Protein turn over as a component in the light/dark regulation of phosphoenolpyruvate carboxylase protein-serine kinase activity in C4 plants

P oc. Nadl. Acad. Sci. USA Vol. 88, pp. 2712-2715, Ap il 1991 Bo any P o ein u no e as a componen in he ligh /da k egula ion o phosphoenolpy u a e ca boxylase p o ein-se ine kinase ac i i y in C4 plan s ( egula o y phospho yla ion/C4 pho osyn hesis/ligh ac i a ion/p o ein kinase/cycloheximide) JIN-AN JIAO*, CRISTINA ECHEVARR A* , JEAN VIDAL , AND RAYMOND CHOLLET*§ *Depa men o Biochemis y, Uni e si y o Neb aska-Lincoln, Eas Campus, Lincoln, NE 68583-0718; Labo a o io de Fisiologia Vege al, Facul ad de Biolog a, Uni e sidad de Se illa, A enida de la Reina Me cedes s/n, 41012, Se illa, Spain; and Labo a oi e de Physiologie V6gd ale Mol6culai e, Uni 6 de Reche che Associde Cen e Na ional de la Reche che Scien i ique 1128, Uni e si de Pa is-Sud, Cen e d'O say, Bi imen 430, O say-Cedex, F ance Communica ed by William L. Og en, Janua y 3, 1991 ( ecei ed o e iew No embe 13, 1990) ABSTRACT Maize lea phosphoenolpy u a e ca boxylase [PEPC; o hophospha e:oxaloace a e ca boxy-lyase (phospho- yla ing), EC 4.1.1.311 p o ein-se ine kinase (PEPC-PK) phos- pho yla es se ine-15 o i s a ge enzyme, hus leading o an inc ease in ca aly ic ac i i y and a concomi an dec ease in mala e sensi i i y o his cy oplasmic C4 pho osyn hesis enzyme in he ligh . We ha e ecen ly demons a ed ha he PEPC-PK ac i i y in maize lea es is slowly, bu s ikingly, inc eased in he ligh and dec eased in da kness. In his epo , we p o ide e idence ha cycloheximide, an inhibi o o cy oplasmic p o- ein syn hesis, when ed o de ached lea es o C4 monoco s (maize, so ghum) and dico s (Po ulaca ole acea) in he da k o ligh , comple ely p e en s he in i o ligh ac i a ion o PEPC-PK ac i i y ega dless o whe he he p o ein kinase ac i i y is assessed in i o o in i o. In con as , chlo am- phenicol, an inhibi o o p o ein syn hesis in chlo oplas s, has no e ec on he ligh ac i a ion o maize PEPC-PK. Simila ly, ea men wi h cycloheximide did no in luence he ligh ac i- a ion o o he pho osyn hesis- ela ed enzymes in maize, in- cluding cy oplasmic suc ose-phospha e syn hase and chlo o- plas s omal NADPH-mala e dehyd ogenase and py u a e,P; dikinase. These and ela ed esul s, in which de ached maize lea es we e ea ed simul aneously wi h cycloheximide and mic ocys in-LR, a po en in i o and in i o inhibi o o he PEPC ype 2A p o ein phospha ase, indica e ha sho - e m p o ein u no e o he PEPC-PK i sel o some o he essen ial componen (s) (e.g., a pu a i e p o ein ha modi ies his kinase ac i i y) is one o he p ima y le els in he complex and unique egula o y cascade e ec ing he e e sible ligh ac i a- ion/se yl phospho yla ion o PEPC in he mesophyll cy o- plasm o C4 plan s. Ligh e e sibly ac i a es a numbe o pho osyn hesis- ela ed enzymes in plan s ia se e al di e en mechanisms (1-4). Among hese is he ligh ac i a ion o lea cy oplasmic phosphoenolpy u a e ca boxylase [PEPC; o hophos- pha e:oxaloace a e ca boxy-lyase (phospho yla ing), EC 4.1.1.31] in C4 plan s by e e sible p o ein phospho yla ion (5, 6). P e ious in i o (7, 8) and in i o (9) s udies wi h maize lea PEPC demons a ed ha he phospho yla ion o a single, N- e minal se yl esidue (Se -15) leads o an inc ease in ca aly ic ac i i y and a dec ease in eedback inhibi ion o he a ge enzyme by L-mala e. Rela ed indings om a