf K (1992) Influence of subunit- specific antibodies on the activity of the Fo complex of the ATP game, 1976; Nelson et al., 1977; Sigrist et al., 1977) or synthase of Escherichia coli. 11. Effects of subunit c-specific poly- preparative SDS-PAGE (Fromme et al., 1987a). Remova1 of clonal antibodies. J Biol Chem 267: 12370-12374 CFI from the CFI-III complex in solution by Triton X-100 Deckers-Hebestreit G, Simoni RD, Altendorf K (1992) Influence (this paper) or by EDTA (this paper) or NaBr treatment of subunit-specific antibodies on the activity of the Fo complex of the ATP synthase of Escherichia coli. I. Effects of subunit b-specific (Grotjohann and Graber, 1990) of CFl-I11 proteoliposomes polyclonal antibodies. J Biol Chem 267: 12364-12369 provides milder conditions for obtaining a potentially Deno H, Shinozaki K, Sugiura M (1984) Structure and transcription more physiologically intact complex for the study of proton pattem of a tobacco chloroplast gene coding for subunit 111 of translocation. proton-translocation ATPase. Gene 32 195-201 Feng Y, McCarty RE (1990a) Chromatographic purification of the That CFl remains bound to subunit I11 and can be incor- chloroplast ATP synthase (CFo-CFI)and the role of CFo subunit porated into liposomes in the absence of other CFo subunits IV in proton conduction. J Biol Chem 265 12474-12480 does not preclude other CFl-CFo connections. In addition to Feng Y, McCarty RE (1990b) Subunit interactions within the chlo- the evidence described in the introduction to this paper, roplast ATP synthase (CFo-CF1)as deduced by specific depletion of CFo polypeptides. J Biol Chem 265: 12481-12485 results of other studies have supported the idea of multiple Fillingame RH (1976) Purification of the carbodiimide-reactive pro- subunit interactions. Apley and Wagner (1990) found that, tein component of the ATP energy transducing system of Esche- when CFI-CFo is treated with eosin-5-isothiocyanate in arti- richia coli. J Biol Chem 251: 6630-6637 ficial membranes, subunit I11 is specifically labeled and dis- Fling SP, Gregerson DS (1986) Peptide and protein molecular weight distribution by electrophoresis using a high-molarity tris sociates from the ATP synthase complex into monomers. CF1 buffer system without urea. Anal Biochem 155: 83-88 remains associated with the membrane, presumably bound Fromme P, Boekema EJ, Graber P (1987a) Isolation and character- to the other CFo subunits. Feng and McCarty (1990a) deter- ization of a supramolecular complex of subunit I11 of the ATP- mined that subunit IV was required for ATP synthesis in the synthase from chloroplasts. Z Naturforsch 42c: 1239-1245 Fromme P, Graber P, Salnikow J (1987b) Isolation and identification reconstituted enzyme complex. In E. coli, purified subunit c of a fourth subunit in the membrane part of the chloroplast ATP- (equivalent to subunit 111) is capable of limited binding of Fl synthase. FEBS Lett 218: 27-30 (Schneider and Altendorf, 1985). Also in E. coli, anti-b anti- Futai M, Noumi T, Maeda M (1989) ATP synthase (H+-ATPase): bodies (b is equivalent to subunit I) disrupt binding of F1 to results by combined biochemical and molecular biologcal ap- proaches. Annu Rev Biochem 58: 111-136 Fo (Deckers-Hebestreit et al., 1992). The cumulative evidence Gao Z, Bauerlein E (1987) ldentifying subunits of ATP synthase from studies of CFI-CFo interactions indicates a model with TFo-F1in contact with phospholipid head groups. FEBS Lett 223: severa1 points of contact. It seems likely that there are differ- 366-370 ent functions for each region of association: regulation of Grotjohann I, Graber P (1990) Isolation and properties of the membrane-integrated part of the ATP-synthase from chloroplasts, proton flow, transduction of electrochemical