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Free subunits of ribulose-1,5-bisphosphate carboxylase in pea leaves
Plant Science Letters, 11 (1978) 159--168
159
© Elsevier/North-Holland Scientific Publishers Ltd.
FREE SUBUNITS OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE IN PEA LEAVES* HARRY ROY**, KRIS ANN COSTA and HEDY ADARI
Biology Department, Rensselaer Polytechnic Institute, Troy, N. Y. 12181 (U.S.A.) (Received September 8th, 1977) (Revision received and accepted October 15th, 1977)
SUMMARY
Pea leaves, supplied with [3sS] methionine, were homogenized and a crude hypotonic soluble fraction was centrifuged on sucrose gradients to separate fully assembled ribulose-l,5-bisphosphate (RuBP) carboxylase from any free or partially assembled carboxylase subunits. Slowly sedimenting subunits of the enzyme were identified in upper fractions of the sucrose gradient, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), isoelectric focussing, and immune precipitation. The presence of these subunits in low molecular weight form was shown not to be due to artefactual dissociation of the enzyme. It is suggested that these subunits are related to the assembly of RuBP carboxylase.
INTRODUCTION
RuBP carboxylase (3 phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39) has a molecular weight of about 550 000, and is thought to be composed of eight large, 55 000 dalton (L), and eight small 13 000 dalton (S) subunits [ 1]. It has been demonstrated that the large subunit is synthesized in chloroplasts [2] on 70S ribosomes [3], while the small subunit is synthesized in the cytoplasm on 80S ribosomes [3--5]. The ribosomes engaged in synthesizing each of these polypeptides appear to be localized in the soluble cell fraction [2,6--8], despite earlier evidence suggesting a membrane-bound location for those engaged in small subunit synthesis [3]. The assembly of the enzyme from partially dissociated subunits has been reported to be a spontaneous process in vitro [9], but there is evidence suggesting that in vivo assembly may be complex: for example, small subunit anti* Supported by Grant No. GM-23353 to Harry Roy from the National Institutes of Health. ** To whom correspondence should be sent. Abbreviation: RuBP, ribulose-l,5-bisphosphate; SDS, sodium dodecyl sulfate.
160 bodies were found to react with a 20 000 dalton protein of unknown function made by wheat cytoplasmic polysomes [4] ; a similarly reactive protein made by poly A + RNA of Chlamydomonas was shown to be cleaved by an endoprotease to yield a protein with the same electrophoretic mobility as small subunits, and it was proposed that cleavage of this putative precursor of small subunits accompanies transport of the small subunit into the chloroplast [6] ; we have observed a similarprotein produced by pea polyribosomes and poly A + R N A (unpublished).In addition, comparative enzymatic assays and isotopically labeled amino acid incorporation studieshave suggested the existence of partiallyassembled molecules of this enzyme in greening barley plants [10]. W e report here the detection of newly synthesized subunits of this enzyme sedimenting in sucrose gradientsat rates far below those exhibited by the native enzyme. The presence of these slowly sedimenting subunits appears not to be due to dissociationof the enzyme. METHODS All chemicals were reagent grade or of the highest purity obtainable. [3sS] Methionine (300--600 Ci/mmole) was obtained from New England Nuclear, Waltham, MA. Nonidet P-40 (NP-40) was obtained from Particle Data Laboratories, Elmhurst, Ill. Pea seeds (Pisum sativum, var. "Progress No. 9") were obtained from Agway, Inc., Buffalo, NY. RuBP carboxylase was prepared as described [4] and modified [11]. This material gives a single band in nondenaturing polyacrylamide gel electrophoresis, and 99% of the Coomassie-Blue positive material is found in the characteristic positions of large and small subunit in SDS-polyacrylamide gel electrophoresis [8]. Antiserum to the nondenatured enzyme was prepared as described by Gooding et ah [3]. Antiserum to the small subunit of the enzyme was prepared by injecting homogeneous small subunit isolated by gel filtration in urea [4] or SDS [12] into Dutch Belted rabbits. Subcutaneous injection of 1 mg electrophoretically pure small subunit in 2 ml of 8 M urea mixed 1 : 1 with Freund's adjuvant was followed at bioweekly intervals with 1 to 2 mg of electrophoretically pure small subunit in 0.5% SDS injected in ear veins. The antiserum