Protein turnover as a component in the light/dark regulation of phospho<i>enol</i>pyruvate carboxylase protein-serine kinase activity in C4 plants

Maize leaf phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.311 protein-serine kinase (PEPC-PK) phosphorylates serine-15 of its target enzyme, thus leading to an increase in catalytic activity and a concomitant decrease in malate sensitivity of this cytoplasmic C4 photosynthesis enzyme in the light. We have recently demonstrated that the PEPC-PK activity in maize leaves is slowly, but strikingly, increased in the light and decreased in darkness. In this report, we provide evidence that cycloheximide, an inhibitor of cytoplasmic protein synthesis, when fed to detached leaves of C4 monocots (maize, sorghum) and dicots (Portulaca oleracea) in the dark or light, completely prevents the in vivo light activation of PEPC-PK activity regardless of whether the protein kinase activity is assessed in vivo or in vitro. In contrast, chloramphenicol, an inhibitor of protein synthesis in chloroplasts, has no effect on the light activation of maize PEPC-PK. Similarly, treatment with cycloheximide did not influence the light activation of other photosynthesis-related enzymes in maize, including cytoplasmic sucrose-phosphate synthase and chloroplast stromal NADPH-malate dehydrogenase and pyruvate,P; dikinase. These and related results, in which detached maize leaves were treated simultaneously with cycloheximide and microcystin-LR, a potent in vivo and in vitro inhibitor of the PEPC type 2A protein phosphatase, indicate that short-term protein turnover of the PEPC-PK itself or some other essential component(s) (e.g., a putative protein that modifies this kinase activity) is one of the primary levels in the complex and unique regulatory cascade effecting the reversible light activation/seryl phosphorylation of PEPC in the mesophyll cytoplasm of C4 plants. Light reversibly activates a number of photosynthesisrelated enzymes in plants via several different mechanisms (1-4). Among these is the light activation of leaf cytoplasmic phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31] in C4 plants by reversible protein phosphorylation (5, 6). Previous in vitro (7, 8) and in vivo (9) studies with maize leafPEPC demonstrated that the phosphorylation ofa single, N-terminal seryl residue (Ser-15) leads to an increase in catalytic activity and a decrease in feedback inhibition of the target enzyme by L-malate. Related findings from a reconstituted phosphorylation system indicated that the activity of the protein-serine kinase that catalyzes this regulatory phosphorylation of PEPC is not affected by a number of putative, light-modulated cytoplasmic effectors (e.g., reduced thioredoxin h, Ca2+, PPj, fructose 2,6-bisphosphate) and autophosphorylation (6, 7). However, more recent work has established that the phosphoenolpyruvate carboxylase proteinserine kinase (PEPC-PK) is activated by light and inactivated by darkness in vivo (10). Moreover, this striking regulatory process appears independent of SH status, Ca2l levels, and a putative, tight-binding PEPC-PK effector (10). One of the distinguishing features of the reversible light activation of PEPC-PK and its target enzyme, PEPC, in C4 plants is its sluggishness in vivo; when compared to the in vivo activation of photoregulated mesophyll chloroplast stromal enzymes such as pyruvate,Pi dikinase (PPDK) and NADPHmalate dehydrogenase (MDH) (2, 6), the former are both relatively slow processes, taking up to 1 hr, rather than minutes, for completion (10-12). To gain more insight into this difference and the specific mechanism(s) by which the PEPC-PK activity in vivo is slowly, but strikingly, increased in the light and decreased in darkness (10), detached maize leaves were fed two widely used inhibitors of protein synthesis. PEPC-PK activity was subsequently assessed either in vivo [malate IC50 values for inhibition of the target enzyme (11, 12)] or in vitro [32p phosphorylation of purified dark-form PEPC (7, 10)]. Whereas chloramphenicol (CAP), a 70S ribosome-specific inhibitor of chloroplastic protein synthesis, had no effect on the light activation of PEPC-PK, cycloheximide (CHX), an inhibitor of cytoplasmic protein synthesis, completely blocked the light activation of this protein-serine kinase. In contrast, the in vivo activation of several other photoregulated cytoplasmic [sucrose-phosphate synthase (SPS)] and chloroplastic (PPDK, MDH) photosynthesisrelated enzymes was not influenced by CHX treatment. These results indicate that the synthesis and degradation of PEPC-PK per se or some other essential component(s) are involved at one of the primary levels in the regulatory cascade effecting the reversible light activation/seryl phosphorylation ofPEPC in the mesophyll cytoplasm ofC4 plants. MATERIALS AND METHODS Materials. Maize (Zea mays L., cv. Golden Cross Bantam) plants were grown as described (7, 10). [y-32P]ATP [specific activity, 3000 Ci (111 TBq)/mmol] was purchased from Amersham. Dark-form maize leaf PEPC was purified by the procedure described (7, 8). All biochemical reagents were obtained from Sigma except for microcystin-LR (MC) (CalAbbreviations: PEPC, phosphoenolpyruvate carboxylase; PEPCPK, phosphoenolpyruvate carboxylase protein-serine kinase; SPS, sucrose-phosphate synthase; PPDK, pyruvate,P1 dikinase; MDH, NADPH-malate dehydrogenase; CHX, cycloheximide; CAP, chloramphenicol; MC, microcystin-LR. §To whom reprint requests should be addressed at: University of Nebraska-Lincoln, Department of Biochemistry, 210 BcH, East Campus, Lincoln, NE 68583-0718. 2712 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 88 (1991) 2713 biochem). Stock solutions of 10 mM CHX and 310 mM CAP were prepared in absolute ethanol, while 0.5 mM MC was dissolved in 20% (vol/vol) methanol. Feeding of Protein Synthesis and Protein Phosphatase Inhibitors. Preilluminated leaves (-2 g fresh wt each) from 4to 6-week-old maize plants were excised underwater, inserted into 150-ml beakers containing 100 ml of distilled water (control), 5 ILM CHX, 310 ,uM CAP, or 10 nM MC in water and maintained at room temperature. When feeding was done in the dark, the beakers were placed in a darkened fume hood overnight. The dark sample was then prepared from these leaves and the corresponding light sample was collected after a 90-min illumination of the tissue. When feeding was done in the light, detached control leaves that had been preilluminated for 1.5 hr in water were either maintained in water or fed inhibitors for 4 hr in continued light, followed by preparation of leaf extracts. Illumination was provided by a forced-air cooled 300-W, low-temperature lamp at an incident light intensity of 600-800 /iE m 2 s-' (E, einstein) (400-700 nm). Preparation of Leaf Extracts. Samples (0.3 g fresh wt) from the control or inhibitor-treated leaf material were chopped and ground at 40C in a prechilled mortar containing washed sand, 2% (wt/vol) insoluble polyvinylpyrrolidone, and 1.5 ml of the appropriate extraction buffer. Buffer A [0.1 M Tris HC1, pH 8.0/20% (vol/vol) glycerol/10 mM MgCl2/14 mM 2-mercaptoethanol/1 mM EDTA] was used for preparation ofPEPC and its protein-serine kinase; buffer B (buffer A plus 2 mM pyruvate) was used for PPDK; buffer C (50mM Mops-NaOH, pH 7.5/15 mM MgCl2/2.5 mM dithiothreitol/1 mM EDTA/0.1% Triton X-100) was used for SPS; and buffer D (0.1 M Tris-HCl, pH 8.0/1 mM EDTA/14 mM 2-mercaptoethanol) was used for MDH. The crude leaf homogenates were filtered through an 80-,tm nylon net and centrifuged for 1.5 min at 8700 x g. The supernatant fluid was either used immediately (PPDK, MDH) or after a 0.2-ml aliquot was rapidly desalted at 4°C on a Sephadex G-25 column (1 x 5 cm) equilibrated with 0.1 M Tris-HCI, pH 7.5/10 mM MgCI2/20% (vol/vol) glycerol for PEPC and PEPC-PK or buffer C minus Triton X-100 for SPS. Activity Assays. PEPC activity was determined spectrophotometrically at 340 nm and 30°C. The assay mixture (12) contained, in a total vol of 1 ml, 50mM Hepes-KOH (pH 7.3), 2.5 mM phosphoenolpyruvate, 5 mM MgCI2, 1 mM NaHCO3, 0.2 mM NADH, 10 units of malate dehydrogenase, various concentrations of L-malate, and 10 ,ul of desalted extract (added last). Malate IC50 values were taken as the malate concentration required for 50%o inhibition of PEPC activity under these assay conditions. PEPC-PK activity was measured by 32p