membrane po- CFo. Biochim Biophys Acta 1017: 177-180 tential, and conformational changes associated with activa- Hammes GG (1983) Mechanism of ATP synthesis and coupled tion or deactivation of the enzyme complex are possibilities. proton transport: studies with purified chloroplast coupling factor. Now that methods exist for depletion and isolation of each Trends Biochem Sci 8 131-134 Howe CJ, Auffret AD, Doherty A, Bowman CM, Dyer TA, Gray CFo subunit, reconstitution of enzymically active ATP syn- JC (1982) Location and nucleotide sequence of the gene for the thase complexes should begin to reveal more information proton-translocating subunit of wheat chloroplast ATP synthase. about the influence of the subunits on CFl function. Proc Natl Acad Sci USA 79 6903-6907 Hudson GS, Mason JG, Holton TA, Koller B, Cox G, Whitfeld PR, Bottomley W (1987) A gene cluster in the spinach and pea ACKNOWLEDCMENTS chloroplast genomes encoding one CF1 and three CFOsubunits of the H+-ATP synthase complex and the ribosomal protein 52. J Mo1 The authors acknowledge Yu Feng, Patricia Soteropoulos, Adam Bioll96 283-298 Shapiro, Jian-Ping Xiao, Xenia Young, and Michael Dann for their Hurt E,,Hauska G (1981) A cytochrome f/b, complex of five poly- assistance with these experiments and advice concerning them. We peptides with plastoquinol-plastocyanin-oxidoreductaseactivity would also like to thank Drs. Andr6 Jagendorf, Peter Hinkle, Thomas from spinach chloroplasts. Eur J Biochem 117: 591-599 lsolation of a Chloroplast Coupling Factor 1-Subunit III Complex 249

Junge W (1989) Protons, the thylakoid membrane, and the chloro- Pick U, Racker E (1979) Purification and reconstitution of the N,N'- plast ATP synthase. Ann NY Acad Sci 574: 268-286 dicyclohexylcarbodiimide-sensitiveATPase complex from spinach Kagawa Y, Racker E (1971) Partia1 resolution of the enzymes cata- chloroplasts. J Biol Chem 254 2793-2799 lyzing oxidative phosphorylation. XXV. Reconstitution of vesicles Richter ML, Lampton JD, McCarty RE (1987) Comparison of meth- catalyzing "P,-adenosine triphosphate exchange. J Biol Chem 246: ods of activation of the ATPase activity of the chloroplast coupling 5477-5487 factor 1. In J Biggins, ed, Progress in Photosynthesis Research, Vol Kessler RJ, Fanestil DD (1986) Interference by lipids in the deter- 111. Martinus Nijhoff, Dordrecht, The Netherlands, pp 57-60 mination of protein using bicinchonic acid. Anal Biochem 159 Richter ML, Patrie WJ, McCarty RE (1984) Preparation of the e 138-142 subunit and e subunit-deficient chloroplast coupling factor 1 in Krupinski J, Hammes GG (1986) Steady-state ATP synthesis by reconstitutively active forms. J Biol Chem 259 7371-7373 bacteriorhodopsin and chloroplast coupling factor co-reconstituted Rott R, Nelson N (1983) Conservation and organization of subunits into asolectin vesicles. Proc Natl Acad Sci USA 834233-4237 of the chloroplast proton ATPase complex. In C Sybesma, ed,

Lemaire C, Wollman F-A (1989a) The chloroplast ATP synthase in Advances in Photosynthesis Research, Vol 2. Martinus Nijhoff/ Downloaded from https://academic.oup.com/plphys/article/102/1/241/6066835 by guest on 23 September 2021 Chlamydomonas reinhardtii. I. Characterization of its nine consti- Dr. W. Junk, Dordrecht, The Netherlands, pp 501-510 tutive subunits. J Biol Chem 264 10228-10234 Schmidt G, Graber P (1985) The rate of ATP synthesis by reconsti- Lemaire C, Wollman F-A (1989b) The chloroplast ATP synthase in tuted CFoCFl liposomes. Biochim Biophys Acta 808 46-51 Chlamydomonas reinhardtii. 