obtained after three injections gave a single band on immunodiffusion [3] with soluble alkylated small subunits [13]. Antiserum to native carboxylase and to small subunit gave single immunodiffusion lines with crude pea leaf homogenates. Pea seedlings were grown on a 16 h light--8 h dark cycle at 25 ° C for enzyme isolation, or in continuous darkness for 10 days followed by 24 h in the light for labeling studies, Greening seedlings were cut with a razor blade about 2 mm below the leaves. The leaf-bearing plant top was labeled by transpiration of about 150/J1 of water containing [3sS] methionine. Driven by a stream of air from a hair dryer, this amount of fluid was taken up in about I h. Just before this time, the labeled plant top (0.1 g) was ground at 4 ° C in 1 ml~of 0.05 M Tris--0.02 M ascorbate--0.007 M mercaptoethanol
161 (pH 8.0) (this buffer was sufficient to maintain the pH of the homogenate). The homogenate was then centrifuged at 10 000 g for 10 min. The supernatant was transferred to the top of a linear 5--20% sucrose gradient in 0.01M Tris-HC1 (pH 8.0) and centrifuged at 105 000 g for 12 h at 4 ° C. The gradient was fractionated by displacement from the bottom, using a Sage syringe pump and an ISCO gradient fractionator. Gradient polyacrylamide gel electrophoresis in the presence of SDS was performed as described by Studier [14] on samples prepared as follows: 30 #1 of the sucrose gradient fraction was mixed with 10 #1 of a solution containing 2.3% (w/v) of SDS--5% (v/v) mercaptoethanol--0.02 M Na2CO3--0.05 M dithiothreitol and heated 1 min at 65 ° C. 25 ~1 of this mixture was then applied to a sample well with an automatic pipette, and electrophoresis carried out as described [14], using a 7 . 5 t o 12% polyacrylamide gradient. Immune precipitation was carried out on samples containing 12--15 #g pure RuBP carboxylase, using 50 #1 anti-native enzyme serum, or 120 #1 antismall subunit serum, in the presence of I to 2% Triton X-100. After 6 h, the immune precipitates were washed 3 times and dissolved in SDS as described above, or in 30 #1 9.5 M urea--2.5% NP-40-- 2% Ampholines (LKB pH 5--7 to pH 3.5--10, 4 : 1)--5% mercaptoethanol. In that case, samples were subjected to isoelectric focussing overnight, equilibrated 2 h with 2.3% SDS--5% mercaptoethanol---0.0541 M Tris--H2SO4 (pH 6.1)--20% glycerol, and then subjected to second dimension electrophoresis as described by O'Farrel [15] with a gradient polyacrylamide gel in the presence of SDS. Gels were stained by the method of Weber and Osbom [16], dried by the method of Studier [ 14], and autoradiographed. RESULTS Polypeptides with electrophoretic mobilities corresponding to large and small subunits of RuBP carboxylase were found in sucrose gradient analyses of the supernatants of homogenates of the labeled plants, not only in the fractions corresponding to the 18S holoenzyme (Ffg. 1, fractions i7--25), but also in more slowly sedimenting fractions, particularly in fractions 2--5 (Fig. 1). The location of the holoenzyme was determined by the co-sedimentation of the large and small subunit bands, as judged by visual inspection of the Coomassie-Blue stained gels. The labeled carboxylase subunits in gradient positions other than those ill which the holoenzyme occurred were identified by a two-step procedure. First, antisera specific for small subunit or holoenzyme were used in the presence of carrier holoenzyme to precipitate the labeled proteins; then the labelled proteins were analysed by one dimensional electrophoresis in SDS polyacrylamide gels (Fig. 2), or by the two dimensional electrophoretic system developed by O'Farrel [15] (Fig. 3). The antisera to small subunit and holoenzyme appeared to precipitate the same spectrum of labeled polypeptides, as judged from the one~iimenslonal
S
-3
#O J.J
/9
Fig. 1. Distribution of labeled polypeptides in sucrose gradients. The crude 10 000 g supernatant of two plant tops, homogenized in ~)ml after inhibition of a total of 500 ~Ci of [3SS]methionine, was centrifuged on sucrose gradients which were fractionated as tescribed in METHODS. Samples (30 ul) from the top through the 18S carboxylase peak were denatured and electrophoresed on SDS ~olyacrylamide slab gels. Exposure time of autoradiogram: 14 h. Numerals indicate fraction numbers in the sucrose gradient, and L md S refer to electrophoretic dimension positions of large and small subunits, respectively. Dotted arrow: faint, non-reproducible ~and apparently co-sedimenting with carboxylase.
