Plant
Planta (1989) 178:297 302
9 Springer-Verlag 1989
Localization of ribulose-bisphosphate carboxylase-oxygenase and its putative binding protein in the cell envelope of Chromatium vinosum Bruce A. McFadden a *, Jose A. Torres-Ruiz a **, and Vincent R. Franceschi 2 1 Biochemistry-Biophysics Program and z Department of Botany, Washington State University, Pullman, WA 99164-4660, USA
Abstract. Antibodies to the large and small sub-
units of ribulose-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1-1.39) and a putative binding protein (PBP) for RuBisCO from Chromatiurn vinosum have been used to localize these proteins in thin sections. Immunogold techniques employing single and double antibodies establish that RuBisCO and the RuBisCO PBP are concentrated in the cell envelope of C. vinosum. Key words: Chromatium - Immunogold labeling
Membrane localization - Ribulose bisphosphate carboxylase-oxygenase - Ribulose-bisphosphate carboxylase-oxygenase-binding protein
Introduction
Ribulose-bisphosphate carboxylase-oxygenase (RuBisCO) is a ubiquitous, abundant dual-function enzyme which initiates photosynthetic carbondioxide fixation or the opposing process of photorespiration (for a review, see McFadden 1980). Among the prokaryota and eukaryota, the dominant form of RuBisCO consists of eight 55-kDa (kilodalton) large (L) and eight 15-kDa small (S) subunits (summarized in McFadden et al. 1986). In higher plants, L and S subunits are encoded by the chloroplast and nuclear genome, respectively, and the S subunit is synthesized on cytoplasmic ribosomes as a precursor which is taken up by chloroplasts. After removal of a 5-kDa leader se* To whom correspondence should be addressed ** P r e s e n t a d d r e s s : Department of Biochemistry, Ponce School of Medicine, Ponce, Puerto Rico kDa = kilodalton; L = large subunit of RuBisCO; PBP =putative binding protein of RuBisCO; RuBisCO = ribulose-bisphosphate carboxylase-oxygenase; S=small subunit of RuBisCO Abbreviations:
quence, mature S subunits assemble with L subunits in the chloroplast in a process which may require an L-subunit-binding protein (Musgrove and Ellis 1986). The resultant LsSs complex is catalytically competent. Recently, our research has focused upon RuBisCO in Chromatium vinosum. Two highly active forms of this enzyme, LsSs and Ls, were isolated (Torres-Ruiz and McFadden 1985), and it was found that the LsSs form is converted to the Ls form at high centrifugal forces (Torres-Ruiz and McFadden 1987a). In the process, S subunits are concentrated in the membrane fraction of C. vinosum (Torres-Ruiz and McFadden 1987 b). In purification of the LsSs form of RuBisCO from this source, a 700-kDa protein consisting of 12 apparently identical subunits copurifies with RuBisCO. This protein from C. vinosum is a homolog of the RuBisCO L-subunit-binding protein from higher plants (Torres-Ruiz and McFadden 1988) and has been designated as a putative binding protein (PBP) of RuBisCO. In higher plants, the dodecameric L-subunit-binding protein consists of two similar 61- and 60-kDa subunits which have been designated e and fl, respectively (for a review see Hemmingsen et al. 1988), and which are strongly cross-reactive immunologically (Torres-Ruiz and McFadden 1988). In a recent report, a striking similarity between the sequence of cDNAs for the c~-subunit of RuBisCO L-subunit-binding proteins from castor bean and wheat and that of the gene for g r o E L protein from Escherichia coli has been noted (Hemmingsen et al. 1988). In E. coli, g r o E L and groES proteins are required for cell viability and the assembly of bacteriophage capsids. The homologous plant and bacterial proteins have been termed chaperonins (Hemmingsen et al. 1988) and are found in chloroplasts, mitochondria and pro-
298
B.A. McFadden et al. : Membrane localization of RuBisCO in Chromatium
karyotes. On the basis of its homology with the RuBisCO L-subunit-binding protein, PBP from C. vinosum should be designated as a chaperonin (Torres-Ruiz and McFadden 1988). We now describe electron-microscopic studies of thin sections of C. vinosum carried out using gold-labeled antibodies raised against L and S subunits of RuBisCO and PBP. The results establish that all three proteins are localized at the innerouter membrane (cell envelope) of C. vinosum. These results may have a bearing on the intracellular localization of RuBisCO and chaperonins in other prokaryota and in eukaryota as well. Material and methods Bacterial strain and growth conditions. Chromatium vinosum was obtained from the American Type Culture Collection in Rockville, Md., USA, through Professor R. Chollet, University of Nebraska, Lincoln, Neb., USA. Cells were grown anaerobically for 5 d at 30~ in the light using the HCO~-S20~--Na2S medium of Hurlbert and Lascelles (1963). Illumination was provided by placing culture tubes 20cm from two incandescent 100-W lamps with an interposed container of water as a heat trap. Preparation of polyclonal antibodies. The RuBisCO L and S subunits were purified as described by Torres-Ruiz and McFadden (1985) and L subunits freed of excess sodium dodecyl sulfate prior to use as an antigen (Torres-Ruiz and McFadden 1987 a). Antigenic S subunits were prepared by excising the corresponding gel band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maceration of the gel band before injection. The 700-kDa PBP was separated from RuBisCO by anion-exchange chromatography on diethylaminoethyl (DEAE)-Sephadex A50 (Torres-Ruiz and McFadden 1988). For antibody development, 50 100 Ixg of a given antigen in complete Freund's adjuvant (or incomplete Freund's adjuvant in the case of the macerated gel band