32 Plant Phytoene Synthase Complex

8/4/2019 32 Plant Phytoene Synthase Complex

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3 5 2 M O L E C U L A R A N D C E L L B I O L O G Y [32]p re sen t d u r in g p re in cu b a t io n a l i g h t i n cu b a t io n o f 3 0 min i s su f f i c i en t ,b ecau se ti s su e b reak d o w n b eg in s a ft e r 3 0 r a in an d a f t er 6 h r t h e t is su e isb r o w n a n d n e c r o ti c .l : A n A L A c o n c e n t ra t io n o f 10 m M d u r i n g pr e in c u -b a t io n ,~ r e su lt s in t o t a l c a ro ten o id l ev el s t h a t a r e 2 - 3 t imes o f t h o se o fc o n t r o l c o t y l e d o n s e i th e r w h e n d a r k i n c u b a t e d f o r 6 h r o r l ig h t i n c u b a t e df o r 30 m i n . I n t h e d a r k , A L A s t i m u l a t i o n o f c a r o t e n o i d a c c u m u l a t i o n w a su n a f f ec ted b y A T ( 1 r aM ) , b u t i n t h e l ig h t (30 m in ) A T (1 r aM ) h a lv ed th eAL A s t imu la t io n . A l th o u g h lo wer AL A co n cen t r a t io n s may b e e f f ec t iv e ,h ig h e r o n es t en d to b e to xic . S o lu t io n s co n ta in in g AL A a re a l so p a r t i cu -la r ly p rone to bac te r ia l p ro l i fe ra t ion , so ex t ra ca re shou ld be taken tom i n i m i z e c o n t a m i n a t io n .C a ro ten o id s a r e m easu re d a f t e r an ap p ro p r i a t e i n cu b a t io n t ime .1 1 T i s -su e can b e f ro zen in th e d i sh w i th l i q u id N 2 to s to p th e l i g h t i n cu b a t io n a n dplaced in a f reezer un t i l neede d . Al te rna t ive ly , sam ples can be s to red fo r ash o r t t ime in th e d a rk , p r io r t o p ig m en t ex tr ac t io n .T h i s s y s te m l e n d s i ts e lf w e ll t o t h e a d d i t i o n o f m a n y e x o g e n o u s c o m -p o u n d s b e s id e s AT an d AL A. I t i s s imp le an d r e l i ab le ( co e f f i c i en t o fvar ia t ion o f genera l ly be tw een 5 an d 10%) an d re la tive ly inexpens ive .lo p. A. C astelfranco,P. M. Rich, and S. I. Beale,Plant Physiol. 53, 615 (1974).l~ Th is series, Volum e213.

[ 3 2 ] P l a n t P h y t o e n e S y n t h a s e C o m p l e x : C o m p o n e n tE n z y m e s , I m m u n o l o g y , a n d B i o g e n e s i s

B y BILAL CAMARAI n t r o d u c t i o n

In p lan ts , th e sy n thes is o f phy toen e , the f ir st C4o caro teno id , requ i rest h e c o o r d i n a t e d a c ti v it y o f t h e d i f fe r en t c o m p o n e n t e n z y m e s o f t h e p h y -to en e sy n th ase co m p lex wh ich ca t a ly ze th e s eq u en ce o f reac t io n s p re sen tedin F ig . 1. T h e en t i r e p a th wa y is read i ly d em o n s t r a t ed in v i t ro u s in g i so l a tedp las tids . 1-4 H ow ever , d esp i te the con serv a t ion o f th is pa th wa y , i so la ted' B . Ca ma ra , F . Bardat , and R . Mon~ger, E u r . J . B i o c h e m . 127, 255 (1982).2 . Dog bo, F . Bardat , A. Lafer ri~ l'e , J . Qu en ne m et , J . Brang eon, and B. Cam ara , P l a n t S c i .( L i m e r i c k , l r e l . ) 4 9 , 89 (1987).3 B. Cam ara , this series, Vo l . 110A, p. 244.4 H . K lein ig an d P. Beyer , this ser ies , Vol . 110A, p. 267 .

