Ribulose-1,5-bisphosphate carboxylase, a marker for chloroplast species specificity in Acetabularia

60

Biochimica et Bioplo'stca Acta, 699 (1982) 60-66 Elsevier Biomedical Press

BBA 91134

R I B U L O S E - I , 5 - B I S P H O S P H A T E CARBOXYLASE, A MARKER FOR CHLOROPLAST S P E C I E S SPECIFICITY IN ACETABULARIA M I C H A E L B. LEIBLE, R O B E R T L. S H O E M A N and H A N S - G E O R G S C H W E I G E R

Max-Planck-lnstitut fiir Zellblologie, Rosenho]~ 6802 l~denburg bei Heidelberg (F. R. G,) (Received May 25th, 1982)

Key words: Ribulose-bisphosphate carbo.~vlase," Species specificity; lsoelectric cariant," (Acetabularia chloroplast)

lnterspecific and intergeneric nucleo-cytoplasmic hybrids may be readily produced under controlled conditions from Acetabularia and other related Dasycladaeeae. The role of the nuclear genome has been well documented, whereas the lack of a suitable chloroplast marker has hindered the analysis of the possible interaction of the two plastid genomes in these hybrids. To this end, the species specificity of the chloroplast genome-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1. !.39) was studied by two-dimensional electrophoresis in nine species of Dasycladaceae. The large subunit has a molecular weight ( ± S.D.) of 52000 + 2000 and multiple isoelectric variants with isoelectric points between pH 5.3 and 5.7. The nine species could be divided into two distinct groups on the basis of the isoelectric focusing patterns. In both groups, individual isoelectric variants were found to give rise to the other isoelectric variants characteristic for that group upon re-electrofocusing. Mixing experiments confirmed the observed uniqueness of the two groups of species. These results indicate that the two-dimensional pattern of the large subunit of ribulose-l,5-bisphosphate carboxylase is a valid marker for chloroplast species specificity in Acetabularia.

Introduction The unicellular and uninuclear alga Acetabularia and other related Dasycladaceae have proved to be good objects for studying nucleo-cytoplasmic interactions [1]. These organisms allow the production of nucleo-cytoplasmic hybrids under controlled conditions [1]. Such hybridization experiments have shown that a number of plastid proteins are encoded by the nuclear genome [2-5]. A major problem in some hybridization experiments may be the behaviour of heterologous chloroplast populations. In order to study this problem it is necessary to have a species-specific chloroplast-encoded marker. Ribulose-l,5-bisphosphate

Abbreviation: Mes, 4-morpholineethanesulphonic acid. 0167-4781/82/0000-0000/$02.75 ~; 1982 Elsevier Biomedical Press

carboxylase/oxygenase (EC 4.1.1.39) apparently fulfills these requirements. Ribulose- 1,5-bisphosphate carboxylase is one of the major chloroplast stroma proteins. The holoenzyme ( M r 550000) is composed of two types of subunits. The large subunit ( M r 55000) is encoded by the chloroplast genome [6-8]. In contrast, the small subunit ( M r 12000-15000) is encoded by the nuclear genome [9]. Isoelectric focusing of this enzyme in the presence of 8 M urea resolves the large as well as the small subunit into polypeptides with different isoelectric points [10]. This multiple polypeptide composition has been used extensively for the iden[ification of somatic hybrids and the establishment of evolutionary relationships [11 13]. In this study, species specificity in the isoelectric variants of the large subunit of ribulose-1,5-bisphosphate carboxylase by two-dimensional elec-

61 trophoresis of various species of Dasycladaceae, in particular Acetabularia, were analysed and compared. Materials and Methods