containing S subunits) was injected subcutaneously in the neck region of New Zealand rabbits (Garvey et al. 1977, pp. 183 188). Additional booster injections prepared with incomplete Freund's adjuvant were administered as necessary every two weeks. Final blood was withdrawn either by cardiac puncture or from the vena cava after making an incision in the abdominal cavity of the rabbit. After blood clotting, the serum obtained was chromatographed on CM Affi-Gel Blue (Bio-Rad, Richmond, Cal., USA) to remove serine proteases. The immunoglobulin G (IgG) fraction was collected by precipitation (at 20 ~ C) by adjusting the solution to 50% saturation with (NH4)2SO4. The precipitate was then dissolved in and dialyzed against a borate-saline buffer, pH 8.3 (Garvey et al. 1977). Antisera against S subunits gave a cross reaction with PBP but the other antisera were monospecific (Torres-Ruiz and McFadden 1987a, 1988). Nevertheless, antibodies to the L and S subunits of RuBisCO and to PBP from C. vinosum were further purified by affinity chromatography on an Affi-Prep 10 medium-pressure affinity chromatography matrix which h a d been conjugated with L subunits, S subunits or PBP (see BioRad Bull. No. 1251 and Hill 1972). Resultant affinity-purified antibody preparations were monospecific. Preparation of cells for electron microscopy. Chromatium vinosum ceils were fixed for 16 h at 4 ~ C by resuspension of pelleted
cells in bacterial medium containing 2.5% (v/v) glutaraldehyde, 2% (v/v) freshly depolymerized paraformaldehyde and 50 mM 1,4-piperazinediethanesulfonic acid (Pipes), pH 7.2. The cells were washed by repeated pelleting and resuspension in bacterial medium. After dehydration in an ascending ethanol concentration series, the cells were infiltrated with L.R. White resin (Ted Pella, Redding, Cal., USA) which was later polymerized at 60 ~ C. Thin sections were cut with a diamond knife and picked up onto uncoated nickel grids. Irnmunogold localization. All incubations described below were done at room temperature on a mechanical shaker. The grids were incubated for 5 min by immersion in 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-buffered saline-Tween (TBST; 1 0 m M Tris, 500mM NaC1, 0.3% polyoxyethylenesorbitan (Tween 20), pH 7.2) plus 1% (w/v) bovine serum albumin (BSA) and then transferred to rabbit antibody against L subunit, S subunit, PBP, or rabbit preimmune serum, each of which had been diluted 1:50 in TBST + BSA. The grids were incubated for 2 h, then rinsed four times by immersion (5 min) in TBST + BSA, and finally immersed for 1 h in Protein A-gold (15 or 5 nm gold particle size; Janssen, Ted Pella) which had been diluted 1 : 50 (or 1 : 20 for S subunit) in TBST + BSA. After five rinses in TBST + BSA, followed by three rinses in TBST and three in glass-distilled water, the sections were poststained with 2% (w/v) uranyl acetate and 2% (w/v) lead citrate or 10% (w/v) phosphotungstic acid in 10% HC1 and examined on an Hitachi 300 or HS-8 transmission electron microscope (Hitachi Scientific Instruments, Nissei Sangyo American, Mountain Home, Cal., USA). For double-labelling, a similar procedure was followed except that the grids were floated on solutions so that each side was exposed to a different antibody. Phosphotungstic acid gave the best staining of the outer and inner membrane (cell envelope). All micrographs shown in this paper were stained with phosphotungstic acid except for Fig. 4.
Figs. 1-7. Transmission electron micrographs of thin sections through Chromatium vinosum after immunogold labelling
Fig. 1. Section treated with preimmune serum. Only one gold particle (arrow) can be seen. x 69600; bar=0.2 gm Fig. 2. Section treated with antibody against the putative binding protein (PBP). Label (gold particles) can be seen along the cell envelope but is also distributed in the cytoplasm, x 63700; bar = 0.2 gm Fig. 3. Localization of PBP along the cell envelope, x 142300; bar=0.1 gm
Figs. 4, 5. Immunogold localization of the small subunit of RuBisCO. Label is highly concentrated along the cell envelope. Fig. 4 x64700; bar=0.2 ~tm. Fig. 5 x 195000; bar=0.1 gm. In Fig. 4, vesicular intracytoplasmic membrane (for a review see Remsen 1978) is especially evident as are several electrontransparent sulfur deposits (Vatter and Wolfe 1958) in the upper portion of a dividing cell. These deposits may also be seen in Figs. 1-3 Figs. 6, 7. Immunogold localization of the large subunit of RuBisCO. Label can be found on and slightly interior to the cell envelope. Fig. 6 x 81 000; bar=0.2 gm. Fig. 7 x 141 300; bar = 0.1 gm. In Fig. 7, the inner and outer membranes comprising the cell envelope can be seen
B.A. M c F a d d e n et al. : M e m b r a n e localization o f R u B i s C O in Chromatium
299
300
B.A. McFadden et al. : Membrane localization of RuBisCO in Chromatium
Labelling density along the cells from the envelope inwards was estimated after immunolocalization of RuBisCO L and S subunits. Gold particles were counted in 20-nm segments parallel to the cell surface where the membrane had been cut in nearly perfect cross-section. The first segment was centered on the cell envelope (inner and outer membrane) which was 20 nm wide; then particles in two segments interior to the membrane were also counted. Samples from 15 to 20 cells were analysed for distribution of each antigen, giving total cell envelope lengths analyzed of 105 pm for the L subunit and 93 pm for the S subunit. The data are presented as percent of total label in each segment for all samples used for each antigen. A direct comparison of label density for each antigen could not be made because of differences in antibody titer.