7
;EDIMENTATION
SERUM
H
S
H
S
H
S
ii¸ ¸¸7¸¸
H
3 ITII
S
Fig. 2. Immune precipitation of polypeptides from selected sucrose gradient fractions. Approx. 15 ~g RuBP carboxylase was precipitated with 30 ~1 samples of sucrose gradient fractions from the experiment shown in Fig. 1, using saturating amounts o f antiholoenzyme or anti-small subunit serum in the presence of 1% Triton X-100. The immune precipitates were dissolved in SDS and mercaptoethanol, electrophoresed on polyacrylamide gels, and autoradiographed as described in METHODS. Tracks lettered " S " , anti-small subunit serum; tracks lettered " H " , anti-holoenzyme serum. Antibodies were added to sucrose gradient fractions 21, 15, 7 and 3.
S
L
15
164
Fig. 3. Two-dimensional eiectrophoresis of immune precipitated labeled proteins. I m m u n e precipitates prepared as described in Fig. 2 and METHODS were dissolved in 9,5 M urea, 2% NP-40, 5% mercaptoethanol, 0.5% Ampholines, and subjected to ~ e c t r i e focussing followed by SDS polyaerylamide slab gel electrophoresis as described by O'Farrel [15]. (A) Anti-mnall subunit serum precipitate of fraction 3 of sucrose gradient shown in Fig. 1. (B) Anti-holoenzyme serum precipitate of fraction 21 of the same gradient. No difference was observed when anti-small subunit serum was employed on this fraction. Arrows mark labeled spots corresponding exactly in size, form, and location to visible stained marker polypeptides in the dried gel itself.
165
electrophoresis (Fig. 2); in the precipitates from gradient fraction 3, these included primarily small subunit, but also some large subunit and some lesser, unidentified bands. In the holoenzyme peak (fraction 21) both sera precipitated primarily large and small subunits in the characteristic ratios in which they are present in the holoenzyme, as judged by visual inspection of the X-ray film. It should be noted that a faint, ca. 20 kD band apparently cosedimenting with holoenzyme in Fig. 1 (see dotted arrow), did not appear in the immune precipitates of these same fractions. All of the fractions analysed in Fig. 2 were also analysed by two dimensional electrophoresis after immune precipitation. The results were independent of the specificity of the sera. We have shown [8] that in two-dimensional electrophoresis, pea RuBP carboxylase yields 3--5 large subunit spots, clustered together, in about the same isoelectric region as 2 small subunit spots. With anti-small subunit (Fig. 3A) each small subunit spot is distinctly labeled, and the large subunit spots are also faintly labeled. Fig. 3B shows the label from the holoenzyme peak precipitated by anti-holoenzyme, where the distribution of label corresponds with that of large and small subunits on the stained gel. (There is a small amount of label at the edge of the gel, corresponding to the top of the isoelectric focussing gel. This is an artefact caused by incomplete solubilization of the immune precipitate in urea. This artefact is also responsible for some streaking of the radioactive material in the isoelectric dimension [8] .) The labeled small subunits in the upper sucrose gradient fractions were resolved also by two-dimensional electrophoresis without prior use of immune precipitation. In that case (not shown), we estimated that the small subunits represent more than half of the radioactivity in the 13 000 dalton band, with only one or two other proteins contributing detectable radioactivity. This observation suggested a test for dissociation: extracts from plants labeled in [ass] methionine for 30 rain or for 30 rain followed by a period of several hours' duration in unlabeled methionine were centrifuged in parallel on sucrose gradients, fractionated, and analysed by electroph~resis as described for the experiment in Fig. 1. Autoradiography (Fig. 4) revealed the small subunit~band in fractions near the top of the sucrose gradient only during exposure of the plant to labeled methionine. This band was not detectable in the top fractions 3 h after feeding the unlabeled methionine. By contrast, the grain density over the small subunit bands in the 18S enzyme peak continued to increase even after feeding the unlabeled methionine. The visual impression gained here was verified by liquid scintillation counting of small subunit bands from each fraction of each gradient in a parallel experiment. The contrast in the labeling behavior of the two populations of small subunit shows that the appearance of the slowly sedimenting form of the small subunit was not due to dissociation of previously assembled, labeled carboxylase. This conclusion was directly substantiated in control reconstruction experiments where radioactive enzyme mixed in the homogenate of an unlabeled plant sedimented in a monodisperse fashion.
