Biochimie 70 (1988) 769-772 t~) Soci6t6 de Chimie biologique / Elsevier, Paris
In vivo phosphorylation of sorghum leaf phosphoenolpyruvate carboxylase Marie-Th6r6se G UIDICI-ORTICONP, Jean V I D A L l*, Pierre LE MARI~CHAL 1, Martine THOMAS 1, Pierre G A D A L 1 and Ren6 RI~MY 2
l Laboratoire de Physiologie Vdg~tale Moldculaire, CNRS (UAI128), Universit~ de Paris-Sud, 91405 Orsay Cedex, and ZLaboratoire de Photosynth~se, CNRS, 91190 Gif-sur-Yvette, France (Received 30-6-1987, accepted after revision 12-1-1988)
Summary - - The use of immunological techniques allowed us to purify close to homogeneity phosphoenolpyruvate carboxylase (PEPc, EC 22.214.171.124) from sorghum leaf. It was thus established that: 1) this protein is phosphorylated in vivo on seryl residues; 2) in C4-type photosynthesis, the phosphorylation process mainly concerns t h e PEPC isozyme form G; 3) enzyme phosphorylation displays significant variations through a day-night alternation which therefore suggests light control of the process.
phosphoenolpyruvate carboxylase/ phosphorylation/ C4 plant / sorghum
Introduction In leaves of C4 and CAM (Crassulacean acid metabolism) plants, two main forms of phosphoenolpyruvate carboxylase (PEPc) (E and G forms) were found. The G isozyme catalyzes the primary step of COz fixation in photosynthesis [ 1]. Different investigations have helped to clarifi functional, regulatory and physicochemical properties of this enzyme at the molecular level. Moreover, due to the fact that its activity was found to be light-controlled, this form attracted more attention than any other member of the PEPc isozymic family. As a molecular basis for this phenomenon, it was established that light induced the corresponding g e , e to become activated and to produce PEPc messenger RNA through phytochrome mediation [2, 3]. In addition, it has been recently reported that a posttranslational modification process could account for the modulation of catalytic activity a n d / o r regulatory properties of the enzyme. We have
*Author to whom all correspondence should be addressed.
shown that the leaf enzyme from the Crassulacean plant Kalanchoe daigremontiana is phosphorylated in vivo through a process which converts a dephosphorylated day form into a phosphorylated night one, mainly on the serine residue . Nimmo et aL [5, 6] reached very similar conclusions for PEPc with another CAM plant, Bryophyllura fedtschenkoi. Interestingly, it was observed that in vitro dephosphorylation increased the sensitivity of the enzyme to malate. This result strengthened the metabolic control hypothesis according to which retroinhibition exerted by malate was the causative effect of circadian rhythmicity. More recently, Nimmo et al.  have extended these results to a C4 plant (maize), establishing that changes in its kinetic properties were associated with a specific state of protein phosphorylation. Again, a rhythmic phenomenon was suggested. Supporting these data, Budde and Chollet  observed that phosphorylation of the maize enzyme could also occur in vitro using leaf extracts. However,
M.- 7". Guidici-Orticoni et al'.
the authors also stressed the tact that P P D K (pyruvate-P~ dikinase, EC 126.96.36.199), another phosphorylated enzyme of the C4 cycle [9-11] was most likely contaminating PEPc, as molecular weights and physicochemical properties were very similar for both enzymes. Consequently, immune sera which were raised previously by several groups working on PEPc were thought to contain, in addition to anti-PEPc antibodies, a certain amount of anti-PPDK antibodies. The present study was undertaken with sorghum leaf PEPc to investigate whether or not this enzyme is actually phosphorylated in vivo, paying particular attention to the possible interference of PPDK. Questions as to whether phosphorylation is dependent upon photoperiodic conditions, or is acting on G and E isozymes, have also been considered.
phosphate. Immunoprecipitated enzyme was then subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Fluorography of the corresponding dried gel detected a well-marked radioactive signal which in all cases superimposed perfectly on the PEPc subunit, thus suggesting that labeled phosphorus had been covalently linked in vivo to the PEPc protein. In order to confirm the reality of this posttranslational modification process, radiolabeled PEPc purified by immunochromatography was hydrolyzed with hydrochloric acid and the phosphoamino acids were resolved by two-dimensional chromatography on celhdose plate. Identification of the phosphoamino acid was inferred from autoradiography of the plate. Fig. I clearly shows a major radioactive spot at the position of phosphoserine.
Characterization o f p h o s p h o r y l a t e d P E P c Materials and methods
Plant material Sorghum seeds (Sorghum vulgare var. Tamaran), (100-200), were sown a:~, germinated on vermiculite imbibed with water (about 100 ml), in darkness for 4 days at 30°C. Etiolated shoots were then transferred to photoperi¢~dic conditions for 5 additional days as described elsewhere . Incubation with 32p 560 #Ci of labeled orthophosphate (200 mCi / mol) were added to the culture medium when plants were transferred to photoperiodic conditions. 2 - 4 days later, when most of the label had been incorporated inlo the plants, green leaves were sampled (1 g fresh material) at the indicated time. Extraction and immunopurification of PEPc Extraction, purification, measurements of activity and immunoprecipitation of PEPc were as previously described . Immunochromatography was essentially as in . Mono- and r,vo-dimensional (2D) gel electrophoresis Immunologically purified PEPc was analyzed in both SDS-ptJyaerylamide gels and 2D-gel electrophoresis according to  and 114], respectively. Western blot experiments were performed as described previously .
