Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different

Thio edoxin-linked p ocesses in cyanobac e ia a e as nume ous as in chlo oplas s, bu a ge s a e di e en Ma ika Lindahl* and F ancisco J. Flo encio Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Consejo Supe io de In es igaciones Cien ı´ icas, Uni e sidad de Se illa, Cen o de In es igaciones Cien ı´ icas Isla de la Ca uja, A enida Ame ico Vespucio s兾n, E-410 92 Se illa, Spain Edi ed by Bob B. Buchanan, Uni e si y o Cali o nia, Be keley, CA, and app o ed Oc obe 10, 2003 ( ecei ed o e iew July 15, 2003) Ligh -dependen egula ion o a g owing numbe o chlo oplas enzyma ic ac i i ies has been ound o occu h ough he e e sible educ ion o in a- o in e molecula disulphides by hio edoxins. In cyanobac e ia, despi e hei simila i y o chlo oplas s, no p o eins ha e hi he o been shown o in e ac wi h hio edoxins, and he ole o he cyanobac e ial e edoxin兾 hio edoxin sys em has e- mained obscu e. By using an immobilized cys eine 35- o-se ine si e-di ec ed mu an o he Synechocys is sp. PCC 6803 hio edoxin T xA as bai , we sc eened he Synechocys is cy osolic and pe iph- e al memb ane p o ein complemen s o p o eins in e ac ing wi h T xA. The co alen bond be ween he isola ed a ge p o eins and mu a ed T xA was con i med by non educing兾 educing wo- dimensional SDS兾PAGE. Thus, we ha e iden i ied 18 cy osolic p o eins and 8 memb ane-associa ed p o eins as candida e hio e- doxin subs a es. Twen y o hese p o eins ha e no p e iously been associa ed wi h hio edoxin-media ed egula ion. Phospho- glucomu ase, one o he p e iously uncha ac e ized hio edoxin- linked enzymes, has no ea lie been conside ed a a ge o me abolic con ol h ough disulphide educ ion. In his a icle, we show ha phosphoglucomu ase is inhibi ed unde oxidizing con- di ions and ac i a ed by DTT and educed wild- ype T xA in i o. The esul s imply ha hio edoxin-media ed edox egula ion is as ex ensi e in cyanobac e ia as in chlo oplas s bu ha he subjec s o egula ion a e la gely di e en . Di hiol兾disulphide exchange ca alyzed by hio edoxin (T x) o ms he molecula basis o ligh -dependen egula ion o many enzymes in he chlo oplas s o highe plan s and algae (1–3). Fe edoxin ecei es educing equi alen s om he pho- osyn he ic elec on anspo in he ligh and educes T x mand by means o e edoxin-T x educ ase. The T xs, in u n, con e disulphides o di hiols in hei espec i e a ge enzymes, he eby modula ing hei ac i i ies. The ea lies disco e ed a ge s o T x-media ed egula ion belong o he Cal in cycle o CO 2 assimila ion (1). Since he ini ial disco e ies, se e al mo e chlo oplas enzymes ha e been ecognized as subs a es o T x (3). A b eak h ough in he in es iga ion o chlo oplas edox egula ion came wi h he in oduc ion o a new me hod o isola e T x a ge p o eins (4–6). The me hod in ol es mu a ion o he bu ied edox-ac i e cys eine o T x, which a o s he o ma ion o s able mixed disulphides wi h i s a ge p o eins. This expe - imen al app oach con i med he in e ac ion be ween T x and i s known a ge s and mo e han doubled he numbe o po en ial T x- egula ed p o eins (6). Cyanobac e ia a e oxygenic pho osyn he ic p oka yo es ha p obably sha e a common ances o wi h he chlo oplas . The comple e sequence o he cyanobac e ium Synechocys is sp. PCC 6803 genome (Cyanobase, www.kazusa.o .jp兾cyano兾cyano. h ml) e eals ha his o ganism, he ea e e e ed o as Syn- echocys is, con ains e edoxin-T x educ ase and a leas ou di e en T xs. Ne e heless, a emp s o demons a e ligh - dependen edox egula ion o h ee cyanobac e ial enzymes o he Cal in cycle (phospho ibulokinase, uc ose-1,6-bisphos- pha ase, and glyce aldehyde-3-phospha e dehyd ogenase) we e unsuccess ul (7), al hough pu i ied phospho ibulokinase om Synechococcus sp. PCC 7942 could be ac i a ed by DTT in i o (8). The s a k con as be ween he g owing numbe o chlo o- plas T x a ge s and he lack o da a indica ing T x unc ion in cyanobac e ia migh lead o he conclusion ha ligh -induced T x-media ed egula ion e ol ed wi h he need o coo dina e chlo oplas ic and ex achlo