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Biochemistry -- Faculty Publications , Department of

1991 Posttranslational Regulation of Phosphoenolpyruvate Carboxylase in C4 and Crassulacean Acid Metabolism Plants Jin-an Jiao University of Nebraska - Lincoln

Raymond Chollet University of Nebraska - Lincoln, [email protected]

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Jiao, Jin-an and Chollet, Raymond, "Posttranslational Regulation of Phosphoenolpyruvate Carboxylase in C4 and Crassulacean Acid Metabolism Plants" (1991). Biochemistry -- Faculty Publications. 334. http://digitalcommons.unl.edu/biochemfacpub/334

This Article is brought to you for free and open access by the Biochemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Biochemistry -- Faculty Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Plant Physiol. (1991) 95, 981-985 Received for publication August 24, 1990 0032-0889/91/95/0981/05/$01 .00/0 Accepted November 15,1990

Review Posttranslational Regulation of Phosphoenolpyruvate Carboxylase in C4 and Crassulacean Acid Metabolism Plants1

Jin-an Jiao and Raymond Chollet* Department of Biochemistry, University of Nebraska-Lincoln, East Campus, Lincoln, Nebraska 68583-0718

ABSTRACT reaction is compartmented either spatially (in Control of C4 and Crassulacean acid metabo- mesophyll cells in the light) or temporally (in the dark) in C4 lism (CAM) is, in part, mediated by the diel regulation of phos- or CAM plants, respectively. phoenolpyruvate carboxylase (PEPC) activity. The nature of this Based on physiological and biochemical considerations, it regulation of PEPC in the leaf cell cytoplasm of C4 and CAM plants is evident that PEPC activity must be regulated by light + is both metabolite-related and posttranslational. Specifically, the dark transitions in C4 and CAM species in order to: (a) regulatory properties of the vary in accord with the coordinate C4- and C3-photosynthetic carbon metabolism in physiological activity of C4 photosynthesis and CAM: PEPC is response to light intensity in C4 plants (7, 10); (b) avoid futile less sensitive to feedback inhibition by L-malate under light (C4 decarboxylation/carboxylation cycling during the day in plants) or at night (CAM plants) than in darkness (C4) or during CAM plants (21); and (c) minimize uncontrolled utilization the day (CAM). While the view that a light-induced change in the aggregation state of the holoenzyme is a general mechanism for of glycolytic PEP during the dark in C4 plants (2, 7). While the diel regulation of PEPC activity in CAM plants is currentiy in light-induced changes in the cytoplasmic levels of known dispute, there is no supportive in vivo evidence for such a tetra- metabolite effectors (e.g. glucose 6-P, L-malate, triose-P) likely mer/dimer interconversion in C4 plants. In contrast, a wealth of in contribute to the overall regulation of PEPC activity (5, 7, vitro and in vivo data has accumulated in support of the view that 21), recent attention has focused on the diel regulation ofthis the reversible of a specific, N-terminal regulatory enzyme's activity in C4 and CAM species by posttranslational serine residue in PEPC (e.g. Ser-15 or Ser-8 in the maize or modification. In this review we summarize the recent devel- sorghum , respectively) plays a key, if not cardinal, role opments in this latter area of research and discuss the molec- in the posttranslational regulation of the carboxylase by light/ ular mechanism(s) involved in this dark or day/night transitions in both C4 and CAM plants, regulatory process. respectively. GENERAL PROPERTIES OF THE REGULATORY PROCESS The initial interest in the posttranslational modulation of PEPC activity came from work on the diel regulation of Phosphoenolpyruvate carboxylase (PEPC2, EC 4.1.1.31) CAM. The enzyme responsible for the initial fixation of catalyzes the irreversible ,B-carboxylation of PEP in the pres- atmospheric CO2 by CAM plants in darkness is PEPC, which, ence of bicarbonate and Me2", (e.g. Mg2", Mn2") to yield like the C4 isoform, is activated allosterically by glucose 6-P oxalacetate and Pi, a reaction that serves a variety of physio- logical functions in plants. This cytoplasmic enzyme com- and feedback inhibited by L-malate (7, 21). A related, phys- prises four identical subunits with monomeric molecular mass iologically important feature of CAM PEPC is its day/night of about 110 kD. During both C4 photosynthesis and CAM, fluctuation in enzymatic properties (29); the rapidly prepared, PEPC is the initial carboxylating enzyme that fixes atmos- desalted enzyme extracted from night leaf tissue has lower pheric CO2 into C4-dicarboxylic acids (oxalacetate, malate, Km(PEP) and higher Ki(malate) values than those from the and aspartate), from which CO2 is subsequently released corresponding day leaftissue. These day/night changes in the internally by various decarboxylating enzymes and photosyn- properties of PEPC lead to a higher enzymatic activity at thetically reassimilated by the Calvin cycle (7, 21). This initial night and a much lower activity during the day (e.g. the night/ day activity ratio is about 8 [15, 29]) which are paralleled by the classical physiological changes in the activity ofCAM (e.g. 'The research described herein from this laboratory was supported in part by grant DMB-8704237 from the National Science Founda- external C02 fixation, titratable acidity [21]). Further detailed tion. This review is published as Paper No. 9323, Journal Series, study of several CAM plants under continuous dark or light Nebraska Agricultural Research Division. conditions indicated that CAM physiology, as well as the 2Abbreviations: PEPC, phosphoenolpyruvate carboxylase; PEP, malate sensitivity of PEPC, are controlled by an endogenous phosphoenolpyruvate; PPDK, pyruvate,Pi dikinase; PP, protein phos- circadian rhythm rather than by light or dark signals per se phatase; PK, protein . (20, 29). 981 982 JIAO AND CHOLLET Plant Physiol. Vol. 95, 1991