econ- s i u ed phospho yla ion sys em indica ed ha he ac i i y o he p o ein-se ine kinase ha ca alyzes his egula o y phos- pho yla ion o PEPC is no a ec ed by a numbe o pu a i e, ligh -modula ed cy oplasmic e ec o s (e.g., educed hio e- doxin h, Ca2+, PPj, uc ose 2,6-bisphospha e) and au ophos- pho yla ion (6, 7). Howe e , mo e ecen wo k has es ab- lished ha he phosphoenolpy u a e ca boxylase p o ein- se ine kinase (PEPC-PK) is ac i a ed by ligh and inac i a ed by da kness in i o (10). Mo eo e , his s iking egula o y p ocess appea s independen o SH s a us, Ca2l le els, and a pu a i e, igh -binding PEPC-PK e ec o (10). One o he dis inguishing ea u es o he e e sible ligh ac i a ion o PEPC-PK and i s a ge enzyme, PEPC, in C4 plan s is i s sluggishness in i o; when compa ed o he in i o ac i a ion o pho o egula ed mesophyll chlo oplas s omal enzymes such as py u a e,Pi dikinase (PPDK) and NADPH- mala e dehyd ogenase (MDH) (2, 6), he o me a e bo h ela i ely slow p ocesses, aking up o 1 h , a he han minu es, o comple ion (10-12). To gain mo e insigh in o his di e ence and he speci ic mechanism(s) by which he PEPC-PK ac i i y in i o is slowly, bu s ikingly, inc eased in he ligh and dec eased in da kness (10), de ached maize lea es we e ed wo widely used inhibi o s o p o ein syn- hesis. PEPC-PK ac i i y was subsequen ly assessed ei he in i o [mala e IC50 alues o inhibi ion o he a ge enzyme (11, 12)] o in i o [32p phospho yla ion o pu i ied da k- o m PEPC (7, 10)]. Whe eas chlo amphenicol (CAP), a 70S ibo- some-speci ic inhibi o o chlo oplas ic p o ein syn hesis, had no e ec on he ligh ac i a ion o PEPC-PK, cyclohex- imide (CHX), an inhibi o o cy oplasmic p o ein syn hesis, comple ely blocked he ligh ac i a ion o his p o ein-se ine kinase. In con as , he in i o ac i a ion o se e al o he pho o egula ed cy oplasmic [suc ose-phospha e syn hase (SPS)] and chlo oplas ic (PPDK, MDH) pho osyn hesis- ela ed enzymes was no in luenced by CHX ea men . These esul s indica e ha he syn hesis and deg ada ion o PEPC-PK pe se o some o he essen ial componen (s) a e in ol ed a one o he p ima y le els in he egula o y cascade e ec ing he e e sible ligh ac i a ion/se yl phos- pho yla ion o PEPC in he mesophyll cy oplasm o C4 plan s. MATERIALS AND METHODS Ma e ials. Maize (Zea mays L., c . Golden C oss Ban am) plan s we e g own as desc ibed (7, 10). [y-32P]ATP [speci ic ac i i y, 3000 Ci (111 TBq)/mmol] was pu chased om Ame sham. Da k- o m maize lea PEPC was pu i ied by he p ocedu e desc ibed (7, 8). All biochemical eagen s we e ob ained om Sigma excep o mic ocys in-LR (MC) (Cal- Abb e ia ions: PEPC, phosphoenolpy u a e ca boxylase; PEPC- PK, phosphoenolpy u a e ca boxylase p o ein-se ine kinase; SPS, suc ose-phospha e syn hase; PPDK, py u a e,P1 dikinase; MDH, NADPH-mala e dehyd ogenase; CHX, cycloheximide; CAP, chlo - amphenicol; MC, mic ocys in-LR. §To whom ep in eques s should be add essed a : Uni e si y o Neb aska-Lincoln, Depa men o Biochemis y, 210 BcH, Eas Campus, Lincoln, NE 68583-0718. 