C o py ri gh t 1 99 3 b y A c t i n i c l ~ I nc .METHODS IN ENZYMOLOGY , VOL. 214 Al l r lgh t s o f r q~ xlu c t io n in ~ny fo rm re . f r ed .


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[32] PLANT PHYTOENE SYNTHASE COMPLEX 3532 IPP

DIMETHYLALLYLPP ~ GERANYLPP1 l 3 IPP ~

FARNESYLPPISOPENTENYL PP (IPP) |4 IP P

2 x ( GERANYLGERANYLPP )PREPHYTOENE PP- 6 - PHYTOENE

Fro . 1 . Pathw ay o f phy toene synthesis in plants . The different s teps are catalyzed byope ration ally soluble enzymes: isopen tenyl PP isom erase (1) , geranylgeranyl PP synthase (2 ,3 , 4) , and phytoene synthase (5, 6) , col lec tive ly e rm ed the phy toene syn thase complex .

no ng reen plastids are by far mo re act ive tha n chloroplasts . This cha pterreports general considerat ions con cern ing the isolat ion of chloroplasts an dchromop las ts f rom Capsicumfruits and their use for three specific pur-poses, nam ely , the charac ter iza t ion o f the phy toene synthase com po ne ntenzymes , the i r imm uno chem is t ry , and the i r b iogenesis .I s o l a ti o n o f P l a s t i d s a n d S u b f r a c t i o n s

Th e plast ids are puri f ied according to th e pro cedures out l in ed in Fig. 2.Th e p uri ty o f these fractions is i llust rated in Figs. 3 and 4. At th e co m ple-t ion, th e pu ri f ied plast id suspensions are di lu ted to 0.4 M sucrose a ndpel le ted af ter cent r ifugat ion for 10 min a t 4000 g . T he pel let i s homo ge-nized in a P ot ter homo genizer wi th 50 m M Tr is -HCl (pH 7 .6) conta in ing5 m M di thiothre i tol (DT T). Th e plastic s t rom a ob taine d af ter centr ifuga-t ion for 1 hr a t 100,000 g i s saved and used for the pur i f ica t ion ofp hy toe nesynthase com po ne nt enzymes . This prepara t ion i s s table for up to 1 year a t- 2 0 , especia lly i f the p rote in conce nt ra t ion is h igh (> 10 mg /ml) .C o m p o n e n t E n z y m e s o f CapsicumP h y t o e n e S y n t h a s e C o m p l e x

Init ia lly , the individual com po ne nt enzym es of the phy toene synthasecom plex [ isopen tenyl pyrop hosp hate (PP) isomerase (EC 5.3.32) , geranyl-geranyl PP synthase (EC 2.5.1.1) , a nd ph yto ene synthase (EC 2.5.1.32)]were separa ted af ter conc ent ra t ing the s t romal ext rac t wi th polyethyleneglycol 6000 fol lowed by D EAE-Sephacel (Pharm acia, Piscataway, N J) an da m i n o p h e n e t h y l c h r o m a t o g ra p h y ? 6 O w i n g to t h e t i m e c o n s u m e d i n m a k -

5 0 . Do g b o a n d B . Ca m a r a , B i o c h i m . B i o p h y s . A c t a 920, 140 (1987).6 0 . Do g b o a n d B . Ca ma r a , P ro c. N a t l . A c a d . S c i . U . S . A . 85, 7054 (1988).


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354 M O L E C U L A R A N D C E L L B IO L O G Y [32]HOM OGE NIZA T IONP er icar p t i ssu e of f i rm green or red fru i t (2 kg f r esh we ig h t ) is ch o p p ed in to 2 l ite r s o f[ c h i l l e d s o l a t io n m e d i u m (1 m M 2-mercaptoethano l, 1 mM EDTA, 0 .4 M sucrose, Tr i s -,~ HC 1, pH 8)DISRUPTION (2 t imes for 1 eec each in a War in g b len d er ) ; p H r ead ju s ted t o 8 w i t h c onc en-t rated Tr isFILTRATION ( through 4 l ayers o f B lu tex , 50 gm ap er tu r es )CE NTRIFUGA TIONSorval l GS A rotor , 150 g for 5 ra inP k , tSorve ll GS A ro tor (2200 g fo r 30 sec for c h l o ro p l a s ts a n d 1 0 m i n for chromoplasts)