With respect to C4 PEPC, intensive investigation ofits light/ concentration of CAM PEPC would be about 1.6 mg/mL (or dark regulation was initiated only recently. Despite this limi- about 0.1 mg PEPC per g fresh weight of leaf tissue). This tation, it is now established unequivocally that light reversibly estimate is manyfold higher than the PEPC concentrations induces a two- to threefold increase in catalytic activity and used in vitro for the size-exclusion studies (e.g. less than 0.05 decrease in malate sensitivity of the enzyme from a variety of mg/mL after the HPLC chromatography step [calculations C4 plants when assayed at suboptimal, but physiological levels from ref. 31]). Thus, whether a dimer-tetramer interconver- of pH and PEP and in the presence of L-malate. Typically, sion of PEPC takes place in vivo during day/night transitions such assay conditions result in a light/dark activity ratio of of CAM leaf tissue and, if so, whether it is in any way about 5 (8-10, 19, and references therein). Darkness reverses influenced by other posttranslational modifications of the these effects of illumination on C4-leaf PEPC activity. More- enzyme (see below) are questions certainly worthy of further over, light activation of PEPC in C4 plants is related, either investigation. directly or indirectly, to photosynthetic electron transport An alternative mechanism for the diel regulation of CAM and/or photophosphorylation and is modulated by several PEPC activity was proposed initially by Nimmo's group (3, photosynthesis-related environmental factors, including light 17, 18, 20). By a combination of feeding Bryophyllum fedt- intensity, CO2 concentration, and temperature (19, 23, 24 schenkoi leaf tissue with 32Pi and the subsequent SDS-PAGE and references therein). It is notable that when compared to analysis of PEPC immunoprecipitates, they found (17) that the light induction of C4 photosynthesis and the activation of PEPC was seryl-phosphorylated in vivo at night when the photoregulated C4 mesophyll-chloroplast stromal enzymes enzyme is relatively malate insensitive [apparent Ki(malate), (NADPH-malate dehydrogenase, PPDK), light activation of 3 mm] and dephosphorylated during the day when it is highly this cytoplasmic enzyme is relatively slow, taking 30 to 60 malate sensitive [Ki(malate), 0.3 mM]. Furthermore, when the min for completion (9, 19, 24). purified, malate-insensitive night form of 32P-labeled PEPC In contrast to the situation in CAM and C4 plants, recent was preincubated withl exogenous alkaline in reports indicate that there is no such light/dark modulation vitro, dephosphorylation was correlated with a marked in- of PEPC activity in C3 leaftissue (4) and guard cells (25). crease in malate sensitivity [i.e. a 10-fold decrease in the apparent Ki(malate)] of the enzyme (18). Notably, under various in vitro conditions (e.g. high or low protein concen- MOLECULAR MECHANISMS OF PEPC tration, in the absence or presence of malate or Mg2+), both POSTTRANSLATIONAL REGULATION the phosphorylated, malate-insensitive PEPC and the dephos- phorylated, malate-sensitive enzyme-form from B. fedtschen- CAM-PEPC koi existed as a tetramer (cf. 30, 31), suggesting no direct relationship between the enzyme's