2712 The publica ion cos s o his a icle we e de ayed in pa by page cha ge paymen . This a icle mus he e o e be he eby ma ked "ad e isemen " in acco dance wi h 18 U.S.C. §1734 solely o indica e his ac . P oc. Na l. Acad. Sci. USA 88 (1991) 2713 biochem). S ock solu ions o 10 mM CHX and 310 mM CAP we e p epa ed in absolu e e hanol, while 0.5 mM MC was dissol ed in 20% ( ol/ ol) me hanol. Feeding o P o ein Syn hesis and P o ein Phospha ase In- hibi o s. P eillumina ed lea es (-2 g esh w each) om 4- o 6-week-old maize plan s we e excised unde wa e , in- se ed in o 150-ml beake s con aining 100 ml o dis illed wa e (con ol), 5 ILM CHX, 310 ,uM CAP, o 10 nM MC in wa e and main ained a oom empe a u e. When eeding was done in he da k, he beake s we e placed in a da kened ume hood o e nigh . The da k sample was hen p epa ed om hese lea es and he co esponding ligh sample was collec ed a e a 90-min illumina ion o he issue. When eeding was done in he ligh , de ached con ol lea es ha had been p eillumi- na ed o 1.5 h in wa e we e ei he main ained in wa e o ed inhibi o s o 4 h in con inued ligh , ollowed by p ep- a a ion o lea ex ac s. Illumina ion was p o ided by a o ced-ai cooled 300-W, low- empe a u e lamp a an inciden ligh in ensi y o 600-800 /iE m 2 s-' (E, eins ein) (400-700 nm). P epa a ion o Lea Ex ac s. Samples (0.3 g esh w ) om he con ol o inhibi o - ea ed lea ma e ial we e chopped and g ound a 40C in a p echilled mo a con aining washed sand, 2% (w / ol) insoluble poly inylpy olidone, and 1.5 ml o he app op ia e ex ac ion bu e . Bu e A [0.1 M T is HC1, pH 8.0/20% ( ol/ ol) glyce ol/10 mM MgCl2/14 mM 2-me cap oe hanol/1 mM EDTA] was used o p epa- a ion o PEPC and i s p o ein-se ine kinase; bu e B (bu e A plus 2 mM py u a e) was used o PPDK; bu e C (50 mM Mops-NaOH, pH 7.5/15 mM MgCl2/2.5 mM di hio h ei ol/1 mM EDTA/0.1% T i on X-100) was used o SPS; and bu e D (0.1 M T is-HCl, pH 8.0/1 mM EDTA/14 mM 2-me cap- oe hanol) was used o MDH. The c ude lea homogena es we e il e ed h ough an 80-, m nylon ne and cen i uged o 1.5 min a 8700 x g. The supe na an luid was ei he used immedia ely (PPDK, MDH) o a e a 0.2-ml aliquo was apidly desal ed a 4°C on a Sephadex G-25 column (1 x 5 cm) equilib a ed wi h 0.1 M T is-HCI, pH 7.5/10 mM MgCI2/20% ( ol/ ol) glyce ol o PEPC and PEPC-PK o bu e C minus T i on X-100 o SPS. Ac i i y Assays. PEPC ac i i y was de e mined spec o- pho ome ically a 340 nm and 30°C. The assay mix u e (12) con ained, in a o al ol o 1 ml, 50 mM Hepes-KOH (pH 7.3), 2.5 mM phosphoenolpy u a e, 5 mM MgCI2, 1 mM NaHCO3, 0.2 mM NADH, 10 uni s o mala e dehyd ogenase, a ious concen a ions o L-mala e, and 10 ,ul o desal ed ex ac (added las ). Mala e IC50 alues we e aken as he mala e concen a ion equi ed o 50%o inhibi ion o PEPC ac i i y unde hese assay condi ions. PEPC-PK ac i i y was mea- su ed by 32p inco po a ion om [y32P]ATP in o pu i ied da k- o m PEPC (10). The phospho yla ion mix u e con- ained 35 ,ul o desal ed ex ac , 10 ug o pu i ied da k- o m maize PEPC, an adenyla e kinase inhibi o plus a c ea ine kinase/phosphoc ea ine ADP-sca enging sys em (10), 25 ,uM ATP, and 3 ,Ci o [y-32P]ATP in a inal ol o 60 p1. A e 45 min o incuba ion a 30°C, he eac ion was s opped by adding 20 ,ud o SDS sample bu e (0.25 M T is-HCl, pH 6.8/8% SDS/40o glyce ol/20% 2-me cap oe hanol), ol- lowed by immedia e boiling o 2 min. Ve ical SDS/PAGE was pe o med as desc ibed (13, 14), and au o adiog aphs we e p epa ed om he d ied gels wi h Kodak X-Oma AR ilm and wo Ligh ning Plus in ensi ying sc eens (DuPon ) a -800C. SPS, PPDK, and MDH ac i i ies we e measu ed acco ding o e . 15 (a limi ing subs a e concen a ions plus 10 mM P1 a 250C), e . 16 ( o wa d di ec ion plus 2.5 mM glucose 6-phospha e and 2 uni s o pu i ied maize PEPC a 300C), and e . 