Lateral Distribution of the Cytochrome b 6 / f and Coupling Factor ATP Synthetase Complexes of Chloroplast Thylakoid Membranes

Abstract

We have visualized directly the distribution of the cytochrome b./fand coupling factor ATP synthetase complexes in thylakoid membranes of embedded, thin-sectioned, intact chloroplasts by using rabbit antibodies directed against each complex, folowed by ferritin-conjugated goat anti-(rabbit immunoglobulin G) antibodies. The labeling patterns indicate that in spinach (Spinacia okrcea) chloroplasts the cytochrome blfcomplex is distributed laterally throughout both stacked grna and unstacked stroma membrane regions, whereas the coupling factor ATP synthetase complex is found exclusively in strom thylakoids and in the marginal and end membranes of grana. Three major hypotheses have been forwarded regarding the spatial distribution of Cyt b6/lf in chloroplast thylakoid membranes: (a) random distribution between grana and stroma membranes (1, 2, 5, 8); (b) specific localization within the membrane region interfacing grana and stroma membranes (i.e. fret region) (7, 9); and (c) localization within stroma membranes only (1 1). All three hypotheses are backed by a certain amount of experimental evidence. The evidence in all cases involved somewhat indirect observations, and in some cases even involved different interpretations of the same basic result. To avoid the pitfalls inherent to indirect observations of what is undoubtedly a dynamic distribution, we have directly visualized the location of the Cyt b6ff and CFo/CF1 ATP synthetase complexes using immunocytochemical labeling of intact and broken chloroplasts. Our data confirm the distribution of the CFo/CF1 complexes in unstacked thylakoid membranes only. In contrast, we found the Cyt b6/fcomplex throughout both stroma and grana membrane regions in similar quantities. MATERIALS AND METHODS All chemicals used were either reagent grade or of the highest quality available. Chloroplast Isolation. Washed, deveined leaves of commer-'Supported by National Science Foundation PCM-8 118627. 2 Abbreviations: CYT b6/f cytochrome f and b6 complex; IgG, im-munoglobulin class G; CFo/CFI, coupling factor ATP synthetase complex ; TBST, Tris-buffered saline with Tween-20. cially grown spinach (Spinacea oleracea) were homogenized in a blender equipped with razor blades, in an isolation buffer made of 0.4 M sucrose, 50 mM Tricine, 10 mm NaCl, 5 mM MgCI2, pH 8.0. The brei was filtered through four layers of Miracloth (Calbiochem-Behring) to remove large debris. The filtrate was sedimented at 300g for 4 min, and the pellet discarded. The supernatant was then sedimented at 2000g for 10 min to collect the crude chloroplast pellet. All steps were performed at 0 to 4°C. Immocytochemistry. The chloroplasts were fixed for I h at room temperature in 2% (w:v) glutaraldehyde, in 7.5 mM sodium phosphate, 3.75 mM NaCl, 3.75 mM MgC92, pH 7.4. Dehydration was carried out in an ascending ethanol series consisting of 30, 50, 70, 95, and 100% (v:v). Infiltration was performed by incu-bating for 20 min each in 1:1, then 1:3 (v:v) ethanol:Lowicryl K4M, followed by absolute Lowicryl K4M (Polysciences, Inc., Warrington, PA). Polymerization was done under a N2 atmosphere in Beem capsules at-20°C using longwave UV light as the initiator (Ultra-violet Products, Inc., San Gabriel, CA). Silver sections were picked up on nickel grids (200 or 300 mesh) for immunocytochemical labeling. Sections were first incubated on a blocking solution of 10 mg/ml BSA (fatty acid-free, Sigma Chemical Co.) for 15 min. The sections were then exposed to the primary antibody, a rabbit anti-Cyt b6/fantiserum (described in 18), for 2 h at room temperature. The IgG fraction, collected by DEAE-cellulose column chromatography (16), was used to reduce nonspecific background staining. By electroblot analysis, the anti-Cyt b6/frecognizes only Cytfand b6, without detectable reactivity toward the other polypeptides in the complex (data not shown). Controls included at this step consisted of sections given further exposure to blocking solution, exposure to preimmune rabbit serum, anti-Cyt b6/f preadsorbed against isolated Cyt b6/fcomplex (13), or with buffer alone. As a positive control for the efficacy of the immunostaining procedure, we used a rabbit anti-CFo/CF1 antibody to localize the ATP synthe-tase complex; these antibodies are also described in Morschel and Staehelin (18). Following exposure to the primary antibody, the grids were rinsed with TBST buffer (20 mm Tris, 0.5 M NaCI, 1.0% Tween-20 [Sigma Chemical Co.], pH 7.4) and incubated with the secondary antibody, a ferritin-conjugated goat anti-rabbit IgG (H and L chains) antibody (IgG fraction; Cappel Laboratories, Downingtown, PA), for 20 min. The grids were again rinsed with TBST buffer, then with water. Sections were counter-stained with 2% (w:v) aqueous uranyl acetate for I min. All immunoreagents and blocking solution were diluted in TBST buffer and all steps were carried out at room temperature. The labeling densities were determined by carefully delineating on 199