oplas ic me abolism in pho osyn- he ic euka yo es. Su p isingly, Synechocys is T xA was ound essen ial o su i al unde pho oau o ophic as well as he e o- ophic g ow h condi ions (9). Among he di e se Synechocys is T xs, T xA is he one ha mos esembles he chlo oplas m- ype T x. Aiming a cla i ying he oles o T xA in Synechocys is, we ha e sc eened he Synechocys is cy osolic and pe iphe al memb ane p o ein complemen s o p o eins in e ac ing wi h T xA. To his end, we used a s a egy simila o ha desc ibed in e s. 5 and 6. Thus, we ha e iden i ied 18 cy osolic p o eins and 8 memb ane- associa ed p o eins as candida e subs a es o T xA. One o hese a ge s, phosphoglucomu ase (PGM), which ep esen s a me abolic b anch poin be ween s o age and u iliza ion o ca bohyd a es, was shown o be ac i a ed by T xA in i o. Ma e ials and Me hods Ma e ials. DEAE Sephacel ma ix, His-Bind esin, and Supe dex 75 we e pu chased om Sigma, No agen, and Ame sham Pha - macia Biosciences, espec i ely. DTT, T i on X-100, and NAD ⫹ we e om Sigma. O he chemicals we e o he highes g ade comme cially a ailable. Mu agenesis, Exp ession, and Pu i ica ion o T xA. The xA gene (ORF sl 0623, Cyanobase) was mu a ed, exchanging cys eine 35 o a se ine. The mu a ed T xA (T xA35) was exp essed wi h a C- e minal His- ag by using he pET22b ec o in Esche ichia coli BL21 DE3 and pu i ied o homogenei y by anion-exchange ch oma og aphy on DEAE Sephacel ma ix, Ni-a ini y ch o- ma og aphy wi h he His-Bind esin, and gel il a ion on Su- pe dex 75. The gel- il a ion s ep was p eceded by a 1-h incu- ba ion wi h 20 mM DTT on ice. Pu e T xA35 was elu ed in 25 mM T is䡠HCl (pH 7.9) and 150 mM KCl. P epa a ion o Cy osolic and Pe iphe al Memb ane P o ein F ac ions. Synechocys is cul u es we e g own pho oau o ophically a 30°C in BG11 medium (10) including 1 g兾l NaHCO 3 and bubbled wi h 1% ( ol兾 ol) CO 2 in ai unde con inuous illumina ion wi h whi e ligh a an in ensi y o 50 ␮ mol pho ons䡠m ⫺2 䡠s ⫺1 . Cells we e ha es ed and b oken wi h glass beads as desc ibed in e . 11 wi h bu e A (25 mM Hepes䡠NaOH, pH 7.0兾15 mM CaCl 2 兾5mM MgCl 2 兾15% ( ol/ ol) glyce ol兾1 mM PMSF). A e emo al o unb oken cells by cen i uga ion a 2,300 ⫻g o 5 min, o al memb anes we e pelle ed by cen i uga ion a 16,000 ⫻g o 20 min, yielding a supe na an con aining cy osolic p o eins. Mem- This pape was submi ed di ec ly (T ack II) o he PNAS o ice. Abb e ia ions: T x, hio edoxin; GS, glu amine syn he ase; GOGAT, glu ama e syn hase; Rubisco, ibulose 1,5-bisphospha e ca boxylase兾oxygenase; RbcL, la ge subuni o Rubisco; PGM, phosphoglucomu ase; P x, pe oxi edoxin. *To whom co espondence should be add essed. E-mail: [email p o ec ed]. © 2003 by The Na ional Academy o Sciences o he USA www.pnas.o g兾cgi兾doi兾10.1073兾pnas.2534397100 PNAS 兩 Decembe 23, 2003 兩 ol. 100 兩 no. 26 兩 16107–16112 PLANT BIOLOGY b anes we e washed h ee imes by esuspension in bu e A and cen i uga ion. Pe iphe al memb ane p o eins we e ex ac ed by 15-min incuba ion on ice wi h bu e A supplemen ed wi h 1 M NaCl and 0.02% ( ol兾 ol) T i on X-100 ollowed by cen i uga- ion a 16,000 ⫻g o 30 min. The supe na an was concen a ed, and NaCl and T i on X-100 we e elimina ed by cen i uga ion il a ion in Ul a ee-MC (Millipo e), 3-kDa cu -o , and suc- cessi e eplacemen wi h bu e A. P o ein concen a ions we e measu ed as desc ibed (12). Isola ion o T xA Ta ge P o eins. We added 1.2 mg o pu e T xA35 o 150 ␮ l o His-Bind esin in a 1.5-ml mic ocen i uge ube and incuba ed i a 4°C unde gen le agi a ion o 1 h. The esin was washed wice wi h 1 ml o bu e A a e emo al o he supe na an . Then 2.8 mg o cy osolic p o ein o 1.8 mg o pe iphe al memb ane p o ein was added ollowed by a 16-h incuba ion a 4°C unde gen le agi a ion. Unbound p o eins we e emo ed, and he esin was washed ou imes in 1 ml o bu e B (20 mM T is䡠HCl, pH 7.9兾0.5 M NaCl) wi h he addi ion o 60 mM imidazole. T xA35– a ge p o ein complexes we e eleased by addi ion o 120 ␮ l o bu e B including 1 M imidazole, and he elua es we e eco e ed by cen i uga ion o 10 sec a 2,000 ⫻g. Non