Results
The results of the immunogold-localization studies are summarized by the micrographs shown in Figs. 1-7. Preimmune controls were virtually free of gold labelling and even the control in Fig. 1 showing one gold particle was a rarity, but is presented to show that sections were exposed to preimmune serum and Protein A-gold. The distribution of labelling for PBP showed considerable variability from cell to cell. Some cells had heavy labelling in the cytoplasm as well as along the envelope (Figs. 2, 3) while in other cells labelling was primarily along the envelope. In all cases, the envelope (inner and outer membrane) had a greater concentration of label than cytoplasm (Fig. 3). This was especially clear if considered on a crosssectional surface-area basis. For example, in Fig. 2 two-thirds of the label is on the envelope or within 20 nm of the envelope, giving a labelling density of about 770 gold particles per 1 pm 2, while onethird of the label is in the cytoplasm, giving a labelling denstiy of 60 particles per I pm 2. We speculate that the variability seen in cytoplasmic labelling reflects the age of cells. The S subunit of RuBisCO appeared to be highly concentrated along the envelope in all cells examined (Figs. 4, 5). In contrast, the L subunit appeared to be distributed along the envelope and 15-30 nm interior to it (Figs. 6, 7). Only small amounts of RuBisCO S and L subunits were seen in the interior of the cells (Figs. 4, 6). Quantitative analysis of the distribution of the S and L subunits supports the visual observation that the L subunit is distributed further inward from the envelope as compared with the S subunit (Table 1). Presence of label outside of cells was negligible. The variability in cytoplasmic labelling for PBP precluded a similar analysis of distribution for this protein. Double-labelling studies employing antibodies to PBP and L subunit, PBP and S subunit, or L and S subunits emphasized the co-localization of
Table 1. Distribution pattern of RuBisCO large and small subunits in C. vinosum Region of cell"
A B C
Label in each region (as % of total in all regions) Small subunit
Large subunit
76% (126) b 20 (34) 4 (7)
52% (323) b 34 (213) 14 (86)
a A = First 20 n m corresponding to the width of the inner-outer membrane; B = 20-nm region interior to A; C = 20-nm region interior to B b Values in 0 are actual counts of gold particles within a given segment
PBP and RuBisCO L and S subunits at the envelope (Figs. 8-11). Tangential sections through cells giving a large sheet of cell envelope provided a different view of the general distribution of the PBP and RuBisCO subunits (Figs. 10, 11). Discussion
As shown in previous research, centrifugation of extracts of C. vinosurn at 175000-g in the presence of polyethylene glycol-6000 converted the LaSs form of RuBisCO to the L8 form (Torres-Ruiz and McFadden 1987a). From the 175000.g pellet, a highly pigmented purple membrane fraction could be isolated by flotation on a sucrose gradient. Immunoblotting with antibodies to the S subunit of RuBisCO established that the purified membrane was highly enriched in S subunits; these could only be removed by extraction at 4-25~ C with 0.40.6 M KC1, buffers o f p H 2.5 or 12 at 0.1 M KC1, 4~ (Torres-Ruiz and McFadden 1987b) or 0.8 m M ethylenediaminetetraacetate at 0.1 M KC1, 4~ (unpublished observation, Torres-Ruiz). It was inferred that the S subunit is a peripheral membrane protein bound through electrostatic forces by way of a metallic-ion bridge although the question of the intracellular site of membrane association remained. The present results provide ultrastructural evidence that RuBisCO is localized at the cell envelope and not on the vesicular intracytoplasmic membrane (Fig. 4) that presumably contains light-harvesting components in C. vinos u m (for a review, see Remsen 1978). Given the present observations that L subunits are more interior and that S subunits are peripheral membrane components, it is likely that RuBisCO is attached to the cytoplasmic (inner) membrane by way of S subunits. In related research, Mori et al. (1984) have re-
B.A. McFadden et al. : Membrane localization of RuBisCO in Chromatium
301
Figs. 8-11. Immunogold labelling of thin sections of Chromatium vinosum using two antibodies. Bars=0.1 ~m. Large and small gold particles had diameters of 15 and 5 nm
Fig. 10. Tangential section near surface of a cell labelled for RuBisCO large subunit (large gold particles) and PBP (small gold particles), x 120000
Fig. 8. Double labeling for RuBisCO large subunit (large gold particles) and PBP (small gold particles), x 200 000
Fig. 11. Tangential section near surface of a cell labelled for RuBisCO small subunit (large gold particles) and PBP (small gold particles), x 120000
Fig. 9. Double labelling for RuBisCO small subunit (large gold particles) and PBP (small gold particles), x 200 000
ported a loose attachment of RuBisCO to chloroplast membranes from young leaves of spinach and pea. At 25 ~ C in the presence of 0.06 M KC1, the association of RuBisCO activity with chloroplast membranes was approx. 18%; at 20 m M MgC12 association of 25% was maximal at p H 8.0 but declined sharply in the p H interval 7.5-6.0. Of interest was the report that chloroplast-membrane association of chloroplastic aldolase and phosp-