SEDIMENTATION
TOP
L
.--) ,I -.J ,I
~L
"V"
Fig. 4. Labeling behavior of free small subunit vs. holoenzyme. Two plants were fed [3sS]methionine as described for Fig. 1, but o n e )f these was allowed to imbibe a solution of ca. 10 ~M unlabeled methionine and other amino acids for 3 h after the initial feeding • ith labeled amino acid. Both plants were homogenized simultaneously and the crude 10 000 g supernatants of the resulting homogenates were centrifuged on sucrose gradients. Alternate fractions were analyzed essentially as described for the experiment shown in Pig. 1, except that carrier carboxylase was added to fractions requiring it. The dried gel was autoradiographed. Left: samples from ;hort-term labeled plant. Right: samples from plant similarly labeled but subsequently fed unlabeled methionine. Direction of ~edimentation: right to left.
FOP
-------
.a
167
DISCUSSION
Smith et al. [10] provided data suggesting the existence of soluble, partially assembled or unassembled subunits of R u B P carboxylase in greening barley. The current studies support their finding, and more clearly identify these polypeptides as being genuine carboxylase subunits not associated with the mature enzyme. This statement is best substantiated by the electrophoretic coincidence of the labeled bands with authentic carriers,not only in gradient polyacrylamide gel electrophoresis, but also in the two dimensional electrophoretic analysis of O'Farrel, which is capable of resolving thousands of different proteins in a single experiment. Our immunological data also tend to support the conclusion, although it is clear that the antisera can precipitate a few radioactive proteins which appear not to be related to the carboxylase. (A similar lack of specificity was observed by Dobberstein et al. [7], and m a y be due to the greater sensitivity of radioimmunoassay over the conventional immunodiffusion criteriaused to characterize antisera.) The experiment described in Fig. 4, and control reconstruction experiments, merely show that the detection of the carboxylase small subunit in a slowly sedimenting peak is not the result of the dissociation of previously assembled enzyme upon homogenization or sedimentation. It is reasonable, however, to assume that these "free" subunits are related to assembly of the mature enzyme. We cannot tell whether they represent actual intermediates in the assembly of the enzyme, or whether they might be products of aborted steps in the assembly process: quantitative pulse-chase experiments will be required to distinguish these alternatives. The relationship between these observations and pS, a proposed precursor polypeptide of the small subunit, made in the cytosol [5,7], has not been established. The ca. 18 kD band apparently co-sedimenting with RuBP carboxy. lase in Fig. 1 was not detected in immune precipitates of the enzyme or other fractions of the same gradient; and it was not seen again in several repetitions of that experiment. It is possible that processing enzymes released during homogenization act upon pS before it can be isolated; or pS may be in the membrane fraction which has not been examined yet; or pS may turn over rapidly, yielding a large pool of processed small subunits in the cytosol or, more likely, the stroma of the chloroplast. Binding between large and small subunits in the free pool, suggested by Fig. 2 and Fig. 3A, may be real, but remains to be investigated by nonimmunological methods. ACKNOWLEDGEMENTS
W e thank L. Mader for assistance in preparing antisera, and A. Grebanier for help with an earlierdraft of this paper. REFERENCES 1 T.S. Baker, D. Eisenberg, and F. Eiserling,Science, 196 (1977) 293.