Cells. Acetabularia caraibica, A. cliftonii, A. crenulata, A. mediterranea, A. polyphysoides, A. ryukyuensis, Acicularia schenckiL Batophora oerstedii and Chalmasia antillana were cultured in modified Mtiller's medium under standard conditions as described elsewhere [ 14,15]. Preparation of ribulose-l,5-bisphosphate earboxylase. Approx. 1000 cells were blotted dry, frozen in liquid nitrogen and ground to a fine powder in a cold mortar. The powder was thawed in 50 ml Mes buffer [16]. The remaining steps in the enzyme isolation were performed at 0-4°C. The total homogenate was filtered through cheesecloth and then centrifuged at 5000 X gmax for 10 min. The green pellet was resuspended in 10 ml Mes buffer and centrifuged at 5000 X gmax for 10 min. The pellet was resuspended in 1 ml of sample buffer (50 mM Tris-HC1, p H 8.0/100 mM NaC1/10 mM MgCI2/5 mM E D T A / 1 0 mM 2mercaptoethanol) supplemented with 40 /~1 Nonidet P-40, 100 /~1 of a D N A a s e / R N A a s e stock solution (1 m g / m l DNAase,/0.5 mg/ml RNAase/0.5 M Tris-HC1, pH 7 / 50 mM MgC12 [17]) and 1 ffl of 100 mM phenylmethylsulfonyl fluoride (dissolved in dimethyl sulfoxide) and shaken for 30 min. The mixture was centrifuged at 13000 X gmax for 2 min and the resulting supernatant was recentrifuged for 3 h at 200000 X gmax in a Beckman LP 42 Ti rotor. The pellet was dissolved in 1 ml sample buffer and used as the ribulose, 1,5-bisphosphate carboxylase preparation. Enzyme assay. The activity of ribulose-l,5-bisphosphate carboxylase was estimated by the incorporation of NaHI4CO3 into acid-stable material. 25 #1 of a substrate mixture containing 2 mM ribulose- 1,5-bisphosphate/ 2 mM NaHI4CO3/2 mM dithioerythritol/100 mM TrisHCI, pH 8.4/20 mM MgC12 were added to either 25 #l enzyme sample or 25 /~l bovine serum albumin (1 m g / m l ) as control. After incubation at 25°C for 15 min, the reaction was stopped by the addition of 100 ffl 2 0 % / ( w / v ) HC104 and the free HI4CO~- removed by incubation at 56°C for 30

min. 50/~1 of this reaction mixture were counted in l0 ml of Packard 299 scintillation cocktail in a Packard 460 C / D TRI CARB calibrated with [J4C]toluene for dpm conversion. Electrophoresis. The SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis were performed essentially as described elsewhere [18], with the exception that pH 4-6 ampholytes were used for the isoelectric focusing. The two-dimensional isoelectric focusing was performed with a second, identical isoelectric focusing gel in slab form substituted for the SDS-polyacrylamide gel in the previously described two-dimensional gel electrophoresis system [18]. Staining of the gels was performed with either Serva blue or silver [ 18]. Materials. NaHI4CO3 (56 m C i / m m o l ) was purchased from Amersham Buchler and stored in aliquots at - 2 0 ° C . Ribulose-l,5-bisphosphate (Cat. No. R 8250) was obtained from Sigma, dissolved in distilled water at a concentration of 100 mM and stored in aliquots at - 2 0 ° C . Ampholine pH 4 - 6 ampholytes (Nr. 1809-116) were purchased from LKB. All other chemicals were either reagent grade or as described in the reference. Results

The kinetics of the ribulose-l,5-bisphosphate carboxylase from Acetabularia (data not shown) were similar to those described for enzymes from other sources [19,20] (see Ref. 21 for a review of problems with the enzyme assay). The yield of enzyme activity of A. mediterranea after the 200000 X gma~ centrifugation was approx. 60% of the activity found in the crude chloroplast homogenate. The molecular weight of the large subunit was estimated by SDS-polyacrylamide gel electrophoresis to be 52000 + 2000 (S.D.) (Fig. 1). Twodimensional electrophoresis resolved four isoelectric variants for the large subunit of A. mediterranea after staining with Serva blue (Fig. 2A) and five isoelectric variants and many additional proteins after silver staining (Fig. 2B). The additional variant towards the acid end was not always detectable since it was present in the lowest concentration. Overloading the gel, with respect to the other four variants, was in each instance sufficient to display this fifth variant. The isoelectric variants

62

Mr

Mr

76000 68000 55O00 43000

55000

25000

25000 17000 12000 A

12000

5.9

.... a

pH

4.7

b

Fig. 1. SDS-polyacrylamide gel electrophoresis of an A. medihomogenate. Electrophoresis was performed (a) after treatment with NP-40 and (b) on the sediment obtained after 2 0 0 0 0 0 × g centrifugation of the homogenate. The gel was stained with Serva Blue G-250. The standards for molecular weight were as in Materials and Methods. The arrows indicate the positions of the large and small subunits of the ribulose- 1,5-bisphosphate carboxylase. terranea chloroplast

showed isoelectric points between p H 5.3 and 5.7 and the individual variants were separated from each other by 0.02-0.06 pH units. A comparison of the large subunit patterns of various species of Acetabularia and B. oerstedii after two-dimensional electrophoresis and silver staining permits the subdivision of these species into two groups (Fig. 3). One group consisting of A. mediterranea, A. ryukyuensis, A. caraibica, A. crenulata, A. cliftonii, C. antillana and B. oerstedii showed identical patterns of four isoelectric variants. Depending on the amount of sample applied to the gels, a fifth, faintly-stained variant towards the acid end was resolved. A second group consisting of A. polyphysoides and Acicularia schenckii displayed a pattern of three isoelectric variants. Likewise, a fourth, faintly-stained variant towards the acid end could be resolved in some gels. Co-electrophoresis of preparations from A. mediterranea and Acicularia sehenckii confirmed