17 a 30TC, espec i ely. RESULTS AND DISCUSSION E ec s o CHX, CAP, and MC on he Ligh -Induced Changes in Mala e Sensi i i y o Maize Lea PEPC. The IC50 alues o PEPC inhibi ion by L-mala e we e used as an indi ec means o ollowing he e ec o da k o ligh ansi- ions on he appa en in i o ac i i y o he PEPC-PK since hese alues e lec he se yl-phospho yla ion s a us o he a ge enzyme bo h in i o (7, 8) and in i o in esponse o ligh and da k (9-12). Feeding 5 ,uM CHX o de ached p eillumina ed maize lea es in he da k o e nigh comple ely and ep oducibly p e en ed he subsequen ligh -induced inc ease in he mala e IC50 alue o PEPC wi hou ha ing any signi ican e ec on he da k- o m enzyme (Table 1). In con as , CAP ea men had no e ec on he ligh -induced changes in mala e sensi i i y o PEPC (Table 1). O e nigh eeding o 5 AM CHX in he da k o p eda kened maize lea es had he same inhibi o y e ec on ligh ac i a ion o PEPC. Resul s simila o hose p esen ed in Table 1 we e ob ained when de ached lea es o so ghum, ano he C4 g ass, and hal ed lea es o Po ulaca ole acea, a C4 dico , we e ed CHX (da a no shown). Gi en ha such inhibi o s a e known no o be absolu ely speci ic, hus possibly causing de imen al side e ec s (18), and ha he 3-(3 ,4-dichlo ophenyl)-1, 1-dime hylu ea- sensi i e ligh ac i a ion/phospho yla ion o PEPC occu s in he cy oplasm and is somehow ela ed o pho osyn he ic elec on anspo and/o pho ophospho yla ion (5, 6, 19), i was impe a i e o examine he e ec o CHX ea men on he in i o ligh ac i a ion o o he pho osyn hesis- ela ed enzymes in maize. Cy oplasmic SPS and chlo oplas s omal PPDK a e, like PEPC, ligh -ac i a ed by e e sible phos- pho yla ion/dephospho yla ion cycles (2, 3, 6, 15, 20). In con as , s omal MDH is ligh ac i a ed by 3-(3,4- dichlo ophenyl)-1,1-dime hylu ea-sensi i e changes in i s SH edox s a us media ed by noncyclic elec on low and he chlo oplas ic e edoxin/ hio edoxin m sys em (1, 2, 17, 21). No ably, he esul s (Table 2) indica e ha he ligh ac i a ion o hese h ee enzymes was no signi ican ly a ec ed by eeding 5 A&M CHX o de ached maize lea es unde condi- ions iden ical o hose desc ibed in Table 1. Simila ly, CHX ea men o de ached lea es had no ob ious e ec on ei he hei o al soluble p o ein con en (mg/g esh w ) o poly- pep ide pa e n (e.g., see Fig. lA, lane 2 e sus 3 and lane 5 e sus 6) o e he du a ion o hese ela i ely sho - e m expe imen s. Thus, he inhibi o y e ec o CHX on he appa en in i o ac i i y o he PEPC-PK (Table 1) appea s a he selec i e o he ligh ac i a ion o his speci ic con- e e enzyme. F om he esul s desc ibed abo e, i is clea ha de no o syn hesis o PEPC-PK o some o he essen ial componen (s) (e.g., a pu a i e modi ying p o ein ha ac i a es his p o ein- se ine kinase in i o) is induced du ing a 1.5-h exposu e o ligh . Thus, i was an icipa ed ha i CHX we e ed o illumina ed de ached lea issue a e a poin a which su i- cien p o ein (i.e., ei he PEPC-PK o he pu a i e modi ying Table 1. E ec s o p o ein syn hesis inhibi o s on ligh -induced inc ease in he mala e IC50 alue o maize lea PEPC Mala e IC50, mM Inhibi o Ligh (L) Da k (D) L/D None (con ol) 0.45 0.18 2.5 CHX 0.20 0.17 1.2 CAP 0.46 0.18 2.6 P eillumina ed maize lea es we e excised and ed wa e (con ol), 5 gM CHX, o 310 jLM CAP in he da k o e nigh . Da k- o m PEPC ex ac s we e hen p epa ed om hese lea es and he co esponding ligh - o m ex ac