hoglycerate kinase was also favored at 2 5 ~ by increasing concentrations of MgC12 up to 20 m M (Mori et al. 1984). The present indication that RuBisCO is localized on the cytoplasmic membrane of a purple sulfur bacterium should stimulate probes for a similar association with chloroplast membranes of higher plants. In all but the final step in purifying the L8S8 form of RuBisCO from C. vinosum, a 700-kDa pro-
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B.A. McFadden et al. : Membrane localization of RuBisCO in Chromatiurn
rein consistently co-purified with this enzyme in a fixed mole ratio. In the final step of anion-exchange chromatography this protein was eluted before RuBisCO. Analysis of the 700-kDa protein established that it was probably composed of 12 identical subunits (Torres-Ruiz and McFadden 1988). These subunits showed considerable N-terminal sequence similarity to those sequences for the similar c~- and fl-subunits of RuBisCO L-subunit-binding protein of higher plants (Torres-Ruiz and McFadden 1988). Subunits of the large 700kDa C. vinosurn protein also showed considerable immunological crossreaction with the plant e- and fl-subunits of the RuBisCO L-subunit binding protein. The C. vinosum protein was designated a putative binding protein (PBP) of RuBisCO (TorresRuiz and McFadden 1988). Recently, strong similarities in sequence have been noted between esubunits of the RuBisCO L-subunit binding protein of higher plants and the groEL protein of E. coli (Hemmingsen et al. 1988). These homologous proteins have been termed chaperonins. In general, they may assist in the post-translational assembly of oligomeric proteins such as RuBisCO (Hemmingsen et al. 1988). Indeed it has been found very recently that groE protein, consisting ofgroEL and groES subunits, promotes assembly of foreign prokaryotic RuBisCO oligomers in E. coli (Goloubinoff et al. 1989). The present evidence indicates that in the photosynthetic prokaryote C. vinosum, PBP, a chaperonin, is localized at the cytoplasmic membrane; this should stimulate studies of the localization ofgroE proteins in E. coll. Perhaps these bacterial chaperonins function by assisting posttranslational assembly of oligomeric proteins at the membrane. Whether chaperonins function in an analogous manner in eukaryotes remains to be determined. Certainly, localization of the RuBisCObinding protein in chloroplasts will be of interest.
We gratefully acknowledge support by the National Institutes of Health (GM-19,972) and the technical assistance of Xing Xiang Li. Facilities for electron microscopy were provided by the Electron Microscope Center of Washington State University.
References Garvey, J.S., Cremer, N.E, Sussdorf, D.H. (1977) Adjuvantmodified antigens. In: Methods in immunology. A laboratory text for instruction and research, 3rd edn., pp. 183-188, W.A. Benjamin, Reading, Mass., USA Goloubinoff, P., Gatenby, A.A., Lorimer, G.H. (1989) GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Eseherichia coli. Nature 337, 44-47 Hemmingsen, S.M., Woolford, C., van der Vies, S.M., Tilly, K., Dennis, D.T., Georgopoulos, C.P., Hendrix, R.W., Ellis, R.J. (1988) Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333, 330-334 Hill, R.J. (1972) Elution of antibodies from immunoadsorbents: effect of dioxane in promoting release of antibody. J. Immunol. Meth. 1, 231-234 Hurlbert, R.E., Lascelles, J. (1963) Ribulose-l,5-diphosphate carboxylase in Thiorhodaceae. J. Gen. Microbiol. 33, 445 448 McFadden, B.A. (1980) A perspective of ribulose bisphosphate carboxylase/oxygenase, the key catalyst in photosynthesis and photorespiration. Acc. Chem. Res. 13, 394-399 McFadden, B.A., Torres-Ruiz, J.A., Daniell, H., Sarojini, G. (1986) Interaction, functional relations and evolution of large and small subunits in RuBisCO from Prokaryota and Eukaryota. Phil. Trans. R. Soc. Lond. Ser. B 313, 347-358 Mori, H., Takabe, T., Akazawa, T. (1984) Loose association of ribulose 1,5-bisphosphate carboxylase/oxygenase with chloroplast thylakoid membranes. Photosynth. Res. 5, 1728 Musgrove, J.E., Ellis, R.J. (1986) The RuBisCO large subunit binding protein. Phil Trans. R. Soc. Lond. Ser. B 313, 419428 Remsen, C.C. (1978) Comparative subcellular architecture of photosynthetic bacteria. In: The photosynthetic bacteria, pp. 31-60, Clayton, R.K., Sistrom, W.R., eds. Plenum Press, New York Torres-Ruiz, J.A., McFadden, B.A. (1985) Isolation of L8 and LsSs forms of ribulose bisphosphate carboxylase/oxygenase from Chrornatium vinosum. Arch. Microbiol. 142, 55 60 Torres-Ruiz, J.A., McFadden, B.A. (1987a) The nature of L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase from Chromatium vinosum. Arch. Biochem. Biophys. 254, 63-68 Torres-Ruiz, J.A., McFadden, B.A. (1987b) Association of RuBisCO small subunits with a membrane fraction from Chromatiurn vinosum. In: Progress in photosynthesis research, vol. 3, pp. 5.419-5.422, Biggins, J., ed. Martinus Nijhoff Publishers, Dordrecht, The Netherlands Torres-Ruiz, J.A., McFadden, B.A. (1988) A homolog of ribulose bisphosphate carboxylase/oxygenase-bindingprotein in Chromatium vinosum. Arch. Biochem. Biophys. 261, 196 204 Vatter, A.E., Wolfe, R.S. (1958) The structure of photosynthetic bacteria. J. Bacteriol. 75, 480-488 Received 26 October 1988; accepted 3 Februar 1989
Planta (1989) 178:297 302
9 Springer-Verlag 1989
Localization of ribulose-bisphosphate carboxylase-oxygenase and its putative binding protein in the cell envelope of Chromatium vinosum Bruce A. McFadden a *, Jose A. Torres-Ruiz a **, and Vincent R. Franceschi 2 1 Biochemistry-Biophysics Program and z Department of Botany, Washington State University, Pullman, WA 99164-4660, USA