168
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
G.E. Blair and R.J. Ellis, Biochim. Biophys. Aeta, 319 (1978) 223. L.R. Gooding, H. Roy and A.T. Jagendorf, Arch. Biochem. Biophys., 159 (1973) 324. H. Roy, R. Patterson and A.T. Jagendorf, Arch. Bioehem. Biophys., 172 (1975) 64. J.C. Gray and R.G.O. Kekwiek, FEBS Left., 38 (1973) 67. R.J. Ellis, Biochem. Soc. Trans., 2 (1974) 179. B. Dobberstein, G. Blobel and N-H Chua, Proc. Natl. Acad. Sci. USA, 74 (1977) 1082. H. Roy, L.C. Cheong and B. Terenna, Plant Physiol., 60 (1977) 532. M. Niehimura and T. Akazawa, J. Biochem. (Tokyo), 78 (1974) 169. M.A. Smith, R.S. Criddle, L. Peterson and R.C. Huffaker, Arch. Biochem. Biophys., 165 (1974) 494. H. Roy, O. Alvarez and L. Mader, Biochem. Biophys. Res. Commun., 70 (1976) 914. A.C. Ruiner and M.D. Lane, Bioehem. Biophys. Res. Cornmun., 28 (1967) 531. M. Nishimura, T. Takabe, T. Sugiyama and T. Akazawa, J. Biochem. (Tokyo), 74 (1973) 945. F.W. Studier, J. Mol. Biol., 79 (1973) 237. P.H. O'Farrel, J. Biol. Chem., 250 (1975) 4007. K. Weber and M. Osborn, J. Biol. Chem., 244 (1969) 4406. H. Roy, L.R. Gooding and A.T. Jagendorf, Arch. Biochem. Biophys., 159 (1973) 312.
159
© Elsevier/North-Holland Scientific Publishers Ltd.
FREE SUBUNITS OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE IN PEA LEAVES* HARRY ROY**, KRIS ANN COSTA and HEDY ADARI
Biology Department, Rensselaer Polytechnic Institute, Troy, N. Y. 12181 (U.S.A.) (Received September 8th, 1977) (Revision received and accepted October 15th, 1977)
SUMMARY
Pea leaves, supplied with [3sS] methionine, were homogenized and a crude hypotonic soluble fraction was centrifuged on sucrose gradients to separate fully assembled ribulose-l,5-bisphosphate (RuBP) carboxylase from any free or partially assembled carboxylase subunits. Slowly sedimenting subunits of the enzyme were identified in upper fractions of the sucrose gradient, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), isoelectric focussing, and immune precipitation. The presence of these subunits in low molecular weight form was shown not to be due to artefactual dissociation of the enzyme. It is suggested that these subunits are related to the assembly of RuBP carboxylase.
INTRODUCTION
RuBP carboxylase (3 phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39) has a molecular weight of about 550 000, and is thought to be composed of eight large, 55 000 dalton (L), and eight small 13 000 dalton (S) subunits [ 1]. It has been demonstrated that the large subunit is synthesized in chloroplasts [2] on 70S ribosomes [3], while the small subunit is synthesized in the cytoplasm on 80S ribosomes [3--5]. The ribosomes engaged in synthesizing each of these polypeptides appear to be localized in the soluble cell fraction [2,6--8], despite earlier evidence suggesting a membrane-bound location for those engaged in small subunit synthesis [3]. The assembly of the enzyme from partially dissociated subunits has been reported to be a spontaneous process in vitro [9], but there is evidence suggesting that in vivo assembly may be complex: for example, small subunit anti* Supported by Grant No. GM-23353 to Harry Roy from the National Institutes of Health. ** To whom correspondence should be sent. Abbreviation: RuBP, ribulose-l,5-bisphosphate; SDS, sodium dodecyl sulfate.
160 bodies were found to react with a 20 000 dalton protein of unknown function made by wheat cytoplasmic polysomes [4] ; a similarly reactive protein made by poly A + RNA of Chlamydomonas was shown to be cleaved by an endoprotease to yield a protein with the same electrophoretic mobility as small subunits, and it was proposed that cleavage of this putative precursor of small subunits accompanies transport of the small subunit into the chloroplast [6] ; we have observed a similarprotein produced by pea polyribosomes and poly A + R N A (unpublished).In addition, comparative enzymatic assays and isotopically labeled amino acid incorporation studieshave suggested the existence of partiallyassembled molecules of this enzyme in greening barley plants [10]. W e report here the detection of newly synthesized subunits of this enzyme sedimenting in sucrose gradientsat rates far below those exhibited by the native enzyme. The presence of these slowly sedimenting subunits appears not to be due to dissociationof the enzyme. METHODS All chemicals were reagent grade or of the highest purity obtainable. [3sS] Methionine (300--600 Ci/mmole) was obtained from New England Nuclear, Waltham, MA. Nonidet P-40 (NP-40) was obtained from Particle Data Laboratories, Elmhurst, Ill. Pea seeds (Pisum sativum, var. "Progress No. 9") were obtained from Agway, Inc., Buffalo, NY. RuBP carboxylase was prepared as described [4] and modified [11]. This material gives a single band in nondenaturing polyacrylamide gel electrophoresis, and 99% of the Coomassie-Blue positive material is found in the characteristic positions of large and small subunit in SDS-polyacrylamide gel electrophoresis [8]. Antiserum to the