aggregation state and its Wu and Wedding (30) first reported the purification of malate sensitivity/phosphorylation status in this particular CAM PEPC from day- and night-adapted Crassula argentea CAM species (18). leaves and found that these two enzyme-forms existed as Additional biochemical evidence that reversible regulatory kinetically distinct, but thermodynamically interconvertible, seryl-phosphorylation of PEPC in CAM plants contributes to oligomers. The day enzyme was mainly a malate-sensitive the day/night changes in properties of the target enzyme homodimer (a2 [Ki, 1.6 mM]) and the night enzyme a malate- comes from the recent identification of a okadaic acid-sensi- insensitive homotetramer (a4 [Ki, 3.9 mM]). The results from tive type-2A (PP) that dephosphorylates in vitro studies with the purified day enzyme (30, 31) further PEPC from B. fedtschenkoi (3). Incubation of the in vivo 32p_ showed that the substrate PEP and Mg2+, a bivalent cation labeled PEPC with exogenous mammalian type-2A PP results required for catalysis, favor the conversion ofa2 to a4, whereas in a direct correlation between 32P-release from the target L-malate, a feedback inhibitor, shifts the dimer-tetramer equi- enzyme and the concomitant decrease in apparent Ki(malate). librium toward the dimeric enzyme-form. These in vitro In contrast, mammalian type-1 PP did not have any such findings suggested that interconversion between the malate- effect on CAM PEPC. Notably, a partially purified type-2A sensitive dimer and malate-insensitive tetramer of PEPC protein phosphatase preparation from B. fedtschenkoi leaves might be the molecular mechanism for the diel regulation of converted the malate-insensitive, night-form PEPC to a ma- PEPC activity in CAM plants. However, this hypothesis was late-sensitive form. This diel modulation of CAM PEPC not consistent with subsequent results obtained with other properties by reversible regulatory phosphorylation has been CAM species in which both the day and night enzyme-forms confirmed by both in vivo and in vitro studies with other were found to be in the same aggregation state but still CAM species(l, 14). displayed the characteristic differential sensitivity to feedback inhibition by L-malate (15, 18). In this context, it is notable C4-PEPC that the reported in vitro dimer-tetramer interconversion was influenced by protein concentration (30), with a higher PEPC The first observation that suggested a possible relationship concentration favoring tetramer formation and vice versa. between the light-induced changes in the properties of the C4 Based on our calculations from the PEPC protein- and yield- enzyme and its reversible phosphorylation status came from related data reported by Nimmo et al. (18) and the assump- experiments with an in vitro 32P-phosphorylation system (2). tions that 1 g fresh weight of leaf tissue is roughly equivalent This work demonstrated that PEPC from maize and sugar- to 1 mL in total volume and that the cytoplasmic matrix cane can, indeed, be phosphorylated in vitro exclusively on represents about 5% of the total cell volume (21), the in vivo serine residues by an ATP-dependent, soluble protein ki- POSTTRANSLATIONAL REGULATION OF PEP CARBOXYLASE 983