Abstract. Antibodies to the large and small sub-
units of ribulose-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1-1.39) and a putative binding protein (PBP) for RuBisCO from Chromatiurn vinosum have been used to localize these proteins in thin sections. Immunogold techniques employing single and double antibodies establish that RuBisCO and the RuBisCO PBP are concentrated in the cell envelope of C. vinosum. Key words: Chromatium - Immunogold labeling
Membrane localization - Ribulose bisphosphate carboxylase-oxygenase - Ribulose-bisphosphate carboxylase-oxygenase-binding protein
Introduction
Ribulose-bisphosphate carboxylase-oxygenase (RuBisCO) is a ubiquitous, abundant dual-function enzyme which initiates photosynthetic carbondioxide fixation or the opposing process of photorespiration (for a review, see McFadden 1980). Among the prokaryota and eukaryota, the dominant form of RuBisCO consists of eight 55-kDa (kilodalton) large (L) and eight 15-kDa small (S) subunits (summarized in McFadden et al. 1986). In higher plants, L and S subunits are encoded by the chloroplast and nuclear genome, respectively, and the S subunit is synthesized on cytoplasmic ribosomes as a precursor which is taken up by chloroplasts. After removal of a 5-kDa leader se* To whom correspondence should be addressed ** P r e s e n t a d d r e s s : Department of Biochemistry, Ponce School of Medicine, Ponce, Puerto Rico kDa = kilodalton; L = large subunit of RuBisCO; PBP =putative binding protein of RuBisCO; RuBisCO = ribulose-bisphosphate carboxylase-oxygenase; S=small subunit of RuBisCO Abbreviations:
quence, mature S subunits assemble with L subunits in the chloroplast in a process which may require an L-subunit-binding protein (Musgrove and Ellis 1986). The resultant LsSs complex is catalytically competent. Recently, our research has focused upon RuBisCO in Chromatium vinosum. Two highly active forms of this enzyme, LsSs and Ls, were isolated (Torres-Ruiz and McFadden 1985), and it was found that the LsSs form is converted to the Ls form at high centrifugal forces (Torres-Ruiz and McFadden 1987a). In the process, S subunits are concentrated in the membrane fraction of C. vinosum (Torres-Ruiz and McFadden 1987 b). In purification of the LsSs form of RuBisCO from this source, a 700-kDa protein consisting of 12 apparently identical subunits copurifies with RuBisCO. This protein from C. vinosum is a homolog of the RuBisCO L-subunit-binding protein from higher plants (Torres-Ruiz and McFadden 1988) and has been designated as a putative binding protein (PBP) of RuBisCO. In higher plants, the dodecameric L-subunit-binding protein consists of two similar 61- and 60-kDa subunits which have been designated e and fl, respectively (for a review see Hemmingsen et al. 1988), and which are strongly cross-reactive immunologically (Torres-Ruiz and McFadden 1988). In a recent report, a striking similarity between the sequence of cDNAs for the c~-subunit of RuBisCO L-subunit-binding proteins from castor bean and wheat and that of the gene for g r o E L protein from Escherichia coli has been noted (Hemmingsen et al. 1988). In E. coli, g r o E L and groES proteins are required for cell viability and the assembly of bacteriophage capsids. The homologous plant and bacterial proteins have been termed chaperonins (Hemmingsen et al. 1988) and are found in chloroplasts, mitochondria and pro-
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karyotes. On the basis of its homology with the RuBisCO L-subunit-binding protein, PBP from C. vinosum should be designated as a chaperonin (Torres-Ruiz and McFadden 1988). We now describe electron-microscopic studies of thin sections of C. vinosum carried out using gold-labeled antibodies raised against L and S subunits of RuBisCO and PBP. The results establish that all three proteins are localized at the innerouter membrane (cell envelope) of C. vinosum. These results may have a bearing on the intracellular localization of RuBisCO and chaperonins in other prokaryota and in eukaryota as well. Material and methods Bacterial strain and growth conditions. Chromatium vinosum was obtained from the American Type Culture Collection in Rockville, Md., USA, through Professor R. Chollet, University of Nebraska, Lincoln, Neb., USA. Cells were grown anaerobically for 5 d at 30~ in the light using the HCO~-S20~--Na2S medium of Hurlbert and Lascelles (1963). Illumination was provided by placing culture tubes 20cm from two incandescent 100-W lamps with an interposed container of water as a heat trap. Preparation of polyclonal antibodies. The RuBisCO L and S subunits were purified as described by Torres-Ruiz and McFadden (1985) and L subunits freed of excess sodium dodecyl sulfate prior to use as an antigen (Torres-Ruiz and McFadden 1987 a). Antigenic S subunits were prepared by excising the corresponding gel band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maceration of the gel band before injection. The 700-kDa PBP was separated from RuBisCO by anion-exchange chromatography on diethylaminoethyl (DEAE)-Sephadex A50 (Torres-Ruiz and McFadden 1988). For antibody development, 50 100 Ixg of a given antigen in complete Freund's adjuvant (or incomplete Freund's adjuvant in the case of the macerated gel band containing S subunits) was injected subcutaneously in the neck region of New Zealand rabbits (Garvey et al. 1977, pp. 183 188). Additional booster injections prepared with