nondenatured enzyme was prepared as described by Gooding et ah [3]. Antiserum to the small subunit of the enzyme was prepared by injecting homogeneous small subunit isolated by gel filtration in urea [4] or SDS [12] into Dutch Belted rabbits. Subcutaneous injection of 1 mg electrophoretically pure small subunit in 2 ml of 8 M urea mixed 1 : 1 with Freund's adjuvant was followed at bioweekly intervals with 1 to 2 mg of electrophoretically pure small subunit in 0.5% SDS injected in ear veins. The antiserum obtained after three injections gave a single band on immunodiffusion [3] with soluble alkylated small subunits [13]. Antiserum to native carboxylase and to small subunit gave single immunodiffusion lines with crude pea leaf homogenates. Pea seedlings were grown on a 16 h light--8 h dark cycle at 25 ° C for enzyme isolation, or in continuous darkness for 10 days followed by 24 h in the light for labeling studies, Greening seedlings were cut with a razor blade about 2 mm below the leaves. The leaf-bearing plant top was labeled by transpiration of about 150/J1 of water containing [3sS] methionine. Driven by a stream of air from a hair dryer, this amount of fluid was taken up in about I h. Just before this time, the labeled plant top (0.1 g) was ground at 4 ° C in 1 ml~of 0.05 M Tris--0.02 M ascorbate--0.007 M mercaptoethanol
161 (pH 8.0) (this buffer was sufficient to maintain the pH of the homogenate). The homogenate was then centrifuged at 10 000 g for 10 min. The supernatant was transferred to the top of a linear 5--20% sucrose gradient in 0.01M Tris-HC1 (pH 8.0) and centrifuged at 105 000 g for 12 h at 4 ° C. The gradient was fractionated by displacement from the bottom, using a Sage syringe pump and an ISCO gradient fractionator. Gradient polyacrylamide gel electrophoresis in the presence of SDS was performed as described by Studier [14] on samples prepared as follows: 30 #1 of the sucrose gradient fraction was mixed with 10 #1 of a solution containing 2.3% (w/v) of SDS--5% (v/v) mercaptoethanol--0.02 M Na2CO3--0.05 M dithiothreitol and heated 1 min at 65 ° C. 25 ~1 of this mixture was then applied to a sample well with an automatic pipette, and electrophoresis carried out as described [14], using a 7 . 5 t o 12% polyacrylamide gradient. Immune precipitation was carried out on samples containing 12--15 #g pure RuBP carboxylase, using 50 #1 anti-native enzyme serum, or 120 #1 antismall subunit serum, in the presence of I to 2% Triton X-100. After 6 h, the immune precipitates were washed 3 times and dissolved in SDS as described above, or in 30 #1 9.5 M urea--2.5% NP-40-- 2% Ampholines (LKB pH 5--7 to pH 3.5--10, 4 : 1)--5% mercaptoethanol. In that case, samples were subjected to isoelectric focussing overnight, equilibrated 2 h with 2.3% SDS--5% mercaptoethanol---0.0541 M Tris--H2SO4 (pH 6.1)--20% glycerol, and then subjected to second dimension electrophoresis as described by O'Farrel [15] with a gradient polyacrylamide gel in the presence of SDS. Gels were stained by the method of Weber and Osbom [16], dried by the method of Studier [ 14], and autoradiographed. RESULTS Polypeptides with electrophoretic mobilities corresponding to large and small subunits of RuBP carboxylase were found in sucrose gradient analyses of the supernatants of homogenates of the labeled plants, not only in the fractions corresponding to the 18S holoenzyme (Ffg. 1, fractions i7--25), but also in more slowly sedimenting fractions, particularly in fractions 2--5 (Fig. 1). The location of the holoenzyme was determined by the co-sedimentation of the large and small subunit bands, as judged by visual inspection of the Coomassie-Blue stained gels. The labeled carboxylase subunits in gradient positions other than those ill which the holoenzyme occurred were identified by a two-step procedure. First, antisera specific for small subunit or holoenzyme were used in the presence of carrier holoenzyme to precipitate the labeled proteins; then the labelled proteins were analysed by one dimensional electrophoresis in SDS polyacrylamide gels (Fig. 2), or by the two dimensional electrophoretic system developed by O'Farrel [15] (Fig. 3). The antisera to small subunit and holoenzyme appeared to precipitate the same spectrum of labeled polypeptides, as judged from the one~iimenslonal
S
-3
#O J.J
/9
Fig. 1. Distribution of labeled polypeptides in sucrose gradients. The crude 10 000 g supernatant of two plant tops, homogenized in ~)ml after inhibition of a total of 500 ~Ci of [3SS]methionine, was centrifuged on sucrose gradients which were fractionated as tescribed in METHODS. Samples (30 ul) from the top through the 18S carboxylase peak were denatured and electrophoresed on SDS ~olyacrylamide slab gels. Exposure time of autoradiogram: 14 h. Numerals indicate fraction numbers in the sucrose gradient, and L md S refer to electrophoretic dimension positions of large and small subunits, respectively. Dotted arrow: faint, non-reproducible ~and apparently co-sedimenting with carboxylase.