s we e made a e a 90-min illumina ion o he issue. Bo any: Jiao e al. P oc. Na l. Acad. Sci. USA 88 (1991) Table 2. E ec s o CHX ea men on in i o ligh ac i a ion o SPS, PPDK, and MDH in de ached maize lea es Ligh /da k ac i i y a io Enzyme Con ol* + 5 IAM CHX SWP 6.5 5.5 PPDK 7.0 6.9 MDH 11.7 12.7 See Table 1 and Ma e ials and Me hods o expe imen al de ails. *The ligh -ac i a ed ac i i ies (in ,&mol pe min pe mg o soluble p o ein) o SPS, PPDK, and MDH we e 0.14, 0.43, and 0.16, espec i ely. Ac i i y was de e mined a limi ing subs a e concen a ions in he p esence o he inhibi o Pi (15). p o ein) had al eady been syn hesized in he ligh so ha PEPC was phospho yla ed and in i s high mala e IC50 o m, con inued illumina ion o he inhibi o - ed issue would main- ain he a ge enzyme in i s high ac i a ion (phospho yla ion) s a e. This easoning assumes ha he ac i i y o he PEPC ype 2A p o ein phospha ase (22) is ela i ely low in he ligh and/o ha apid deg ada ion o he newly syn hesized PEPC-PK o pu a i e modi ying p o ein does no ake place in he ligh . Howe e , when p eillumina ed (1.5 h in he ligh ) con ol lea issue was placed in wa e o 5 ,M CHX and main ained in he ligh o an addi ional 4 h , he mala e IC50 alue emained cons an in he absence o he inhibi o bu dec eased o a le el cha ac e is ic o da k- o m PEPC in he p esence o CHX (Table 3). These da a sugges ha (i) he PEPC ype 2A p o ein phospha ase is ac i e in he ligh , and (ii) he e is ne u no e o he PEPC-PK o pu a i e modi- ying p o ein in he ligh in he absence o i s syn hesis, he eby leading o a ne dephospho yla ion o PEPC and he esul ing dec ease in i s mala e IC50 alue. Based on hese obse a ions and sugges ions, a mo e c i ical and e ealing expe imen was designed. Con ol de ached lea es ha had been p eillumina ed o 1.5 h we e ed 5 AsM CHX and 10 nM MC, alone and in combina ion, o 4 h in he ligh . This la e cyclic hep apep ide is a po en and speci ic inhibi o o plan and animal ype 1 and 2A p o ein phospha ases bo h in i o and in i o (23-25). In he absence o he p o ein syn hesis inhibi o , bo h he con ol and MC- ea ed illumina ed issue main ained PEPC in i s high mala e IC50 o m (Table 3). In con as , in he p esence o CHX alone, he ac i a ion (phospho yla ion) s a e o he a ge enzyme collapsed back o a mala e IC50 alue cha ac e is ic o he da k- o m enzyme (see abo e and Table 3). Howe e , in he p esence o bo h inhibi o s, he CHX-induced dec ease in he mala e IC50 alue was la gely p e en ed, clea ly indica ing ha he p o- ein phospha ase ha dephospho yla es ligh - o m PEPC in i o emains o ally ac i e in he ligh and is o he ype 1 o ype 2A class (23-25), as p e iously implica ed by in i o s udies (22). Thus, he egula o y phospho yla ion s a us o PEPC is mainly de e mined by i s ligh -ac i a ed p o ein- se ine kinase (10), he la e o which appea s o be ligh /da k Table 3. E ec s o p o ein syn hesis and p o ein phospha ase inhibi o s on he main enance o he high mala e IC50 o m o PEPC in he ligh Inhibi o (s) Mala e IC50, mM None (con ol) 0.46 CHX 0.17* MC 0.42 CHX + MC 0.35 De ached con ol lea es, p eillumina ed o 1.5 h in wa e , we e A 1 2 3 4 5 6 7 8 9 modula ed by he ela i e a es o i s syn hesis and deg ada- ion in he cy oplasm o bo h C4 monoco s (maize, so ghum) and dico s (P. ole acea). E ec s o P o ein Syn hesis Inhibi o s on PEPC-PK Ac i i y. One ques ion a ising om he mala e-sensi i i y