incomplete Freund's adjuvant were administered as necessary every two weeks. Final blood was withdrawn either by cardiac puncture or from the vena cava after making an incision in the abdominal cavity of the rabbit. After blood clotting, the serum obtained was chromatographed on CM Affi-Gel Blue (Bio-Rad, Richmond, Cal., USA) to remove serine proteases. The immunoglobulin G (IgG) fraction was collected by precipitation (at 20 ~ C) by adjusting the solution to 50% saturation with (NH4)2SO4. The precipitate was then dissolved in and dialyzed against a borate-saline buffer, pH 8.3 (Garvey et al. 1977). Antisera against S subunits gave a cross reaction with PBP but the other antisera were monospecific (Torres-Ruiz and McFadden 1987a, 1988). Nevertheless, antibodies to the L and S subunits of RuBisCO and to PBP from C. vinosum were further purified by affinity chromatography on an Affi-Prep 10 medium-pressure affinity chromatography matrix which h a d been conjugated with L subunits, S subunits or PBP (see BioRad Bull. No. 1251 and Hill 1972). Resultant affinity-purified antibody preparations were monospecific. Preparation of cells for electron microscopy. Chromatium vinosum ceils were fixed for 16 h at 4 ~ C by resuspension of pelleted
cells in bacterial medium containing 2.5% (v/v) glutaraldehyde, 2% (v/v) freshly depolymerized paraformaldehyde and 50 mM 1,4-piperazinediethanesulfonic acid (Pipes), pH 7.2. The cells were washed by repeated pelleting and resuspension in bacterial medium. After dehydration in an ascending ethanol concentration series, the cells were infiltrated with L.R. White resin (Ted Pella, Redding, Cal., USA) which was later polymerized at 60 ~ C. Thin sections were cut with a diamond knife and picked up onto uncoated nickel grids. Irnmunogold localization. All incubations described below were done at room temperature on a mechanical shaker. The grids were incubated for 5 min by immersion in 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-buffered saline-Tween (TBST; 1 0 m M Tris, 500mM NaC1, 0.3% polyoxyethylenesorbitan (Tween 20), pH 7.2) plus 1% (w/v) bovine serum albumin (BSA) and then transferred to rabbit antibody against L subunit, S subunit, PBP, or rabbit preimmune serum, each of which had been diluted 1:50 in TBST + BSA. The grids were incubated for 2 h, then rinsed four times by immersion (5 min) in TBST + BSA, and finally immersed for 1 h in Protein A-gold (15 or 5 nm gold particle size; Janssen, Ted Pella) which had been diluted 1 : 50 (or 1 : 20 for S subunit) in TBST + BSA. After five rinses in TBST + BSA, followed by three rinses in TBST and three in glass-distilled water, the sections were poststained with 2% (w/v) uranyl acetate and 2% (w/v) lead citrate or 10% (w/v) phosphotungstic acid in 10% HC1 and examined on an Hitachi 300 or HS-8 transmission electron microscope (Hitachi Scientific Instruments, Nissei Sangyo American, Mountain Home, Cal., USA). For double-labelling, a similar procedure was followed except that the grids were floated on solutions so that each side was exposed to a different antibody. Phosphotungstic acid gave the best staining of the outer and inner membrane (cell envelope). All micrographs shown in this paper were stained with phosphotungstic acid except for Fig. 4.
Figs. 1-7. Transmission electron micrographs of thin sections through Chromatium vinosum after immunogold labelling
Fig. 1. Section treated with preimmune serum. Only one gold particle (arrow) can be seen. x 69600; bar=0.2 gm Fig. 2. Section treated with antibody against the putative binding protein (PBP). Label (gold particles) can be seen along the cell envelope but is also distributed in the cytoplasm, x 63700; bar = 0.2 gm Fig. 3. Localization of PBP along the cell envelope, x 142300; bar=0.1 gm
Figs. 4, 5. Immunogold localization of the small subunit of RuBisCO. Label is highly concentrated along the cell envelope. Fig. 4 x64700; bar=0.2 ~tm. Fig. 5 x 195000; bar=0.1 gm. In Fig. 4, vesicular intracytoplasmic membrane (for a review see Remsen 1978) is especially evident as are several electrontransparent sulfur deposits (Vatter and Wolfe 1958) in the upper portion of a dividing cell. These deposits may also be seen in Figs. 1-3 Figs. 6, 7. Immunogold localization of the large subunit of RuBisCO. Label can be found on and slightly interior to the cell envelope. Fig. 6 x 81 000; bar=0.2 gm. Fig. 7 x 141 300; bar = 0.1 gm. In Fig. 7, the inner and outer membranes comprising the cell envelope can be seen
B.A. M c F a d d e n et al. : M e m b r a n e localization o f R u B i s C O in Chromatium
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Labelling density along the cells from the envelope inwards was estimated after immunolocalization of RuBisCO L and S subunits. Gold particles were counted in 20-nm segments parallel to the cell surface where the membrane had been cut in nearly perfect cross-section. The first segment was centered on the cell envelope (inner and outer membrane) which was 20 nm wide; then particles in two segments interior to the membrane were also counted. Samples from 15 to 20 cells were analysed for distribution of each antigen, giving total cell envelope lengths analyzed of 105 pm for the L subunit and 93 pm for the S subunit. The data are presented as percent of total label in each segment for all samples used for each antigen. A direct comparison of label density for each antigen could not be made because of differences in antibody titer.