Following the results described above, it seemed evident that PEPc from sorghum leaves (a C4 plant), was phosphorylated in vivo. However, by the time these data were obtained, Budde and Chollet  had pointed out that inconclusive identification or misidentification of PEPc could result from the interference of P P D K , another phosphorylated enzyme from the C4 cycle of photosynthesis, whose properties are similar to those of the carboxylase. They also established that P P D K was in fact distinguishable from the
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Results and Discussion
In vivo phosphorylation o f P E P c PEPc was extracted from greened 6 day old sorghum plants fed for 4 days with [32p]sodium
Fig. 1.2D-Separation of phosphorylatcd amino acids from 3zP-labeled PEPc. The gel was dried and autoradiographed; dotted circles indicate standard' amino acids. S-O-P: serine phosphate, Thr-2P: threonine phosphate.
Phosphorylation of sorghum PEPc
MW Kd 94
e? a 43
14 J 1
ed). Autoradiography of the dried gel showed that the protein spot was radiolabeled (Fig. 2B). Furthermore, it has been firmly established that the phosphorylated residue on PPDK was threonine [8, 9], whereas phosphoserine was mainly found in sorghum leaf PEPc, in good agreement with the experimental results of Nimmo et al. and Budde and Chollet [7, 8]. Taken together, the results allow us to rule out the interference of PPDK and to conclude that the PEPc subunit was the major protein involved in the phosphorylation in this system. As two main isozymes (G and E fo_rms) exist in green sorghum leaf (generally in a proportion of 2 0 - 3 0 to 1, respectively), the second question which arose concerned the identification of the phosphorylated form. It was demonstrated that the immunosorbent used in these experiments had a high selectivity for the G-PEPc isozyme. In a typical experiment, the column was shown to retain less than 0.3 units of activity of the EPEPc form, while it could adsorb nearly 30 units of the G form (data not shown). In a similar experiment, cold exogenous E form was added to the extract (resulting in a 10-fold dilution of endogenous E form). The E form was obtained
Fig. 2. 2D-Gel electrophoresis of proteins purified by immunochromatography. A. Coomassie blue staining of proteins. Lane 1: molecular weight markers; lane 2: protein sample run in one dimension (SDS-PAGE) as control. B. Corresponding autoradiogram of the top part of the gel (arrows mark the location of radioactive and corresponding protein spots).
carboxylase on the basis of its isoelectric point and of the phosphorylated residue, found in that particular case to be phosphothreonine. Therefore, since PPDK was reported to be phosphorylated, contamination of the PEPc band by even very small amounts of this enzyme would result in erroneous conclusions. Consequently, it was necessary to check the specificity of the immune serum for PEPc. Using our experimental conditions, attention was carefully focused on the possible presence of anti-PPDK antibodies. When immunopurified PEPc was subjected to 2D-gel electrophoresis, either Coomassie blue staining or silver staining detected a single peptide spot at the level of the PEPc subunit (Fig. 2A). This peptide corresponded to the PEPc band (immunologically characterized) of the SDS-dimension step and displayed an isoelectric point of approximately 5.5, a value previously found for the sorghum leaf enzyme (unpublish-
Fig. 3. Competition experiment between G- and E-PEPc forms on an immunosorbent column. Assays were either extracts from radiolabeled green sorghum leaves (30 enzymatic units) or the same supplemented with extracts from cold etiolated leaves (30 + 7 enzymatic units, respectively). 10-20 ~g of purified proteins were subjected to SDS-gel eleetrophoresis. 1. Coomassie blue staining of proteins. Lane A: green leaf extract; lane B: green + etiolated leaf extracts. 2. Lanes A, B: corresponding fluorography.
M.-T. Guidici-Orticoni et al.
from etiolated leaves which contain only this PEPc form. Since it was found that the radioactivity incorporated into PEPc was not significantly decreased by the addition of the E form (Fig. 3), the results clearly indicated that the phosphorylation process mainly concerns the G-PEPc form. Nothing is known about the behavior of the E form. Finally, it was observed that PEPc isolated from sorghum leaves was labeled less during the night than during the day, however, the amplitude was not so marked as that which was reported by Nimmo et aL  in the case of maize. In summary, the present study confirms the results of Nimmo et al.  and Budde and Chollet . By eliminating the possibility of interference by PPDK, it demonstrates unambiguously that C4 plant PEPc is a phosphorylated protein. Furthermore, it extends these data by identifying the isozymic form which is phosphorylated.
Acknowledgments Thanks are due to E. Keryer and F. Ambard-Bretteville for excellent technical assistance, Dr. J. Brangeon for correcting the manuscript and M. Weimbaun for photographs.
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