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Integral Membrane Portion (CFO) of Chloroplast ATP-Synthase

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Integral Membrane Portion (CFO) of Chloroplast ATP-Synthase Proc. Nati. Acad. Sci. USA Vol. 81, pp. 3078-3082, May 1984 Biophysics Cooperative transient trapping of photosystem II protons by the integral membrane portion (CFO) of chloroplast ATP-synthase after mild extraction of the four-subunit catalytic part (CF1) (photosynthesis/water oxidation/photophosphorylation/proton pumping/proteolipid) WOLFGANG JUNGE*, YU QUN HONG*t, Lu PING QIAN*t, AND ALEXANDRO VIALEt *Biophysik, Fachbereich Biologie/Chemie, Universitaet Osnabrueck, Postfach 4469, D-4500 Osnabrueck, Federal Republic of Germany; and tCentro de Estudios Fotosinteticos y Bioquimicos, Consejo Nacional de Investigaciones Cientificas y Technicas, Fundacion Miguel Lillo, Universidad de Rosario, Suipacha 536, 2000 Rosario, Argentina Communicated by Pierre Joliot, November 21, 1983 ABSTRACT The ATP-synthase in chloroplasts is built that 8 alone can bind to CF0, without aiding the binding of from two blocks, CF0, which is integral to the thylakoid mem- CF1, in agreement with ref. 14. brane and which serves as a proton channel, and CF1, at- The properties of CFO were reviewed by Nelson (3). Fur- tached to CFO, which is catalytically active. This study is ther details also for FO from other membranes may be found aimed at understanding proton conduction through CF0. By a in an article by Sebald and Hoppe (16). CFO, which was nev- mild procedure we extracted <10% of total CF1, predomi- er purified per se, could be recovered together with CF1 (17, nantly the four-subunit CF1 without the 6 subunit. Extracted 18). It is composed of at least three subunits I, II, and III, chloroplasts were excited with short flashes of light and the with molecular masses of 15, 12.5, and 8 kDa (19). Subunit time course of the transmembrane potential and of the pH III, a proteolipid, was isolated and purified. It is one of the changes in both phases was measured spectrophotometrically. N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteins of Mild extraction of CF1 caused two effects. ('l Up to 50% of the thylakoids. Its reconstitution into vesicles from chloroplast protons rapidly released from water oxidation transiently es- lipids produced enhanced proton conduction (20). In spinach caped detection in the thylakoid interior. (it) The initial extent the proton-translocating ATP-synthase could be deactivated of the transmembrane potential was decreased by some 10% if only one of six of the potentially DCCD-binding subunits (20-,us resolution). Protons that were not detected inside ap- III was labeled with DCCD (21). This suggested that six peared in the external phase after having passed the thylakoid copies of subunit III were required to form the proton chan- membrane. pH titrations of the transient loss of protons pro- nel through CFO. Recently, proteolipid from yeast mitochon- duced an extremely sharp transition (near pH 7.5) as if six dna was incorporated into bimolecular lipid membranes to protons were buffered in a strictly cooperative manner. These study the channel-forming properties in a quantitative way effects were reversed upon addition of NN'-dicyclohexylcar- (22). For technical reasons these studies had to be carried bodiimide, which, among other actions, blocks the proton out at low pH (2.2). Under these conditions dimers seemed channel through CF0. We interpret these observations as fol- to be the minimal unit for proton conduction. The role of the lows. (i) CFO incorporates proton binding groups, which can other subunits, I and II, for the binding of CF1 to CFO or for act in a hexacooperative way. These groups are located near regulation of proton flux still awaits elucidation. the middle of the membrane. (id) After extraction of CF1, pro- In previous studies we found high electric conductivity of, tons produced during water oxidation have very rapid access and proton leakage through, the thylakoid membrane after to these groups, but they pass the full span of the membrane extraction of CF1 (23, 24). These studies were carried out by more slowly: buffering precedes conduction through CFO. flash spectrophotometry via the electrochromic absorption changes of certain chloroplast pigments (25) (reviewed in Light-dependent synthesis of ATP of chloroplasts is cata- refs. 26, 27) and via pH-indicating dyes. We found that pro- lyzed by the enzyme complex CF1-CFO. CFO, an intrinsic ton leakage could be blocked by reincorporation of isolated membrane protein, serves as a proton channel. CF1, a pe- CF1 into CF1-depleted membranes or, alternatively, upon ripheral protein, catalyzes phosphorylation of ADP. Togeth- addition of DCCD. However, electric leakage remained er they function as a proton-translocating ATP-synthase (or high. This was attributed to unspecific damage that was per- ATPase). Their structure and function have been reviewed haps unrelated to CFO. recently (1-3). CF1, which can be readily detached from the In this study we attempted to characterize proton binding thylakoid membrane, is composed of five subunits: a to E by membrane-bound CFO and proton conduction through with molecular masses of approximately 59, 56, 37, 17.5, and CFO under mild extraction of CF1 to minimize electric leaks 13 kDa (4). Its subunit stoichiometry is probably 3:3:1:1:1 (5, as side effects of the extraction procedure. 