B Fig. 2. Two-dimensional electrophoresis of a partially-purified ribulose-l,5-bisphosphate carboxylase preparation from A. rnediterranea. The arrows indicate the position of the isoelectric variants of the large subunit. (A) Gel stained with Serva Blue G-250. (B) The same gel as in (A) stained with silver. See Materials and Methods for details of staining and molecular weight and p I determinations.

the differences between the two groups (Fig. 4). The first group, to which A. mediterranea belongs, has an extra, more basic isoelectric variant. Furthermore, this mixing experiment revealed that the first basic isoelectric variant of the large subunit from Acicularia schenckii and the second basic variant of the large subunit from A. mediterranea have different p I values which give rise to a doublet (Fig. 4), demonstrating a second difference between the two groups. A similar result was obtained with a mixture of A. polyphysoides and A.

63 Isoelectric Focusing

4

a

~i!i!!!

5.9 ql ¸

b C

~

d

.....

"-

pFt

Isoelectric Focusing

......

e f

44 .

G 4.7

g

h i

b --

oo

C

Fig. 3. lsoelectric variants of the large subunits from various species of Dasycladaceae. The preparations were subjected to two-dimensional electrophoresis and silver staining. Only the portion of the gel containing the large subunit is displayed. (a) A. mediterranea; (b) A. ryukyuensis; (c) A. caraibica; (d) A. crenulata; (e) A. cliftonii; (f) A. polyphysoides; (g) C. antillana; (h) A. schenckii and (i) B. oerstedii. Fig. 4. Isoelectric variants of the large subunit of the ribulose1,5-bisphosphate carboxylase from A. rnediterranea and A cicularia schenckii. The preparations were subjected to two-dimensional electrophoresis and silver staining. (a) Acicularia schenckii; (b) mixture of Acicularia schenckii and A. mediterranea and (c) A. mediterranea.

5.9

pH

4.7

A

5.9

pH

--

4.7 5.9

m e d i t e r r a n e a ( d a t a n o t shown).

In o r d e r to ascertain the stability of the various isoelectric variants, as well as to check for artifacts in the o b s e r v e d p a t t e r n which might arise f r o m the isoelectric focusing, the e n z y m e p r e p a r a t i o n s from A . m e d i t e r r a n e a a n d A c i c u l a r i a were subjected to isoelectric focUsing in two d i m e n s i o n s (Fig. 5). If a p r o t e i n was m o d i f i e d d u r i n g the isoelectric focusing it w o u l d b e expected to give rise to variants that focus to a p o s i t i o n not on the d i a g o n a l of the s e c o n d isoelectric focusing gel. This was the case in b o t h species where the i n d i v i d u a l isoelectric v a r i a n t s were o b s e r v e d to give rise to o t h e r isoelectric variants c h a r a c t e r i s t i c for that species. Discussion

T h e e s t i m a t e d m o l e c u l a r weights for the large a n d small s u b u n i t of the r i b u l o s e - 1 , 5 - b i s p h o s p h a t e

pH

4.7

B Fig. 5. Isoelectric focusing of ribulose 1,5-bisphosphate carboxylase in two dimensions. The enzyme was isolated from (A) A. rnediterranea and (B) Acicularia schenckii. The gels were stained with silver. Proteins that deviate from the the diagonal of the second dimension gel are supposed to be altered and/or to be heterogenous in their isoelectric points. The arrows point to the individual isoelectric variants of the large subunit of the enzyme. The additional proteins are the isoelectric variants of the small subunit and of the DNAase used to prepare the isoelectric focusing sample.

c a r b o x y l a s e f r o m A c e t a b u l a r i a are similar to the m o l e c u l a r weights of these subunits from o t h e r p l a n t s [22]. Serva blue staining of S D S - p o l y a c r y l a m i d e gels (Fig. 1) revealed that the ribulose1,5-bisphosphate c a r b o x y l a s e was the m a j o r p r o tein c o n s t i t u e n t o f the c r u d e e n z y m e p r e p a r a t i o n . T h e a m o u n t of s a m p l e p e r t w o - d i m e n s i o n a l gel