11. Biochemical studies on its biogenesis Schneider E, Altendorf K (1985) A11 three subunits are required for using mutants defective in photophosphorylation. J Biol Chem 264 the reconstitution of an active proton channel (F,) of Eschevichia 10235-10242 coli ATP synthase (FIF,). EMBO J 4 515-518 Livingston DM (1974) Immunoaffinity chromatography of proteins. Shapiro AB, McCarty RE (1990) Substrate binding-induced altera- Methods Enzymol34 723-727 tion of nucleotide binding site properties of chloroplast coupling Lowry OH, Rosenberg NJ, Farr AL, Randall RJ (1951) Protein factor 1. J Biol Chem 265 4340-4347 measurement with the Folin phenol reagent. J Biol Chem 193 Shively JE (1986) Methods of Protein Microcharacterization:A Prac- 265-275 tical Handbook. The Humana Press, Clifton, NJ, pp 155-193 Markwell MAK, Haas SM, Bieber LL, Tolbert NE (1978) A modi- Sigrist H, Sigrist-Nelson K, Gitler C (1977) Single-phase butanol fication of the Lowry procedure to simplify protein determination extraction: a new tool for proteolipid isolation. Biochem Biophys in membrane and lipoprotein samples. Anal Biochem 87: 206-210 Res Commun 74 178-184 McCarty RE, Carmeli C (1982) Proton translocating ATPase of Sigrist-Nelson K, Azzi A (1980) The proteolipid subunit of the photosynthetic membranes. ln Govindjee, ed, Photosynthesis: En- chloroplast adenosine triphosphatase complex: reconstitution and ergy Conversion by Plants and Bacteria, Vol 1. Academic Press, demonstration of proton-conductive properties. J Biol Chem 255: New York, pp 647-695 10638-10643 McCarty RE, Moroney JV (1985) Functions of the subunits and Sigrist-Nelson K, Sigrist H, Azzi A (1978) Characterization of the regulation of chloroplast coupling factor 1. In AN Martonosi, ed, dicyclohexylcarbodiimide-binding protein isolated from chloro- The Enzymes of Biological Membranes, Ed 2, Vol4. Plenum Press, plast membranes. Eur J Biochem 92: 9-14 New York, pp 383-413 Strotmann H, Bickel-Sandkotter S (1984) Structure, function, and Moroney JV, Lopresti L, McEwen BF, McCarty RE, Hammes GG regulation of chloroplast ATPase. Annu Rev Plant Physiol 35: (1983) The M,-value of chloroplast coupling factor 1. FEBS Lett 97-120 158 58-62 Siiss K-H (1986) Neighbouring subunits of CFO and between CF1 Moroney JV, McCarty RE (1982a) Effect of proteolytic digestion on and CFO of the soluble chloroplast ATPsynthase (CF1-CFO) as the Ca2+-ATPaseactivity and subunits of latent and thiol-activated revealed by chemical protein cross-linking. FEBS Lett 201: chloroplast coupling factor 1. J Biol Chem 257: 5910-5914 63-68 Moroney JV, McCarty RE (1982b) Light-dependent cleavage of the Siiss K-H, Schmidt O (1982) Evidence for an a3,P3, 7,F, I, 11, c, IIIs y subunit of coupling factor 1 by trypsin causes activation of Mg2'- subunit stoichiometry of chloroplast ATP synthetase complex (CFI- ATPase activity and uncoupling of photophosphorylation in spin- CF,). FEBS Lett 144 213-218 ach chloroplasts. J Biol Chem 257: 5915-5920 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of Nelson N, Eytan E, Notsani B-E, Sigrist H, Sigrist-Nelson K, Gitler proteins from polyacrylamide gels to nitrocellulose sheets: proce- C (1977) lsolation of a chloroplast N,N'-dicyclohexylcarbodiimide- dure and some applications. Proc Natl Acad Sci USA 76 binding proteolipid, active in proton translocation. Proc Natl Acad 4350-4354 Sci USA 74 2375-2378 Wetzel CM, McCarty RE (1993) Aspects of subunit interactions in Patrie WJ, McCarty RE (1984) Specific binding of coupling factor 1 the chloroplast ATP synthase. 11. Characterization of a chloroplast- lacking the 6 and e subunits to thylakoids. J Biol Chem 259 coupling factor 1-subunit I11 complex from spinach thylakoids. 11121-11128 Plant Physiol 102: 251-259 Penefsky HS (1977) Reversible binding of Pi by beef heart mito- Wray W, Boulikas T, Wray V, Hancock R (1981) Silver staining of chondrial adenosine triphosphatase. J Biol Chem 252 289 1-2899 proteins in polyacrylamide gels. Anal Biochem 118: 197-203