I P e l l e t S u p em atan t d iscar d ed(crude p l a s ti d s w a s h e d t w i c e w i t h t h e i s o la t io n m e d i u m )S UCRO S E S TE P GRA DIE NTCh lo r o p las t su sp en s io n (10 ml ) i s l ayer ed o n t o p o f a s u c r o s e g r a d ie n t (0.75, 1, 1.5 M , 10ml per f r ac t io n ) co n ta in in g 1 rnM 2-mercaptoethano l and 50 m M Tri s -HC1, pH 7,6Cen t r i fu g at io n : Sorval l Hb4 rotor , 1000 g for 15 m inInter face 0 .75 - 1 M ( b r ok en m e m b r a n e s )In te r face 1 - 1.5 M I n tac t ch lo r o p las ts

Chromoplast suspension (2 ml ) i s l ayer ed o n t o p o f a s u c r o s e g r a d ie n t (0.45, 0.84, 1.45M , 9 m l pe r f r ac t io n ) co n ta in in g 1 m M 2- rnerceptoethano l a nd 50 m M Tds-HC1, pH 7 .6Cen td fu g at io n : Beckman SW 2 7 ro tor, 6 2,000 g (R.,=) fo r I h rJ I n te rface 0 .4 5 - 0 .84 M ( b rok en m e m b r a n e s )J, In ter face 0 . 84 -1 .4 5 M In tac t chromoplas tsF IG . 2 . M e t h o d f o r the isolation o f Capsicumchloroplasts and chromoplasts.

ing the co lumn a nd i t s ins ta b i l i t y a f t er sev era l runs , we ha v e dev i sed am o re ex ped i t io us pro cedure . Acco rd ing ly , so l id N a C1 i s a dded to th e s tro -m al fraction to br ing the so lut ion to 1 M NaC 1. T he result ing so lu t ion i slo a ded o nto a Pheny l-Sepha rose (Pha rm a c ia ) co lu m n (2 .5 2 5 cm ) equi -l ibra ted a n d e lu ted w i th buff er A (5 0 m M Tr is -HC1 , pH 7 .6 , co nta in in g2 0% g ly ce ro l a n d 5 m M 2 - m e r c a p t o e th a n o l ) s u p p l e m e n t e d w i t h 1 MNa CI . Th e flow- thro ugh a nd the fo l lo w ing 1 M Na C 1 wa sh ing rem o v e u pt o 90% o f t h e i s o p e n t e n y l P P i so m e r a s e a n d t h e e n d o g e n o u s p h o sp h a ta seactivities ( fract ion I) . Th e fract ion e nr ich ed w ith geranylgeranyl P P syn-thase act iv i ty i s e luted w ith a decreasing gradient o f I to 0 M NaC 1 ( frac-t io n I I) . Indeed m o st o f the g era nylgerany l PP sy ntha se a c t iv i ty e lu te sbe tween 0 .5 a nd 0 M Na CI . F ina l ly the phy to ene sy ntha se a c t iv i ty s t r i c t os e n s u i s e lu t e d w i t h b u f fe r A c o n t a i n i n g 40 % d i m e t h y l s u l fo x i d e ( D M S O ) .T h e p r e s en c e o f d i m e t h y l s u l f o x id e i n h i b i t s th e p h y t o e n e s y n th a s e a c t iv i ty(80%). Ful l act iv i ty i s restored by apply ing the 40% dimcthyl sul fox ideso lut io n d irect ly to a c o lu m n o f Q-Sepharo se (Pha rm a c ia ) equ i libra tedwi th buf fer A . Af t er wa sh ing wi th buf fer A (4 to 5 bed v o lum es) , theph y to en e sy n tha se a c t iv i ty i s e lu t ed w i th 0 .3 M Na C 1 in buf fer A ( f ra c tionI l l ) .