64 was equivalent to the enzyme from one to two cells and as a result, not all of the isoelectric variants of the large subunit were detectable with Serva blue. However, with silver staining, a reproducible pattern of three or four isoelectric variants for the large subunit could be identified in the region of molecular weight 52000. The individual isoelectric variants were separated by 0.02-0.06 p H units and the p I values were between p H 5.0 and 5.7. A comparison of these patterns revealed that there are two groups, one with four isoelectric variants and a second group with three isoelectric variants. Besides the observation that one group lacks one isoelectric variant towards the basic end, it is striking that the two groups differ also qualitatively in that a pair of corresponding variants possess similar but significantly different p I values. The majority of published data on the large subunit patterns from other sources refer to three, equally-spaced isoelectric variants [10,23,24]. However, in m a n y figures an additional minor variant is often seen which is discounted as an artifact [10]. In the case of rye grass, a pattern of 5 6 isoelectric variants for the large subunit was demonstrated [25]. The number of observed isoelectric variants is thus similar to that observed in other species. In all reports except one [26], the ribulose1,5-bisphosphate carboxylase was carboxymethylated before focusing to prevent the oxidation of SH-groups. Under these conditions, the p I values of the large subunit isoelectric variants from a variety of sources lie between p H 6.0 and 7.0, which means that they are higher than those found for different species of Dasycladaceae. The isoelectric variants of the ribulose-l,5-bisphosphate carboxylase have been used as a tool to clarify evolutionary relationships [11,12]. Relative to different species of Nicotiana, which show divergence into four distinct groups over a period of 75 million years, the species investigated here display a remarkable homogeneity in light of the 500 million years evolutionary record of the family of Dasycladaceae [27]. The highly conservative nature of the large subunit gene has also been confirmed in other species by the high degree of homology in the analysed gene and amino acid sequences [28,29]. A major problem which has not been resolved up to now is the question of the origin of the

different isoelectric variants. A first possibility is that the isoelectric variants might arise as products of separate genes. However, amino acid analysis and fingerprinting techniques did not indicate that there are significant differences in the amino acid sequences of individual variants of the large subunit from Nicotiana tabacum [30]. Furthermore, only one gene could be identified in all cases in which the gene for the large subunit was localized on the chloroplast genome [6-8]. A second possibility is that the different variants are a result of post-translational modifications of a single gene product. In the case of spinach, the large subunit synthesized in an E. coli cell-free system had a molecular weight 1000-2000 larger than the purified large subunit and treatment of this larger protein with soluble extracts from chloroplasts converted it to the same size as the purified protein [31]. This might suggest that the differences in the isoelectric variants are due to variations in the processing of a single polypeptide chain. The different variants might also reflect in vivo modifications like phosphorylation, carboxylation, methylation etc., of a single gene product which to a certain degree should then be species specific. Beside the possibility that the individual isoelectric variants accurately reflect the in vivo heterogeneity, it is also possible that they could be due to a methodical artifact. Some electrofocusing artifacts have been attributed to a binding of carrier ampholytes [32,33]. In other cases, multiple focusing patterns have been explained as a result of macromolecular interactions [34,35]. For bacteriorhodopsin four isoelectric variants have been observed, some of which show instability if the focusing process was extended over a long period, so that one or more variants increased in abundance at the expense of others. In some cases, refocusing of the individual variants led to the production of the other variants. One of these variants remained unchanged by refocusing [36]. Another type of artifact has been demonstrated by the electrofocusing of single, labeled amino acids [37]. Amino acids with a large pI-pKp (pKp designates the p K of an ampholyte most approximate to its p I ) difference showed multiple focusing patterns in the absence as well as in the presence of urea. It is possible that some polypeptides may also show such multiple focusing

65

patterns, although this has not yet been demonstrated. The results of the re-electrophoresis experiment presented here demonstrate that the individual isoelectric variants may give rise to other isoelectric variants during the course of isoelectric focusing. This suggests that the variants are unstable and can be interconverted during the run, as observed for bacteriorhodopsin [36]. The conversion may be due to a methodical artifact such as binding of ampholytes or blocking of specific charged groups, etc. (see above). One may also tule out the assumption that the differences between the two groups arise simply as a result of reactive compounds in the isolation buffers, such as the oxidative impurities found in Triton X-100 and Brij 35 [38], since there are specific differences in the patterns observed and the same buffers were used to prepare the samples. Nonetheless, the pattern of isoelectric variants of the large subunit of the ribulose-l,5-bisphosphate carboxylase is reproducible and species specific. Therefore, regardless of the origin of the variation observed, the system presented here may be used for the unequivocal identification of this chloroplast enzyme and provides a means to elucidate the chloroplast component of hybrids between the two groups of species. However, there is a critical need to establish the chemical nature of the differences in the isoelectric variants of the large subunit of the ribulose-1,5-bisphosphate carboxylase and in particular to show that these differences originate in the chloroplast genome; experiments are currently underway to answer this question.