6) and its molecular mass is -400 kDa (7). Subunits a and p both interact with nucleotides (8) but the catalytic site(s) MATERIALS AND METHODS seems to be located on subunit P (9). Subunit 'y may be in- volved in the binding of the regulatory subunit £ (4) and its Chloroplasts. Chloroplasts were prepared from winter modification alters the proton permeability of the complex spinach, obtained from the local market (exception: pea (10-12). The role of the 8 subunit is not clear. In earlier work chloroplasts in Fig. 5). We prepared "broken chloroplasts" it was suggested that it may help to bind CF1 to CFO (13), but according to standard procedures (see ref. 28), but without in a detailed study on this question Andreo et al. (14) found the opposite. However, Roos and Berzborn (15) reported Abbreviations: CFO and CF1, integral membrane portion and cata- lytic part of chloroplast ATP-synthase, respectively; DCCD, N,N'- dicyclohexylcarbodiimide; PhMeSO2F, phenylmethylsulfonyl fluo- The publication costs of this article were defrayed in part by page charge ride. payment. This article must therefore be hereby marked "advertisement" tPermanent address: Institute of Plant Physiology, Academia Sin- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ica, 300 Fonglin Road, Shanghai, China. 3078 Biophysics: Ju.nge et aL Proc. NatL. Acad. Sci. USA 81 (1984) 3079 addition of divalent cations to the blending medium. Broken pling interval. Its intensity was set reciprocal to the exposure chloroplasts were pelleted by centrifugation at 12,000 x g for interval to yield <5% excitation of reaction centers by mea- 10 min. The pellet was resuspended in 10 mM tricine/NaOH suring light pulse. Repetitive transient absorption changes (pH 8) to yield a chlorophyll concentration of 2-4 mg/ml in were averaged on a TRACOR TN 1500 computer. the stock solution. The stock was stored on ice until needed. The electric potential difference was measured via the Mild Extraction of CF1. Mild extraction of CF1 was in- electrochromic absorption changes at 515 or 522 nm as de- duced as follows. Chloroplasts from the concentrated stock scribed (25, 35). pH transients in the inner phase of thyla- solution were suspended in distilled water and gently stirred koids were measured via the "pHin-indicating absorption for 2 min at room temperature. EDTA was present if indicat- changes of neutral red." The outer phase was selectively ed. The pH was adjusted to 7.5 by addition of NaOH. The buffered by addition of bovine serum albumin at 1.3 g/liter. chlorophyll concentration in the dilute suspension was 100 Absorption changes were recorded at a wavelength of 522 or AtM for the biochemical assay and 10 AuM for the spectropho- 548 nm as the difference between transient signals obtained tometric measurements. After incubation for 2 min, MgCl2 in the presence (13 /xM) and in the absence of neutral red as (1-2 mM) was added to stop extraction. Control samples un- described (36, 37). The exclusive response of neutral red to derwent the same incubation except that MgCl2 was added pH transients in thylakoids and the absence of any response together with EDTA. to other events (e.g., redox changes) have been demonstrat- Amount of CF1 Extracted. The amount of CF1 extracted ed (37). pH transients in the outer phase were measured via was determined for spinach. The sample was centrifuged for the absorption changes of phenol red at 548 nm (38). 10 min at 15,000 x g. To the supernatant, the following chemicals were added from a concentrated stock solution to yield the given final concentrations: Tris sulfate (pH 7.5), 20 RESULTS mM; EDTA, 2 mM; NH4SO4, with NH40H to yield pH 7.5, Extraction of CF1. Extraction of CF1 from spinach was 100 mM; ATP, 1 mM; and phenylmethylsulfonyl fluoride measured as a function of the EDTA concentration present (PhMeSO2F) as protease inhibitor, 10 AM (Sigma; at 100 mM during the 2 min of incubation in distilled water. The extrac- in a 100% dimethyl sulfoxide stock solution). For concentra- tion curve showed a sigmoidal shape with the following tion, the solution was passed over a DEAE-Sephadex col- properties. At a very low EDTA concentration (e.g., 10 gM) umn (A-50, 2.5 x 6 cm) that was equilibrated with the same it was essentially flat, with an extraction of between 1% and buffer (see ref. 29). Protein was eluted with 50 ml of the 5% of total CF1. At 40 gM EDTA a rise became apparent, stock solution and it was concentrated (20-fold) by Amicon which saturated around 100 gM with a midpoint at 70 gM.
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