educing兾Reducing Gel Elec opho esis. Elua es we e elec o- pho esed unde non educing condi ions in he i s dimension, main aining he T xA35– a ge p o ein complexes in ac , and educing condi ions in he second dimension, eleasing T xA35 om i s a ge s. Se en y mic oli e s o elua e was mixed wi h 15 ␮ l o See Blue p es ained p o ein s anda d (In i ogen) and 30 ␮ l o solubilizing bu e (0.25 M T is䡠HCl, pH 6.8兾12.5% ( ol/ ol) glyce ol兾10% (w / ol) SDS). A e sepa a ion by SDS兾 PAGE on a 16 ⫻18 cm, 1.5-mm hick gel, he lane was excised and incuba ed o 1ha 25°C in 2 ml o solubilizing bu e dilu ed 2.5 imes wi h he addi ion o DTT o 100 mM inal concen a- ion. The lane was ixed on op o an ac ylamide gel o iden ical size and, a e sepa a ion a 40 mA, he gel was s ained wi h Coomassie b illian blue R-250. Ac ylamide gels o 12% and 10% (w 兾 ol) we e used o cy osolic and pe iphe al memb ane p o eins, espec i ely. Appa en molecula masses we e de e - mined by using one-dimensional gels, educing condi ions, and nons ained p o ein ma ke s (Bio-Rad) ollowed by Coomassie s aining o maximal accu acy. P o ein Iden i ica ion. Coomassie-s ained p o eins we e excised and diges ed wi h ypsin. Pep ides we e subjec ed o ma ix- assis ed lase deso p ion ioniza ion– ime-o - ligh (MALDI- TOF) analysis pe o med on an Ul a lex appa a us (B uke , Bille ica, MA). P o eins we e iden i ied by using he MASCOT pep ide mass inge p in sea ch engine (Ma ix Science, London). Wes e n Blo . Fo glu amine syn he ase (GS), he an ibody (13) was used a 1:5,000 dilu ion. Fo NADH-glu ama e syn hase (GOGAT), an an ibody aised agains he la ge subuni , Gl B (F. Na a o and F.J.F., unpublished da a), was used a 1:2,000 dilu ion. Signals we e isualized wi h enhanced chemilumines- cence Wes e n blo ing de ec ion eagen s om Ame sham Pha macia Biosciences. Measu emen o PGM Ac i i y. The ac i i y was measu ed by coupling o educ ion o NAD ⫹ ca alyzed by glucose-6- phospha e dehyd ogenase (G6PD) om Leuconos oc mesen- e oides (Sigma). G6PD om L. mesen e oides is de oid o cys eines, and hiol eagen s do no a ec i s ac i i y. Each assay o 1 ml inal olume con ained 50 mM T is䡠HCl (pH 7.6), 4 mM MgCl 2 , 2 mM NAD ⫹ ,40 ␮ M ␣ -D-glucose-1,6-diphospha e, 1.4 uni s G6PD, and 23 ␮ go Synechocys is cy osolic p o eins. The eac ion was s a ed by addi ion o 4 mM ␣ -D-glucose-1- phospha e, and he abso bance a 340 nm was moni o ed. Incuba ions o cy osolic ex ac be o e measu emen we e pe - o med in 25 mM T is䡠HCl (pH 7.6) and 2 mM MgCl 2 a 25°C and a a p o ein concen a ion o 4 mg兾ml wi h addi ions as speci ied. Wild- ype T xA was p epa ed as in e . 14. Resul s and Discussion Isola ion o P o eins Bound o T xA35. Sc eening o he Synecho- cys is ex ac s o p o eins in e ac ing wi h he T xA Cys-35-Se mu an , T xA35, yielded cy osolic a ge p o eins (Fig. 1A)as well as pe iphe al memb ane a ge p o eins (Fig. 1B). The co alen bond be ween hese p o eins and T xA35 was con- i med by non educing兾 educing wo-dimensional SDS兾PAGE (Fig. 1C). The majo i y o he cap u ed Synechocys is p o eins mig a e unde non educing condi ions wi h an appa en molec- ula mass ha is ⬇15 kDa la ge han he appa en molecula mass unde educing condi ions (Fig. 1C). This di e ence in mass co esponds o one co alen ly bound T xA35 molecule, which is eleased a e educ ion. In some cases, he di e ence in mig a ion be ween he i s and second dimension is so g ea ha i implies mo e han one T xA35 molecule joined o a a ge and兾o an oligome ic s uc u e o he a ge p o ein media ed by in e molecula disulphides. T xA35 con ains only one cys eine, hus excluding he possibili y o T x ime s. A la ge amoun o T xA35 o ms homodime s du ing he binding assay and mi- g a es wi h coo dina es 33 kDa兾15 kDa, and some T xA35 emains a monome and mig a es a he diagonal (Fig. 1C). The diagonal is p ac ically de oid o Synechocys is p o eins. Iden i ica ion o Synechocys is T xA-Linked P o eins. Twen y-six o he esol ed p o eins we e posi i ely iden i ied wi h hei co esponding ORFs in he Synechocys is genome. The 18 cy osolic p