7
;EDIMENTATION
SERUM
H
S
H
S
H
S
ii¸ ¸¸7¸¸
H
3 ITII
S
Fig. 2. Immune precipitation of polypeptides from selected sucrose gradient fractions. Approx. 15 ~g RuBP carboxylase was precipitated with 30 ~1 samples of sucrose gradient fractions from the experiment shown in Fig. 1, using saturating amounts o f antiholoenzyme or anti-small subunit serum in the presence of 1% Triton X-100. The immune precipitates were dissolved in SDS and mercaptoethanol, electrophoresed on polyacrylamide gels, and autoradiographed as described in METHODS. Tracks lettered " S " , anti-small subunit serum; tracks lettered " H " , anti-holoenzyme serum. Antibodies were added to sucrose gradient fractions 21, 15, 7 and 3.
S
L
15
164
Fig. 3. Two-dimensional eiectrophoresis of immune precipitated labeled proteins. I m m u n e precipitates prepared as described in Fig. 2 and METHODS were dissolved in 9,5 M urea, 2% NP-40, 5% mercaptoethanol, 0.5% Ampholines, and subjected to ~ e c t r i e focussing followed by SDS polyaerylamide slab gel electrophoresis as described by O'Farrel [15]. (A) Anti-mnall subunit serum precipitate of fraction 3 of sucrose gradient shown in Fig. 1. (B) Anti-holoenzyme serum precipitate of fraction 21 of the same gradient. No difference was observed when anti-small subunit serum was employed on this fraction. Arrows mark labeled spots corresponding exactly in size, form, and location to visible stained marker polypeptides in the dried gel itself.
165
electrophoresis (Fig. 2); in the precipitates from gradient fraction 3, these included primarily small subunit, but also some large subunit and some lesser, unidentified bands. In the holoenzyme peak (fraction 21) both sera precipitated primarily large and small subunits in the characteristic ratios in which they are present in the holoenzyme, as judged by visual inspection of the X-ray film. It should be noted that a faint, ca. 20 kD band apparently cosedimenting with holoenzyme in Fig. 1 (see dotted arrow), did not appear in the immune precipitates of these same fractions. All of the fractions analysed in Fig. 2 were also analysed by two dimensional electrophoresis after immune precipitation. The results were independent of the specificity of the sera. We have shown [8] that in two-dimensional electrophoresis, pea RuBP carboxylase yields 3--5 large subunit spots, clustered together, in about the same isoelectric region as 2 small subunit spots. With anti-small subunit (Fig. 3A) each small subunit spot is distinctly labeled, and the large subunit spots are also faintly labeled. Fig. 3B shows the label from the holoenzyme peak precipitated by anti-holoenzyme, where the distribution of label corresponds with that of large and small subunits on the stained gel. (There is a small amount of label at the edge of the gel, corresponding to the top of the isoelectric focussing gel. This is an artefact caused by incomplete solubilization of the immune precipitate in urea. This artefact is also responsible for some streaking of the radioactive material in the isoelectric dimension [8] .) The labeled small subunits in the upper sucrose gradient fractions were resolved also by two-dimensional electrophoresis without prior use of immune precipitation. In that case (not shown), we estimated that the small subunits represent more than half of the radioactivity in the 13 000 dalton band, with only one or two other proteins contributing detectable radioactivity. This observation suggested a test for dissociation: extracts from plants labeled in [ass] methionine for 30 rain or for 30 rain followed by a period of several hours' duration in unlabeled methionine were centrifuged in parallel on sucrose gradients, fractionated, and analysed by electroph~resis as described for the experiment in Fig. 1. Autoradiography (Fig. 4) revealed the small subunit~band in fractions near the top of the sucrose gradient only during exposure of the plant to labeled methionine. This band was not detectable in the top fractions 3 h after feeding the unlabeled methionine. By contrast, the grain density over the small subunit bands in the 18S enzyme peak continued to increase even after feeding the unlabeled methionine. The visual impression gained here was verified by liquid scintillation counting of small subunit bands from each fraction of each gradient in a parallel experiment. The contrast in the labeling behavior of the two populations of small subunit shows that the appearance of the slowly sedimenting form of the small subunit was not due to dissociation of previously assembled, labeled carboxylase. This conclusion was directly substantiated in control reconstruction experiments where radioactive enzyme mixed in the homogenate of an unlabeled plant sedimented in a monodisperse fashion.