Acknowledgements We thank Mrs. Renate Fischer for the photographs and Mrs. Brigitte Nagel and Mrs. Heidi Klempp for typing the manuscript.

References 1 Schweiger, H.-G. and Berger, S. (1979) Int. Rev. Cytol. Suppl. 9, 11-44 2 Schweiger, H.-G., Master, R.W.P. and Werz, G. (1967) Nature 216, 554-557 3 Reuter, W. and Schweiger, H.-G. (1969) Protoplasma 68, 357-368

4 Apel, K. and Schweiger, H.-G. (1972) Eur. J. Biochem. 25, 229-238 5 Kloppstech, K. and Schweiger, H.-G. (1973) Exp. Cell Res. 80, 69-78 6 Coen, D.M., Bedbrook, J.R., Bogorad, L. and Rich, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5487-5491 7 Gelvin, S., Heizmann, P. and Howell, S.H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3193-3197 8 Whitfeld, P.R. and Bottomley, W. (1980) Biochem. Int. 1, 172-178 9 Wildman, S.G. (1979) Arch. Biochem. Biophys. 196, 598610 10 Kung, S.D., Sakano, K. and Wildman, S.G. (1974) Biochim. Biophys. Acta 365, 138-147 11 Kung, S.D. (1976) Science 191,429-434 12 Wettstein, D. v., Poulson, C. and Holder, A.A. (1978) Theor. Appl. Genet. 53, 193-197 13 Chen, K., Kung, S.D., Gray, J.C. and Wildman. S.G. (1976) Plant Sci. Lett. 7, 429-434 14 Schweiger, H.-G., Dehm, P. and Berger S. (1977) in Progress in Acetabularia Research (Woodcock, C.L.F., ed.), pp. 319-330, Academic Press, New York 15 Berger, S. and Schweiger, H.-G. (1979) in The Handbook of Phycological Methods - Cytological and Developmental Methods (Gantt, E., ed.), pp. 47-50, Cambridge University Press. London 16 Apel, K. and Schweiger, H.-G. (1973) Eur. J. Biochem. 38, 373-383 17 Garrels, J.I. (1979) J. Biol. Chem. 254, 7961-7977 18 Shoeman, R.L. and Schweiger, H.-G. (1982) J. Cell Sci., in the press 19 Sugiyama, T., Nakayama, N. and Akazawa, T. (1968) Arch. Biochem. Biophys. 126, 737-745 20 Sugiyama, T., Matsumoto, C., Akazawa, T. and Miyachi, S. (1969) Arch. Biochem. Biophys. 129, 597-602 21 Lorimer, G.H., Badger, M.R. and Andrews, T.J. (1977) Anal. Biochem. 78, 66-75 22 Kung, S.D. (1977) Annu. Rev. Plant. Physiol. 28, 401-437 23 Melchers, G., Sacristan, M.D. and Holder. A.A. (1978) Carlsberg Res. Commun. 43, 203-218 24 Gatenby, A.A. and Cocking, E.C. (1977) Plant Sci. Lett. 10, 97-101 25 Johal, S. and Chollet, R. (1981) FEBS Lett. 124, 75-78 26 Sagher, D., Grosfeld, H. and Edelman, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 722-726 27 Kamptner, E. (1958) Ann. Naturhist. Mus. Wien 62, 95-122 28 Mclntosh, L., Poulsen, C. and Bogorad, L. (1980) Nature 288, 556-560 29 Zurawski, G., Perrot, B., Bottomley, W. and Whitfeld, P.R. (1981) Nucleic Acids Res. 9, 3251-3270 30 Gray, J.C., Kung, S.D. and Wildman, S.G. (1978) Arch. Biochem. Biophys. 185, 272-281 31 Langridge, P. (1981) FEBS Lett. 123, 85-89 32 Wallevik, K. (1973) Biochim. Biophys. Acta 322, 75-87 33 Rhigetti, P.G. and Gianazza, E. (1978) Biochim. Biophys. Acta 532, 137-146 34 Cann, J.R., Stimpson, D.I. and Cox, D.J. (1978) Anal. Biochem. 86, 34-49

66 35 Guengerich, F.P. (1979) Biochim. Biophys. Acta 577, 132 141 36 Plantner, J.J. and Kean, E.L. (1981) Biochim. Biophys. Acta 670, 32-38

37 Rhigetti, P.G. and Chrambach, A. (1978) Anal. Biochem. 90, 633-643 38 Ashani, Y. and Catravas, G.N.(1980) Anal. Biochem. 109, 55-62