o eins a e lis ed in Table 1, and he 8 pe iphe al memb ane p o eins a e lis ed in Table 2. One o he a ge s, he la ge subuni o ibulose 1,5-bisphospha e ca boxylase兾 oxygenase (Rubisco) (RbcL), was ound bo h in he cy osolic and he pe iphe al memb ane p o ein ac ions. The as majo i y o he a ge p o eins, 20 o 25, ha e no p e iously been associa ed wi h T x-media ed egula ion. Only wo o he Synechocys is a ge s, ansla ion elonga ion ac o s EF-G and EF-Tu (Table 1), ha e been epo ed ea lie o in e ac wi h chlo oplas T xs (6). I is s iking ha he e a e no o he common p o eins be ween he 35 chlo oplas T x a ge s iden i ied by Balme e al. (6) and he 25 Synechocys is T x a ge s epo ed in his s udy. The implica ions o ou indings in ela ion o he cu en knowledge abou he a ge enzymes and he me abolic p ocesses in which hey pa icipa e a e discussed below. Ca bon dioxide ixa ion. Cy osolic (Table 1) and memb ane- associa ed (Table 2) o ms o RbcL we e ound o be a ge s o T x. In bo h cases, RbcL mig a ed wi h much oo high appa en molecula mass unde non educing condi ions o be compa ible wi h an RbcL-T xA35 he e odime (Fig. 1C). Cys eine 247 o spinach RbcL, which co esponds o Synechocys is Cys-242, is known o media e dime o ma ion be ween neighbo ing RbcL subuni s (15). Nex o he ac i e si e o RbcL a e wo o he conse ed cys eines whose edox s a e a ec s enzyme ac i i y in Synechocys is (16). A bond be ween T xA35 and one o hese cys eines in addi ion o a disulphide be ween wo Cys-242 esidues would yield an RbcL 2 -T xA35 2 he e o e ame . Such a polypep ide wi h a complex b anched s uc u e could be mo e e a ded on SDS兾PAGE han would be expec ed om he sum o he molecula masses o he indi idual subuni s. The e is as ye no e idence o T x-media ed egula ion o RbcL in any o ganism, bu he small subuni o Rubisco in chlo oplas s has been iden i ied as a T x- a ge (5, 6). CcmM (Table 2) is a ca bon dioxide-concen a ing mechanism p o ein, which is equi ed o 16108 兩 www.pnas.o g兾cgi兾doi兾10.1073兾pnas.2534397100 Lindahl and Flo encio g ow h a ai le els o CO 2 and is p obably a cons i uen o he ca boxysome (17). We iden i ied CcmM as a p o ein mig a ing on SDS兾PAGE wi h an appa en molecula mass o 54 kDa (Fig. 1B), which is in ag eemen wi h Ogawa e al. (17) bu a a iance wi h Cyanobase, whe e i is epo ed as a 73-kDa p o ein. No s uc u al ca boxysomal p o eins ha e so a been epo ed o Fig. 1. (Aand B) Isola ion o Synechocys is T x a ge p o eins wi h he His- agged si e-di ec ed T x mu an T xA35 immobilized on a Ni-a ini y ma ix. To al p o ein be o e binding (TP), unbound p o eins (UB), and p o eins washed o wi h 60 mM imidazole (W) we e sepa a ed on 12% ac ylamide gels and s ained wi h Coomassie b illian blue. Six mic oli e s om each ac ion o cy osolic p o eins and 10 ␮ l om ac ions o pe iphe al memb ane p o eins we e applied on he gels, co esponding o 45 and 30 ␮ g o ini ial o al p o ein (TP), espec i ely. Se en y mic oli e s each o cy osolic p o eins (E1) and pe iphe al memb ane p o eins (E2) bound o T xA35 and elu ed wi h 1 M imidazole we e sepa a ed on 12% ac ylamide gels unde educing condi ions. The iden i ies o he mos abundan a ge s a e indica ed. (C) Sepa a ion o T x a ge p o eins by non educing兾 educing wo-dimensional SDS兾PAGE. Elu ed p o eins E1 and E2 we e sepa a ed on 12% and 10% ac ylamide gels, espec i ely. Be o e he second dimension, p o eins we e educed wi h 100 mM DTT. The molecula masses o he p es ained s anda d p o eins a e indica ed by numbe s. Table 1. Synechocys is cy osolic T xA-linked p o eins ORF Gene p oduc Appa en molecula mass, kDa Theo e ical molecula mass, kDa Numbe o cys eines conse ed* sll1499 Fe edoxin-GOGAT (GlsF) 200 169.4 16 (15) sll1502 NADH-GOGAT (Gl B) 186 169.0 10 (10) sl 0557 Valyl- RNA syn he ase (ValS) 105 102.7 5 (3) sl 1463 Elonga ion ac o EF-G 90 76.7 2 (2) sll0158 Glucan b anching enzyme (GlgB) 84 89.5 6 (0) sl 2076 60-kDa chape onin G oEL 63 57.5 1 (1) sll0726 Phosphoglucomu ase 58 61.1 1 (0) sl 0009 Rubisco la ge subuni (RbcL) 57 52.5 9 (8) sll1393 Glycogen syn hase (Glg2) 55 56.2 10 (4) sl 1176 ADPglucose py ophospho ylase 54 49.3 4 (3) sl 0585 A gininosuccina e syn he ase 53 44.5 2 (1) sll1099 Elonga ion ac o EF-Tu 51 43.7 1 (1) sll1212 GDP-mannose dehyd a ase 45 41.3 2 sll1994 Po phobilinogen syn hase 42 36.1 4 (0) sll0576 Suga -nucleo ide epime ase 35 34.9 1 (1) sl 1198 1-Cys pe oxi edoxin 29 23.5 1 sll1621 YLR109-homologue 25 21.2 2 (2) ss 3383 Phycobilisome linke (L c ) 13 7.8 1 *To al numbe o conse ed cys eines in homologues om any o ganism and hen, in pa en heses, he numbe o cys eines conse ed in plan chlo oplas homologues. Lindahl and Flo encio PNAS 兩 Decembe 23, 2003 兩 ol. 100 兩 no. 26 兩 16109 PLANT BIOLOGY in e ac wi h T xs, bu a chlo oplas ca bonic anhyd ase was ecen ly disco e ed as a a ge o T x (6). PGM. The in e con e sion o glucose-1-phospha e and glucose- 6-phospha e ca alyzed by PGM (Table 1) b idges glycolysis and glycogen me abolism. Al hough PGM is ecognized o play a key ole in suga me abolism, i s ac i i y has no been conside ed a a ge o me abolic con ol. PGM ac i i y in in ac chlo oplas s was in e es ingly ound o be up- egula ed in he ligh , bu p eincuba ion wi h 10 mM DTT in he da k had no e ec on his ac i i y (18). To es he possibili y o edox egula ion o PGM in Synechocys is, we examined he e ec s o DTT and T xA on PGM ac i i y. Incuba ion o cy osol wi h 5 mM DTT o 45 o 90 min be o e measu emen led o a subs an ial inc ease in PGM ac i i y (Fig. 2). In con as , con ol incuba ion wi hou addi ions esul ed in loss o ac i i y. Fu he loss was obse ed when p eincuba ion was pe o med in he p esence o a low concen- a ion o CuCl 2 , which is known o induce he o ma ion o disulphide bonds (5). Addi ion o DTT a low (0.2 mM) con- cen a ion main ained he ac i i y cons an h oughou he incuba ion and, inally, addi ion o 2.5 ␮ M pu i ied Synechocys is wild- ype T xA in he p esence o 0.2 mM DTT caused a signi ican inc ease in PGM ac i i y (Fig. 2). The ac ha 0.2 mM DTT alone p e en ed loss o ac i i y may be a ibu able o he p esence o endogenous T xs in he cy osolic ex ac . Syn- echocys is PGM con ains a single cys eine esidue, which implies ha he egula o y disulphide is in e molecula . Glycogen syn hesis. ADPglucose py ophospho ylase (Table 1) ca - alyzes he syn hesis o ADPglucose om glucose-1-phospha e and ATP. This e e sible eac ion cons i u es he i s s ep o glycogen syn hesis in bac e ia and s a ch syn hesis in he plas ids o plan s and algae (19). The highe plan enzyme is a he e o e - ame consis ing o wo la ge subuni s and wo small subuni s. Expe imen s in i o (20, 21) and in i o (22) ha e shown ha he N- e minal cys eine (Cys-12) o he small subuni s may o m an in e molecula disulphide bond be ween hem, he eby inac i- a ing he enzyme. The oxidized, inac i e enzyme is slowly educed and, hus, eac i a ed by DTT (20) o T x (21) in i o. This mechanism o educ i e ac i a ion has been p oposed o ope a e in all dico plan s, because Cys-12 is conse ed in hese plan s (20). In cyanobac e ia, ADPglucose py ophospho ylase is encoded by a single gene whose p oduc o ms homo e ame s (23). The cyanobac e ial enzyme closely esembles he small subuni o highe plan s. Glycogen syn hases in bac e ia and s a ch syn hases in plan plas ids ca alyze he ans e o ADPglucose o he non educing end o a g owing ␣ -1,4-linked glucan. The Synechocys is glycogen syn hase (Glg2) (Table 1) is simila o he soluble s a ch syn- hases om plan s and algae (24). Ou inding ha Glg2 is a po en ial a ge o T x is an indica ion o a pos ansla ional egula ion o his enzyme in pho osyn he ic o ganisms. The glycogen-b anching enzyme (Table 1) ca alyzes he inal s ep in glycogen syn hesis, ans e ing a segmen o a p eexis ing ␣ -1,4-linked glucan in ␣ -1,6 posi ion (19). Thus, he comple e pa hway o glycogen syn hesis seems o be linked o T x in Synechocys is. Suga -nucleo ide me abolism. Nei he he pu a i e suga -nucleo ide epime ase兾dehyd a ase no GDP-mannose dehyd a ase (Table 1) p e iously has been linked o T x. The la e ca alyzes he i s s ep o he pa hway ha leads o syn hesis o GDP-L- ucose, a componen o , e.g., bac e ial capsula polysaccha ides. Sul u me abolism. The i s s ep o educ i e sul a e assimila ion is he ac i a ion o sul a e ca alyzed by sul a e adenylyl ans- e ase. We ound his enzyme among he pe iphe al memb ane p o eins bound o T xA35 (Table 2). 