SEDIMENTATION
TOP
L
.--) ,I -.J ,I
~L
"V"
Fig. 4. Labeling behavior of free small subunit vs. holoenzyme. Two plants were fed [3sS]methionine as described for Fig. 1, but o n e )f these was allowed to imbibe a solution of ca. 10 ~M unlabeled methionine and other amino acids for 3 h after the initial feeding • ith labeled amino acid. Both plants were homogenized simultaneously and the crude 10 000 g supernatants of the resulting homogenates were centrifuged on sucrose gradients. Alternate fractions were analyzed essentially as described for the experiment shown in Pig. 1, except that carrier carboxylase was added to fractions requiring it. The dried gel was autoradiographed. Left: samples from ;hort-term labeled plant. Right: samples from plant similarly labeled but subsequently fed unlabeled methionine. Direction of ~edimentation: right to left.
FOP
-------
.a
167
DISCUSSION
Smith et al. [10] provided data suggesting the existence of soluble, partially assembled or unassembled subunits of R u B P carboxylase in greening barley. The current studies support their finding, and more clearly identify these polypeptides as being genuine carboxylase subunits not associated with the mature enzyme. This statement is best substantiated by the electrophoretic coincidence of the labeled bands with authentic carriers,not only in gradient polyacrylamide gel electrophoresis, but also in the two dimensional electrophoretic analysis of O'Farrel, which is capable of resolving thousands of different proteins in a single experiment. Our immunological data also tend to support the conclusion, although it is clear that the antisera can precipitate a few radioactive proteins which appear not to be related to the carboxylase. (A similar lack of specificity was observed by Dobberstein et al. [7], and m a y be due to the greater sensitivity of radioimmunoassay over the conventional immunodiffusion criteriaused to characterize antisera.) The experiment described in Fig. 4, and control reconstruction experiments, merely show that the detection of the carboxylase small subunit in a slowly sedimenting peak is not the result of the dissociation of previously assembled enzyme upon homogenization or sedimentation. It is reasonable, however, to assume that these "free" subunits are related to assembly of the mature enzyme. We cannot tell whether they represent actual intermediates in the assembly of the enzyme, or whether they might be products of aborted steps in the assembly process: quantitative pulse-chase experiments will be required to distinguish these alternatives. The relationship between these observations and pS, a proposed precursor polypeptide of the small subunit, made in the cytosol [5,7], has not been established. The ca. 18 kD band apparently co-sedimenting with RuBP carboxy. lase in Fig. 1 was not detected in immune precipitates of the enzyme or other fractions of the same gradient; and it was not seen again in several repetitions of that experiment. It is possible that processing enzymes released during homogenization act upon pS before it can be isolated; or pS may be in the membrane fraction which has not been examined yet; or pS may turn over rapidly, yielding a large pool of processed small subunits in the cytosol or, more likely, the stroma of the chloroplast. Binding between large and small subunits in the free pool, suggested by Fig. 2 and Fig. 3A, may be real, but remains to be investigated by nonimmunological methods. ACKNOWLEDGEMENTS
W e thank L. Mader for assistance in preparing antisera, and A. Grebanier for help with an earlierdraft of this paper. REFERENCES 1 T.S. Baker, D. Eisenberg, and F. Eiserling,Science, 196 (1977) 293.
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