expe imen s desc ibed abo e (Tables 1 and 3) is whe he CHX inhibi s he appa en in i o PEPC-PK ac i i y by di ec in e ac ion wi h his con e e enzyme, a pu a i e modi ying p o ein, o PEPC pe se. To add ess his and o he issues, in i o 32P phospho yla ion assays o PEPC-PK ac i i y we e pe o med wi h apidly p epa ed, desal ed lea ex ac s and pu i ied da k- o m PEPC as he p o ein subs a e (10). Fig. 1 shows he e ec o CHX and CAP on ligh ac i a ion o PEPC-PK ac i i y when he inhibi o s we e ed o p eillumina ed de- ached maize lea es in da kness o e nigh . While he ac i i y o he PEPC-PK is low in da kness and high in he ligh o he con ol (10) and CAP- ea ed issue (c . lane 2 e sus 5 and lane 4 e sus 7, espec i ely, in Fig. 1B), eeding 5 juM CHX comple ely p e en ed his s iking ligh ac i a ion o he p o ein-se ine kinase (c . lane 3 e sus 6 in Fig 1B). When ei he CHX (5 gM) o CAP (310 LM) was added di ec ly o he phospho yla ion assay mix u e con aining he desal ed ex ac om he illumina ed con ol lea issue, no in i o inhibi ion o PEPC-PK ac i i y was obse ed (c . lanes 5, 8, and 9 in Fig. 1B). Simila ly, when de ached con ol lea es, p eillumina ed o 1.5 h in wa e , we e ed CHX (5 juM) o an addi ional 4 h in he ligh , PEPC-PK ac i i y was o ally inhibi ed (c . lanes 2 and 4 e sus lane 3 in Fig. 2B). The p esence o bo h CHX and MC also led o a comple e inhibi ion o PEPC-PK ac i i y (Fig. 2B, lane 5), e en hough he mala e IC50 alue o he endogenous PEPC was ela i ely PEPC=_. _ - PPDK B 1 2 3 4 5 6 7 8 9 PEPC- FIG. 1. Ligh ac i a ion o PEPC-PK ac i i y and he e ec s o p o ein syn hesis inhibi o s in i o and in i o. P eillumina ed maize lea es we e de ached and ed wa e , 5 ,uM CHX, o 310 ,uM CAP in he da k o e nigh . The da k- o m PEPC-PK was ex ac ed be o e illumina ion o hese lea es and he co esponding ligh - o m p o ein kinase was ex ac ed a e a 90-min illumina ion (c . Table 1). PEPC-PK ac i i y in desal ed lea ex ac s was de e mined by 32P phospho yla ion o pu i ied da k- o m PEPC as desc ibed in Ma e- ials and Me hods and in e . 10. (A) SDS gel s ained wi h Coomassie blue R-250. (B) Co esponding au o adiog aph o A. Desal ed ex- ac s we e p epa ed om da kened (lanes 2-4) and illumina ed (lanes 5-9) lea issue ed wa e (lanes 2, 5, 8, and 9), 5 ,.M CHX (lanes 3 and 6), o 310 ,uM CAP (lanes 4 and 7). Lanes 8 and 9, expe imen s in which 5 ,uM CHX and 310 ,uM CAP, espec i ely, we e added di ec ly o he in i o assay mix u e o PEPC-PK ac i i y ex ac ed om illumina ed con ol lea issue. Lane 1, pu i ied da k- o m PEPC. The 43-kDa c ea ine kinase monome [* (see e . 10)] and he 95-kDa PPDK and 110-kDa PEPC subuni s a e indica ed in A. A ows in B poin o o he ligh -enhanced bu CHX-insensi i e phosphop o eins. subsequen ly main ained in wa e (con ol) o edS ,uM CHX, 10 nM MC, o bo h o an addi ional 4 h in he ligh be o e ex ac ion and assay o PEPC ac i i y wi h o wi hou L-mala e. *Da k- o m PEPC has a mala e IC50 alue o -0.2 mM (see Table 1). 2714 Bo any: Jiao e al. P oc. Na l. Acad. Sci. USA 88 (1991) 2715 A 1 2 3 4 5 PEPC-u* -*i a" PPDK- B 2 3 4 5 Al ; I'liL F ..V FIG. 2. E ec s o CHX and MC on PEPC-PK ac i i y when ed o .4 h in he ligh o de ached con ol lea es ha had been p eillumina ed o 1.5 h in wa e be o e ea men (c . Table 3). PEPC-PK was assayed by 32p phospho yla ion o pu i ied da k- o m PEPC as desc ibed in Ma e ials and Me hods and in e . 