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
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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
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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.
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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.
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