3⬘-Phosphoadenylylsul a e (PAPS) educ ase, which was no ound among he T x a ge s in his s udy, educes ac i a ed sul a e o sul i e and is one o he bes known accep o s o educing equi alen s om T x in E. coli (25). Ins ead, we ound he nex enzyme o he pa hway, e e- doxin-dependen sul i e educ ase (Table 2), which is homolo- gous o he highe plan plas id e edoxin-sul i e educ ase (26). Cys eine syn hase, he las enzyme o he pa hway, ecen ly has been iden i ied as a a ge o chlo oplas T xs (6). Ni ogen me abolism. Ammonium assimila ion occu s h ough a cyclic pa hway in ol ing wo enzymes, GS and GOGAT. In his Table 2. Synechocys is pe iphe al memb ane T xA-linked p o eins ORF Gene p oduc Appa en molecula mass, kDa Theo e ical molecula mass, kDa Numbe o cys eines conse ed* sll1789 RNA polyme ase ␤ ⬘subuni 191 144.7 6 (5) sll1787 RNA polyme ase ␤ subuni 152 123.3 3 (3) sl 0335 Phycobilisome linke (L CM ) 92 100.2 3 sl 0963 Fe edoxin-sul i e educ ase 67 71.4 6 (5) sl 0009 Rubisco la ge subuni (RbcL) 56 52.5 9 (8) sll1031 Ca boxysomal p o ein (CcmM) 54 73.1 (56.9) † 2 sl 1165 Sul a e adenylyl ans e ase 43 43.7 4 (0) sll1804 30S ibosomal p o ein S3 32 27.1 1 (0) *See Table 1 o de ini ion o numbe s. †The numbe in pa en heses co esponds o he molecula mass o CcmM in e . 17. Fig. 2. Ac i i y o Synechocys is PGM. Con e sion o glucose-1-phospha e o glucose-6-phospha e in cy osolic ex ac s was measu ed be o e and a e incuba ion a 25°C o 45 and 90 min wi h he addi ions speci ied. Concen- a ions o CuCl2and T xA we e 25 and 2.5 ␮ M, espec i ely. The alues a e he means o h ee di e en expe imen s, and SDs a e p esen ed as e o ba s. The ini ial ac i i y be o e incuba ion was 0.38 ␮ mol䡠mg o p o ein⫺1䡠min⫺1. 16110 兩 www.pnas.o g兾cgi兾doi兾10.1073兾pnas.2534397100 Lindahl and Flo encio pa hway, GS has been conside ed he exclusi e a ge o egula ion a bo h ansc ip ional and pos ansla ional le els in cyanobac e ia (27). Su p isingly, bo h e edoxin-dependen GOGAT and he la ge subuni o NADH-dependen GOGAT (Gl B) we e cap u ed by T xA35 (Table 1), sugges ing ha glu ama e syn hesis may be edox- egula ed. Wes e n blo using an ibodies agains Gl B con i ms ha his p o ein is p esen in elua e E1, which con ains cy osolic p o eins bound o T xA35 (Fig. 3). In addi ion, we ound ha in i o mos o Gl B pa icipa es in high-molecula weigh complexes c osslinked by disulphides (Fig. 3). The appa en molecula mass o hese complexes canno be de e mined, because i exceeds ha o he la ges ma ke p o ein o 250 kDa, bu i is possible ha i co esponds o a dime o he 186-kDa Gl B subuni s. Howe e , i can be concluded ha T xA35 does no in e ac wi h he pa icula cys eine in ol ed in complex o ma ion, because ha would p oduce a Gl B-T xA35 he e odime o ⬇200 kDa mo- lecula mass (Fig. 3, lane 3). Chlamydomonas einha d ii GS (28) and plan GS (29) a e ac i a ed on educ ion by T x o DTT. In bo h e s. 5 and 6, GS is epo ed as a a ge o chlo oplas T xs. Synechocys is GS ( ype I) has ne e been ound o espond o hiol eagen s bu ins ead is known o be egula ed h ough he e e sible binding o wo small p o eins (27). In his a icle, we show ha Synechocys is GS is absen om elua e E1 despi e i s abundance in he cy osolic ex ac (Fig. 3). A gininosuccina e syn he ase (Table 1) is he a e-limi ing enzyme o bo h he u ea- and a ginine-ci ulline cycles. Like mos cyanobac e ia, Synechocys is s o es excess educed ni ogen in he o m o cyanophycin, a non ibosomally syn hesized poly- me o aspa a e and a ginine (30). Thus, he syn hesis o a ginine is analogous o he syn hesis o ac i a ed glucose o s o age o educed ca bon as glycogen. T ansc ip ion. RNA polyme ase subuni s ␤ and ␤ ⬘we e ound o eac wi h T xA35 (Table 2). In e es ingly, in i o ansc ip ion wi h pu i ied RNA polyme ase om he cyanobac