10. (A) SDS gel s ained wi h Coomassie blue R-250. (B) Co esponding au o a- diog aph o A. Desal ed ex ac s we e p epa ed om p eillumina ed con ol lea issue ha was main ained in wa e (lanes 2) o ed 5 JLM CHX (lanes 3), 10 nM MC (lanes 4), o 5 IAM CHX plus 10 nM MC (lanes 5) o an addi ional 4 h in he ligh . Lane 1, pu i ied da k- o m PEPC. The c ea ine kinase (*), PPDK, and PEPC monome s a e indica ed in A. high unde hese condi ions (Table 3). These collec i e ind- ings u he suppo he iew ha he PEPC ype 2A p o ein phospha ase(s) (22) emains ully ac i e in he ligh , e en in he p esence o CHX, and is e ec i ely inhibi ed by nano- mola concen a ions o MC in i o. Excep in he p esence o bo h CHX and MC, whene e he in i o PEPC-PK ac i i y was low, so was he mala e IC50 alue o he endogenous PEPC om he co esponding lea issue and ice e sa (c . Table 1 e sus Fig. 1B and Table 3 e sus Fig. 2B). I is no able ha while he in i o ligh ac i a ion o PEPC-PK ac i i y is comple ely inhibi ed by eeding CHX o de ached lea es (Fig. 1B), o he soluble lea p o eins ha a e phospho yla ed in i o by endogenous p o ein kinases a e no a ec ed by such ea men ; his includes e en hose polypep ides whose in i o phospho yla ion s a us is g ea e in he ligh han in he da k ex ac s (see a ows in Fig. 1B). The e o e, i is e iden om he p esen s udy ha he inhibi o y e ec o his cy oplasmic p o ein syn hesis inhib- i o is qui e speci ic o he PEPC-PK o some o he essen ial componen (s) (e.g., i s pu a i e modi ying p o ein), wi hou ha ing signi ican e ec s on o he p o ein kinase and/o p o ein phospha ase ac i i ies (Fig. 1B) and ligh -ac i a ion sys ems (Table 2). CONCLUDING REMARKS The p esen s udy demons a es ha he in i o ligh /da k egula ion o PEPC-PK ac i i y in C4 lea es (10) in ol es ne de no o cy oplasmic p o ein syn hesis in he ligh and sub- sequen deg ada ion in da kness. A p esen , i is no known wha essen ial componen (s)-e.g., PEPC-PK i sel o a pu a i e modi ying p o ein-is he a ge o his unique ligh -modula ion sys em. The inc easingly complex PEPC egula o y cascade, in ol ing a leas p o ein syn hesis/ deg ada ion and phospho yla ion/dephospho yla ion cycles, is o ally di e en om o he well-known mechanisms o e e sible ligh ac i a ion o pho osyn hesis- ela ed enzymes (1-4, 6, 15) and may well explain why he ligh /da k modu- la ion o PEPC-PK (10) and i s a ge enzyme PEPC (11, 12, 19) is so much slowe han ha o o he pho oac i a ed enzymes (e.g., PPDK, MDH). I is ob ious ha u he pu i ica ion o he PEPC-PK (7) and he subsequen p oduc- ion o monospeci ic an ibodies agains his p o ein will elucida e whe he , indeed, his speci ic enzyme is he a ge o his unique, p o ein u no e -based egula o y sys em. In addi ion, ou indings aise se e al in e es ing ques ions as o how pho osyn hesis- ela ed (5, 6, 19) ligh and da k signals so speci ically in luence cy oplasmic p o ein syn he- sis and deg ada ion, espec i ely, and whe he sho - e m p o ein u no e is, like o he pos ansla ional co alen modi ica ions, a gene al mechanism o egula ing enzyme ac i i y in plan s in esponse o ex e nal and in e nal s imuli. Clea ly, ou esul s indica e ha his is p obably no he case wi h espec o he ligh -ac i a ion sys ems associa ed wi h o he pho osyn hesis- ela ed cy oplasmic and s omal en- zymes. We hank Ms. Shi ley Condon o he excellen echnical assis - ance. 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