nase(s) in desalted extracts prepared from illuminated green nal methionine reveals that (a) the phosphorylation site (Ser- leaftissue. Subsequent in vivo 32Pi-labeling studies with maize 15) is two residues removed from a basic (Lys-12) (13, 19) and sorghum (13, 26) leaf tissue indicated that C4 in the primary structure, a feature similar to that in various PEPC is more sery phosphorylated in the light when the protein-serine/threonine kinase substrates; (b) C4 PEPC from enzyme is less malate sensitive than in darkness when it is sorghum leaves and CAM PEPC from salt-stressed Mesem- more malate sensitive. Using rapidly purified PEPC from bryanthemum crystallinum leaves contain N-terminal se- maize leaves, Jiao and Chollet (9) reported that the enzyme quences that are highly homologous to this regulatory phos- isolated from light-adapted plants had a higher catalytic activ- phorylation site, including the structural motif of Lys/Arg-X- ity and lower malate sensitivity than the corresponding dark X-Ser; and (c) no such homology is present in the bacterial, enzyme-form. Although both light and dark enzymes con- cyanobacterial or C3 (noninduced) M. crystallinum enzymes tained P-serine, the degree of phosphorylation in the light (1 1, 12, and references therein). form was greater than that in the dark enzyme. As in the case More recently, comparative sequence analyses of the phos- of phosphorylated CAM PEPC (18), preincubation of the phopeptides isolated from in vivo 32P-labeled, purified dark- maize light-form enzyme with exogenous alkaline phospha- and light-form PEPC from maize or sorghum indicate that tase converted the more active, less malate-sensitive PEPC to Ser- 15 or its structural homolog in the sorghum enzyme, Ser- a less active, more malate-sensitive enzyme with properties 8, is the only residue to be 32P-phosphorylated in vivo upon similar to those of the control or phosphatase-preincubated illumination and dephosphorylated in the dark (13). These dark enzyme (9). It is interesting to note that exogenous results, which are in complete agreement with the previous in had little effect on the properties ofdark- vitro studies (11), unequivocally establish that Ser-15 is the form PEPC (9) even though it is partially phosphorylated (9, regulatory site that undergoes light/dark changes in phos- 13, 19). Similar results were later obtained with sorghum leaf phorylation status and, thus, contributes to the regulation of PEPC (26). maize PEPC activity in vivo. To assess critically the effects of phosphorylation on the Other available data also support the view that Ser-15 in properties of C4 PEPC, in vitro experiments were performed maize leaf PEPC is the regulatory phosphorylation site that is (10) with an homologous reconstituted phosphorylation sys- directly involved in the light/dark regulation of this enzyme tem comprised ofpurified, dark-form maize PEPC, a partially in vivo. Along these lines, McNaughton et al. (16) recently purified protein kinase(s) from light-adapted leaves, and ATP. reported that dark-form maize PEPC that had been partially Mg. The results from this phosphorylation system unequivo- degraded by an endogenous protease(s) sensitive to chymosta- cally established that the PK-mediated changes in the catalytic tin, a chymotrypsin inhibitor, had not only lost a -4-kD activity and malate sensitivity of C4 PEPC are directly corre- peptide fragment from its N- or C-terminal, but also its malate lated with the concomitant changes in the seryl-phosphoryl- sensitivity and the ability to be phosphorylated in vitro. ation status of the target enzyme in vitro. Moreover, these Vidal et al. (26) have recently isolated a calcium-dependent changes in the enzymatic properties of dark-form PEPC protein kinase(s) from sorghum leaves that phosphorylates caused by in vitro phosphorylation were in quantitative