Results
The results of the immunogold-localization studies are summarized by the micrographs shown in Figs. 1-7. Preimmune controls were virtually free of gold labelling and even the control in Fig. 1 showing one gold particle was a rarity, but is presented to show that sections were exposed to preimmune serum and Protein A-gold. The distribution of labelling for PBP showed considerable variability from cell to cell. Some cells had heavy labelling in the cytoplasm as well as along the envelope (Figs. 2, 3) while in other cells labelling was primarily along the envelope. In all cases, the envelope (inner and outer membrane) had a greater concentration of label than cytoplasm (Fig. 3). This was especially clear if considered on a crosssectional surface-area basis. For example, in Fig. 2 two-thirds of the label is on the envelope or within 20 nm of the envelope, giving a labelling density of about 770 gold particles per 1 pm 2, while onethird of the label is in the cytoplasm, giving a labelling denstiy of 60 particles per I pm 2. We speculate that the variability seen in cytoplasmic labelling reflects the age of cells. The S subunit of RuBisCO appeared to be highly concentrated along the envelope in all cells examined (Figs. 4, 5). In contrast, the L subunit appeared to be distributed along the envelope and 15-30 nm interior to it (Figs. 6, 7). Only small amounts of RuBisCO S and L subunits were seen in the interior of the cells (Figs. 4, 6). Quantitative analysis of the distribution of the S and L subunits supports the visual observation that the L subunit is distributed further inward from the envelope as compared with the S subunit (Table 1). Presence of label outside of cells was negligible. The variability in cytoplasmic labelling for PBP precluded a similar analysis of distribution for this protein. Double-labelling studies employing antibodies to PBP and L subunit, PBP and S subunit, or L and S subunits emphasized the co-localization of
Table 1. Distribution pattern of RuBisCO large and small subunits in C. vinosum Region of cell"
A B C
Label in each region (as % of total in all regions) Small subunit
Large subunit
76% (126) b 20 (34) 4 (7)
52% (323) b 34 (213) 14 (86)
a A = First 20 n m corresponding to the width of the inner-outer membrane; B = 20-nm region interior to A; C = 20-nm region interior to B b Values in 0 are actual counts of gold particles within a given segment
PBP and RuBisCO L and S subunits at the envelope (Figs. 8-11). Tangential sections through cells giving a large sheet of cell envelope provided a different view of the general distribution of the PBP and RuBisCO subunits (Figs. 10, 11). Discussion
As shown in previous research, centrifugation of extracts of C. vinosurn at 175000-g in the presence of polyethylene glycol-6000 converted the LaSs form of RuBisCO to the L8 form (Torres-Ruiz and McFadden 1987a). From the 175000.g pellet, a highly pigmented purple membrane fraction could be isolated by flotation on a sucrose gradient. Immunoblotting with antibodies to the S subunit of RuBisCO established that the purified membrane was highly enriched in S subunits; these could only be removed by extraction at 4-25~ C with 0.40.6 M KC1, buffers o f p H 2.5 or 12 at 0.1 M KC1, 4~ (Torres-Ruiz and McFadden 1987b) or 0.8 m M ethylenediaminetetraacetate at 0.1 M KC1, 4~ (unpublished observation, Torres-Ruiz). It was inferred that the S subunit is a peripheral membrane protein bound through electrostatic forces by way of a metallic-ion bridge although the question of the intracellular site of membrane association remained. The present results provide ultrastructural evidence that RuBisCO is localized at the cell envelope and not on the vesicular intracytoplasmic membrane (Fig. 4) that presumably contains light-harvesting components in C. vinos u m (for a review, see Remsen 1978). Given the present observations that L subunits are more interior and that S subunits are peripheral membrane components, it is likely that RuBisCO is attached to the cytoplasmic (inner) membrane by way of S subunits. In related research, Mori et al. (1984) have re-
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Figs. 8-11. Immunogold labelling of thin sections of Chromatium vinosum using two antibodies. Bars=0.1 ~m. Large and small gold particles had diameters of 15 and 5 nm
Fig. 10. Tangential section near surface of a cell labelled for RuBisCO large subunit (large gold particles) and PBP (small gold particles), x 120000
Fig. 8. Double labeling for RuBisCO large subunit (large gold particles) and PBP (small gold particles), x 200 000
Fig. 11. Tangential section near surface of a cell labelled for RuBisCO small subunit (large gold particles) and PBP (small gold particles), x 120000
Fig. 9. Double labelling for RuBisCO small subunit (large gold particles) and PBP (small gold particles), x 200 000
ported a loose attachment of RuBisCO to chloroplast membranes from young leaves of spinach and pea. At 25 ~ C in the presence of 0.06 M KC1, the association of RuBisCO activity with chloroplast membranes was approx. 18%; at 20 m M MgC12 association of 25% was maximal at p H 8.0 but declined sharply in the p H interval 7.5-6.0. Of interest was the report that chloroplast-membrane association of chloroplastic aldolase and phosp-