e ium Anabaena sp. PCC 7120 equi es he p esence o hiol- educing agen s (31). Thus, i is possible ha polyme ase ac i i y in he da k is low because o oxida ion o egula o y hiols and ha ac i i y is eco e ed in he ligh a e educ ion by T x. Indeed, cyanobac e ial o e all gene exp ession dec eases d as ically a e a shi om ligh o da kness and e u ns o ini ial le els a e he onse o ligh (32). P o ein syn hesis and olding. A yeas homologue o alyl- RNA syn he ase (Table 1) al eady is known o unde go educ i e ac i a ion in a eac ion ha in ol es hiols (33). The ansla ion elonga ion ac o s EF-G and EF-Tu (Table 1) we e bo h isola ed as a ge s o chlo oplas T xs (6), and EF-Tu possesses p o ein disulphide isome ase ac i i y in E. coli (34). Howe e , i can be ques ioned whe he he 30S ibosomal p o ein S3 (Table 2) is a ue a ge , because i s single cys eine is conse ed in only Cyanopho a pa adoxa bu no o he species. The 60-kDa chap- e onin G oEL (Table 1) closely esembles he chlo oplas Rubisco-binding p o ein subuni s ␣ and ␤ , which we e epo ed ea lie as chlo oplas T x a ge s (6). Fu he mo e, he Hsp70 chape one is egula ed by a T x-like p o ein in plan s (35). Te apy ole biosyn hesis. I has been demons a ed ha syn hesis o p o opo phy in IX om 5-aminole ulinic acid is s imula ed by DTT in chlo oplas ex ac s (36). Th ee enzymes om he chlo oplas heme and chlo ophyll syn hesis pa hway ecen ly we e ound o in e ac wi h T xs (6). In his a icle, we epo ano he enzyme om he same pa hway, po phobilinogen syn- hase (Table 1). Oxida i e s ess esponse. Pe oxi edoxins (P xs) cons i u e an ubiq- ui ous amily o hiol-speci ic pe oxidases (37). A ligh - dependen , hiol-speci ic pe oxidase ac i i y was disco e ed in a ca alase dele ion mu an o Synechocys is (38), sugges ing he p esence o P x. Among ou T x a ge s we ound a 1-Cys P x and a homologue o he yeas pe oxidase YLR109 (Table 1). Yeas YLR109 has been shown o in e ac wi h an A abidopsis haliana T x h(4). T xs, o T x-like p o eins, a e known o educe chlo oplas 2-Cys P x (6, 39) and P x-Q (5). Ligh ha es ing. The phycobilisome co e linke polypep ide (L C ) (Table 1) links h ee allophycocyanin he e odime s, he eby o ming he co e o he phycobilisome (40). The Synechocys is L C con ains a single cys eine esidue, which is conse ed in se e al cyanobac e ial species, such as Synechococcus sp. PCC 6301, bu no in Anabaena sp. PCC 7120. The phycobilisome co e- memb ane linke polypep ide (L CM ) (Table 2) ancho s he en i e phycobilisome o he memb ane (40). One o he h ee abso- lu ely conse ed cys eines in he Synechocys is L CM polypep ide, Cys-190, is p obably in ol ed in ch omopho e binding (40). The p esence o edox-ac i e cys eines in bo h L C and L CM sugges s ha hey may bind each o he when joined unde oxidizing condi ions. This inding means ha he phycobilisome could be co alen ly associa ed o he memb ane unde ligh -limi ing condi ions by a disulphide ha is b oken by T x a e an inc ease in ligh in ensi y, ensu ing g ea e lexibili y in ligh ha es ing a highe ligh in ensi ies. In e es ingly, ea men o Synecho- coccus wi h N-e hylmaleimide dis up s ene gy ans e om phycobilisomes o pho osys em I wi hou pe u bing he in eg i y o ei he phycobilisomes o pho osys ems I o II (41). Conse a ion o Cys eines in Synechocys is T xA-Linked P o eins. Conside ing he possibili y o conse a ion o T x- ela ed eg- ula o y mechanisms h ough e olu ion o pho osyn he ic o gan- isms, we examined he p esence o conse ed cys eines in he Synechocys is amino acid sequences (see Table 3, which is published as suppo ing in o ma ion on he PNAS web si e). All T xA-linked Synechocys is p o eins ha e a leas one cys eine conse ed in homologous p o eins om some o he o ganism (Tables 1 and 2), and 17 o hese Synechocys is T xA a ge s ha e a leas one cys eine conse ed in chlo oplas homologues (Tables 1 and 2). Th ee p o eins wi h no cys eines conse ed be ween Synechocys is and A abidopsis chlo oplas coun e pa s [glucan b anching enzyme, PGM, and sul a e adenylyl ans- e ase (Tables 1 and 2)] display e