agree- dark-form PEPC in vitro but yet does not change the proper- ment with those induced by light in vivo (9, 19). These in ties of the target enzyme. In contrast, the soluble leaf PK(s) vitro results, which have been subsequently confirmed by that phosphorylates (activates) maize PEPC at Ser-15 (10, 1 1) Nimmo's group with maize (see pp. 357-364 in ref. 26) and is not affected by either calcium/calmodulin, EGTA, fructose Vidal's group with sorghum (26), clearly demonstrate that the 2,6-bisP, or reduced cytoplasmic thioredoxin h from spinach. regulatory seryl phosphorylation ofPEPC by an ATP-depend- However, this kinase preparation is inhibited by NaCl (KC1), ent, soluble leaf protein kinase(s) is a key component in the L-malate, and glucose 6-P. Such metabolite-mediated inhibi- posttranslational regulation ofC4 PEPC activity by light/dark tion may be a result of a direct interaction of the PK with transitions in vivo. these carbon compounds or a result of a conformational Deduced primary sequences of various isoforms of PEPC change in the protein substrate, PEPC, induced by these two have been reported from diverse organisms, including Esch- known effectors. It is not clear at present whether the calcium- erichia coli, Anacystis nidulans, and C3, C4, and CAM plants dependent C4-leaf protein kinase(s) (26) phosphorylates the (1 1, 12 and references therein). Comparative analysis ofthese specific, N-terminal regulatory seryl-phosphorylation site sequences has revealed that the C-terminal region ofthe - 10- identified previously (1 1, 13) and, if so, whether the molar kD polypeptide is relatively conserved, perhaps encompassing stoichiometry (i.e. mol Ser-P per 110-kD subunit) is suffi- the active-site domain, whereas the N-terminal region is more ciently low that changes in catalytic activity and malate- variable. Along these lines, the regulatory phosphorylation sensitivity of the target enzyme can not be detected. site from in vitro 32P-phosphorylated/activated dark-form With regard to the possible involvement of changes in the maize PEPC (10) has recently been isolated and sequenced aggregation state of PEPC in the reversible light regulation of (1 1). The amino acid sequence ofthis regulatory phosphopep- the C4 enzyme, no supportive in vivo evidence has yet been tide is His-His-Ser(P)-Ile-Asp-Ala-Gln-Leu-Arg, which cor- reported. In fact, both the light and dark enzyme-forms exist responds exactly to residues 13 to 21 in the deduced primary as tetramers under normal conditions (2, 8, 16). However, structure of the maize leaf enzyme. This N-terminal regula- the in vitro dissociation of the active C4 tetramer into dimers tory site (Ser-15) is far removed from a recently identified, and monomers induced by dilution, NaCl, malate or low species-invariant, active-site (Lys-606) in the C-termi- temperature (16, 22, 28, 32) leads to loss of enzyme activity, nal region of the maize primary structure (12). Extension of but with no change in malate sensitivity (16). Once again, it the regulatory phosphorylation site sequence to the N-termi- must be emphasized that the dissociation of C4 PEPC is 984 JIAO AND CHOLLET Plant Physiol. Vol. 95, 1991

Light (C4) or Night/ Rhythm (CAM) the light/dark regulation ofthe C4- and CAM-leafenzymes in vivo (Fig. 1). Future work on the regulatory properties of the protein-senne kinase(s) (10) and type-2A protein phospha- Protein kinase - ' Protein kinase (Less active) A (More active) tase(s) (3) involved in this cytoplasmic phosphorylation/de- - A phosphorylation cycle should provide much-needed insight into the specific nature of the photosynthesis-related light Dark (C )IOr Day/ Rhythm (CAM) signal (23, 24) and its transduction pathway in the C4 meso- A TrJ A,DP ATP VSE eSERP phyll cytoplasm and of the putative factor that controls the _

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