incorporation from [y32P]ATP into purified dark-form PEPC (10). The phosphorylation mixture contained 35 ,ul of desalted extract, 10 ug of purified dark-form maize PEPC, an adenylate kinase inhibitor plus a creatine kinase/phosphocreatine ADP-scavenging system (10), 25 ,uM ATP, and 3 ,Ci of [y-32P]ATP in a final vol of60 p1. After 45 min of incubation at 30°C, the reaction was stopped by adding 20 ,ud of SDS sample buffer (0.25 M Tris-HCl, pH 6.8/8% SDS/40o glycerol/20% 2-mercaptoethanol), followed by immediate boiling for 2 min. Vertical SDS/PAGE was performed as described (13, 14), and autoradiographs were prepared from the dried gels with Kodak X-Omat AR film and two Lightning Plus intensifying screens (DuPont) at -800C. SPS, PPDK, andMDH activities were measured according to ref. 15 (at limiting substrate concentrations plus 10 mM P1 at 250C), ref. 16 (forward direction plus 2.5 mM glucose 6-phosphate and 2 units of purified maize PEPC at 300C), and ref. 17 at 30TC, respectively. RESULTS AND DISCUSSION Effects of CHX, CAP, and MC on the Light-Induced Changes in Malate Sensitivity of Maize Leaf PEPC. The IC50 values for PEPC inhibition by L-malate were used as an indirect means of following the effect of dark to light transitions on the apparent in vivo activity of the PEPC-PK since these values reflect the seryl-phosphorylation status of the target enzyme both in vitro (7, 8) and in vivo in response to light and dark (9-12). Feeding 5 ,uM CHX to detached preilluminated maize leaves in the dark overnight completely and reproducibly prevented the subsequent light-induced increase in the malate IC50 value ofPEPC without having any significant effect on the dark-form enzyme (Table 1). In contrast, CAP treatment had no effect on the light-induced changes in malate sensitivity of PEPC (Table 1). Overnight feeding of 5 AM CHX in the dark to predarkened maize leaves had the same inhibitory effect on light activation of PEPC. Results similar to those presented in Table 1 were obtained when detached leaves of sorghum, another C4 grass, and halved leaves of Portulaca oleracea, a C4 dicot, were fed CHX (data not shown). Given that such inhibitors are known not to be absolutely specific, thus possibly causing detrimental side effects (18), and that the 3-(3 ,4-dichlorophenyl)-1, 1-dimethylureasensitive light activation/phosphorylation ofPEPC occurs in the cytoplasm and is somehow related to photosynthetic electron transport and/or photophosphorylation (5, 6, 19), it was imperative to examine the effect of CHX treatment on the in vivo light activation of other photosynthesis-related enzymes in maize. Cytoplasmic SPS and chloroplast stromal PPDK are, like PEPC, light-activated by reversible phosphorylation/dephosphorylation cycles (2, 3, 6, 15, 20). In contrast, stromal MDH is light activated by 3-(3,4dichlorophenyl)-1,1-dimethylurea-sensitive changes in its SH redox status mediated by noncyclic electron flow and the chloroplastic ferredoxin/thioredoxin m system (1, 2, 17, 21). Notably, the results (Table 2) indicate that the light activation of these three enzymes was not significantly affected by feeding 5 A&M CHX to detached maize leaves under conditions identical to those described in Table 1. Similarly, CHX treatment of detached leaves had no obvious effect on either their total soluble protein content (mg/g fresh wt) or polypeptide pattern (e.g., see Fig. lA, lane 2 versus 3 and lane 5 versus 6) over the duration of these relatively short-term experiments. Thus, the inhibitory effect of CHX on the apparent in vivo activity of the PEPC-PK (Table 1) appears rather selective for the light activation of this specific converter enzyme. From the results described above, it is clear that de novo synthesis ofPEPC-PK or some other essential component(s) (e.g., a putative modifying protein that activates this proteinserine kinase in vivo) is induced during a 1.5-hr exposure to light. Thus, it was anticipated that if CHX were fed to illuminated detached leaf tissue after a point at which sufficient protein (i.e., either PEPC-PK or the putative modifying Table 1. Effects of protein synthesis inhibitors on light-induced increase in the malate IC50 value of maize leaf PEPC