y low o e all simila i ies o he chlo oplas p o eins. A close look a hese chlo oplas enzymes e eals ha hey a e much mo e simila o euka yo ic p o eins (da a no shown). I mus he e o e be concluded ha a possible T x-media ed egula ion has no been conse ed h ough chlo- oplas e olu ion in hese cases. Po phobilinogen syn hase in bac e ia, including cyanobac e ia, ungi, and animals, is a zinc- dependen enzyme ha uses h ee icinal cys eines o coo dina e he zinc ion (42). In plan chlo oplas s and some bac e ia, hese cys eines ha e been eplaced wi h aspa a es ha pa icipa e in coo dina ing a magnesium ion. The pu i ied zinc-con aining Fig. 3. Examina ion o NADH-GOGAT and GS by Wes e n blo analysis. Lanes 1 and 2 con ain 15 ␮ g o cy osolic Synechocys is p o eins; lanes 3 and 4 con ain 5 ␮ l o elua e E1 (cy osolic p o eins bound o T xA35). Lanes 1 and 3 do no include educing agen s; lanes 2 and 4 include 10 mM DTT. P o eins sepa a ed by SDS兾PAGE we e analyzed by using an ibodies agains GS and he la ge subuni o NADH-GOGAT (Gl B). Molecula mass ma ke s o 250, 64, and 50 kDa a e indica ed, as is he 186-kDa appa en molecula mass o he mono- me ic educed Gl B. Lindahl and Flo encio PNAS 兩 Decembe 23, 2003 兩 ol. 100 兩 no. 26 兩 16111 PLANT BIOLOGY enzyme depends on exogenous hiols o main aining he bound zinc and, consequen ly, o i s ac i i y (43), whe eas he plan enzyme does no (44). Thus, T x may unc ion as an an ioxidan , enabling zinc binding in i o and so con olling he ac i i y. This could be an example o a unc ion o T x los in one b anch h ough e olu ion. Synechocys is ADPglucose py ophospho y- lase sha es h ee cys eines (Cys-55, -325, and -330) wi h i s A abidopsis chlo oplas homologue. Mo e in e es ing, howe e , a e he wo N- e minal cys eines (Cys-2 and -3) ha a e no s ic ly conse ed bu appea only 2 aa ups eam o he chlo o- plas Cys-12, which has been implied in edox egula ion o his enzyme. Hence, he e is a possibili y ha cys eines could be unc ionally equi alen wi hou being conse ed in he sequence. Concluding Rema ks. The pu a i e Synechocys is T xA a ge p o- eins iden i ied in his s udy a e mainly enzymes pa icipa ing in anabolic p ocesses. A pic u e begins o eme ge in which he cyanobac e ial p ocesses o assimila ion and s o age o ca bon, sul u , and ni ogen in glycogen, p o eins, and cyanophycin, espec i ely, a e all possible a ge s o ligh -induced edox egula ion by means o T x. Based on he knowledge o T x ac ion in chlo oplas s (1–3), i is expec ed ha educed T x has a s imula o y e ec on hese anabolic p ocesses, hus signaling he a ailabili y o ligh ene gy. Howe e , he iden i ies and ela i e abundance o T x a ge s a e likely o change depending on g ow h condi ions. The new po en ial T xA a ge p o eins poin o di e ences in he T x-media ed egula ion be ween cyanobac e ia and chlo- oplas s. In con as o plan s, cyanobac e ia depend on he ca bon dioxide-concen a ing mechanism o a unc ioning CO 2 assimila ion and g ow h a physiological le els o CO 2 (45). The inding ha CcmM in e ac s wi h T xA sugges s ha he ca bon dioxide-concen a ing mechanism is subjec o T x-media ed edox egula ion in cyanobac e ia, whe eas he Cal in cycle has become a p ime T x a ge p ocess in chlo oplas s. Each Syn- echocys is enzyme o he comple e pa hway o glycogen syn hesis was ound o in e ac wi h T x and no only he a e-limi ing enzyme, ADPglucose py ophospho ylase, as seems o be he case in plas ids (21). Synechocys is GOGAT, a he han GS, we e ound o in e ac wi h T xA, implying a shi o he ocus o edox egula ion o ammonium assimila ion be ween cya- nobac e ia and chlo oplas s. This s udy pa es he way o u u e wo k on egula ion media ed by T x in cyanobac e ia, which has long been consid- e ed insigni ican . I also gi es an in e es ing pe spec i e o he molecula e olu ion o suscep ibili y owa d educ ion by T x. This wo k was inanced by G an BMC 2001-2635 om he Spanish Minis y o Science and Technology (MCYT). M.L. is a holde o a Ramon-y-Cajal con ac om he MCYT. 1. 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