hoglycerate kinase was also favored at 2 5 ~ by increasing concentrations of MgC12 up to 20 m M (Mori et al. 1984). The present indication that RuBisCO is localized on the cytoplasmic membrane of a purple sulfur bacterium should stimulate probes for a similar association with chloroplast membranes of higher plants. In all but the final step in purifying the L8S8 form of RuBisCO from C. vinosum, a 700-kDa pro-
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rein consistently co-purified with this enzyme in a fixed mole ratio. In the final step of anion-exchange chromatography this protein was eluted before RuBisCO. Analysis of the 700-kDa protein established that it was probably composed of 12 identical subunits (Torres-Ruiz and McFadden 1988). These subunits showed considerable N-terminal sequence similarity to those sequences for the similar c~- and fl-subunits of RuBisCO L-subunit-binding protein of higher plants (Torres-Ruiz and McFadden 1988). Subunits of the large 700kDa C. vinosurn protein also showed considerable immunological crossreaction with the plant e- and fl-subunits of the RuBisCO L-subunit binding protein. The C. vinosum protein was designated a putative binding protein (PBP) of RuBisCO (TorresRuiz and McFadden 1988). Recently, strong similarities in sequence have been noted between esubunits of the RuBisCO L-subunit binding protein of higher plants and the groEL protein of E. coli (Hemmingsen et al. 1988). These homologous proteins have been termed chaperonins. In general, they may assist in the post-translational assembly of oligomeric proteins such as RuBisCO (Hemmingsen et al. 1988). Indeed it has been found very recently that groE protein, consisting ofgroEL and groES subunits, promotes assembly of foreign prokaryotic RuBisCO oligomers in E. coli (Goloubinoff et al. 1989). The present evidence indicates that in the photosynthetic prokaryote C. vinosum, PBP, a chaperonin, is localized at the cytoplasmic membrane; this should stimulate studies of the localization ofgroE proteins in E. coll. Perhaps these bacterial chaperonins function by assisting posttranslational assembly of oligomeric proteins at the membrane. Whether chaperonins function in an analogous manner in eukaryotes remains to be determined. Certainly, localization of the RuBisCObinding protein in chloroplasts will be of interest.
We gratefully acknowledge support by the National Institutes of Health (GM-19,972) and the technical assistance of Xing Xiang Li. Facilities for electron microscopy were provided by the Electron Microscope Center of Washington State University.
References Garvey, J.S., Cremer, N.E, Sussdorf, D.H. (1977) Adjuvantmodified antigens. In: Methods in immunology. A laboratory text for instruction and research, 3rd edn., pp. 183-188, W.A. Benjamin, Reading, Mass., USA Goloubinoff, P., Gatenby, A.A., Lorimer, G.H. (1989) GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Eseherichia coli. Nature 337, 44-47 Hemmingsen, S.M., Woolford, C., van der Vies, S.M., Tilly, K., Dennis, D.T., Georgopoulos, C.P., Hendrix, R.W., Ellis, R.J. (1988) Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333, 330-334 Hill, R.J. (1972) Elution of antibodies from immunoadsorbents: effect of dioxane in promoting release of antibody. J. Immunol. Meth. 1, 231-234 Hurlbert, R.E., Lascelles, J. (1963) Ribulose-l,5-diphosphate carboxylase in Thiorhodaceae. J. Gen. Microbiol. 33, 445 448 McFadden, B.A. (1980) A perspective of ribulose bisphosphate carboxylase/oxygenase, the key catalyst in photosynthesis and photorespiration. Acc. Chem. Res. 13, 394-399 McFadden, B.A., Torres-Ruiz, J.A., Daniell, H., Sarojini, G. (1986) Interaction, functional relations and evolution of large and small subunits in RuBisCO from Prokaryota and Eukaryota. Phil. Trans. R. Soc. Lond. Ser. B 313, 347-358 Mori, H., Takabe, T., Akazawa, T. (1984) Loose association of ribulose 1,5-bisphosphate carboxylase/oxygenase with chloroplast thylakoid membranes. Photosynth. Res. 5, 1728 Musgrove, J.E., Ellis, R.J. (1986) The RuBisCO large subunit binding protein. Phil Trans. R. Soc. Lond. Ser. B 313, 419428 Remsen, C.C. (1978) Comparative subcellular architecture of photosynthetic bacteria. In: The photosynthetic bacteria, pp. 31-60, Clayton, R.K., Sistrom, W.R., eds. Plenum Press, New York Torres-Ruiz, J.A., McFadden, B.A. (1985) Isolation of L8 and LsSs forms of ribulose bisphosphate carboxylase/oxygenase from Chrornatium vinosum. Arch. Microbiol. 142, 55 60 Torres-Ruiz, J.A., McFadden, B.A. (1987a) The nature of L8 and L8S8 forms of ribulose bisphosphate carboxylase/oxygenase from Chromatium vinosum. Arch. Biochem. Biophys. 254, 63-68 Torres-Ruiz, J.A., McFadden, B.A. (1987b) Association of RuBisCO small subunits with a membrane fraction from Chromatiurn vinosum. In: Progress in photosynthesis research, vol. 3, pp. 5.419-5.422, Biggins, J., ed. Martinus Nijhoff Publishers, Dordrecht, The Netherlands Torres-Ruiz, J.A., McFadden, B.A. (1988) A homolog of ribulose bisphosphate carboxylase/oxygenase-bindingprotein in Chromatium vinosum. Arch. Biochem. Biophys. 261, 196 204 Vatter, A.E., Wolfe, R.S. (1958) The structure of photosynthetic bacteria. J. Bacteriol. 75, 480-488 Received 26 October 1988; accepted 3 Februar 1989