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[3 2 ] PLANT PHYTOENESYNTHASECOMPLEX 355

FIO. 3. Electron micrographs showing the integrity and purity o f chloroplasts isolatedfrom Capsicumpedc arp. (A) A ppearance o f a typical purified chloroplast fraction; (B, C )detailed structure o f purified chloroplasts dep icted n (A).

Characterization o f Isopentenyl Pyrophosphate Isom eraseEnzyme Assay Procedure. I s o p e n t e n y l P P i s o m e r a s e i s r o u t i n e l y a s -

s a y e d a t 3 0 b y d im e t h y l a l ly l P P f o r m a t i o n i n a n E p p e n d o f f t u b e c o n t a i n -i n g t h e f o l l o w i n g r e a c t i o n m i x t u r e ( 0.1 m l f in a l v o l u m e ) : 5 0 m M T r i s- H C 1( p H 7 .6 ), 5 m M M gC 12 , 1 m M M n C I 2 , 2 m M d i th i ot h re it o l, 1 0 # M [1 -1 4 C ] is o p en t en y l P P , a n d e n z y m e e x t r ac t . A l t e rn a t i v e ly , 1 0 m M K F i s i n -


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356 MOLECULAR AND CELL BIOLOGY [32]

Fio. 4. Electron micrographs showing the integrity and purity o f chromo plasts isolatedfrom Capsicum peficarp. (A) Appearance o f a typical purified chrom oplast fraction; (B, C )detailed structure o f purified chromoplasts depicted in (A). Note that the high osm olarity ofthe gradient and the high sp eed of centrifugation induce the formation of cond ensed stromabeneath the increased sucrose permeable space.

c l u d e d t o t e s t f o r th e p r e s e n c e o f p h o s p h a t a s e s . T h e r e a c t i o n i s s t o p p e da f te r 15 m i n o f i n c u b a t i o n b y a d d i n g 0 . 5 m l o f c o n c e n t r a t e d H C 1 / m e t h -a n o l ( 1 : 4 , v / v ) f o l l o w e d b y i n c u b a t i o n a t 3 7 f o r 1 5 r a in . T h e r e a c t i o nm i x t u r e i s e x t r a c t e d w i t h 0 .5 m l o f c h l o r o f o r m , a n d a f t e r c e n t ri f u g a t i o n a na l i q u o t o f t h e l o w e r o r g a n i c p h a s e i s u s e d f o r l iq u i d s c i n ti l la t io n c o u n t i n g .

Pur i f i c a t i o n . F r a c t i o n I c o n t a i n i n g i s o p e n t e n y l P P i s o m e r a s e i s d i l u t e dt o 0 . 25 M N a C I w i t h b u f f e r A a n d s u b j e c te d t o h y d r o x y a p a t i te c h r o m a t o g -r a p h y ( c o l u m n 2 .5 X 1 5 c m ) , p r e p a r e d b y m i x i n g d r o p w i s e a 4 . 4 % ( w / v )


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[32] PLANT PHYTOENE SYNTHASECOMPLEX 357

so lu t io n o f C aC1 2 an d a 7 .1 % (w]v ) so lu t io n o f N a2 H P O 4 (R e f . 7 ) fo l lo wedb y r ep ea ted wash in g wi th wa te r . T h e co lu m n i s eq u i l ib r a ted wi th b u f fe r A ,a n d e l u t i o n is d o n e w i t h a 1 0 m M t o 0 . 2 M N a 2 H P O 4 l i n e ar g ra d i e n t i nb u f f e r A . P eak f r ac t io n s a r e p o o led , an d a f t er d i lu t io n (5 t imes ) t h e en zy m eso lu t io n is ap p l i ed to a Q -S ep h a ro se co lu m n (2 .5 X 15 cm) e lu t ed wi th a 0to 0 . 4 M NaC 1 lin ea r g rad ien t i n b u f f e r A . P o o led p e ak f r ac t io n s o f a c tiv itya re co n cen t r a t ed b y d i a ly s is ag a in st b u f fe r A sa tu ra t ed wi th p o ly e th y len eg ly co l 2 0 , 0 0 0 an d ap p l i ed to a S ep h ac ry l S -2 0 0 (P h a rmac ia ) co lu mn(2 .5 X 4 5 cm ) eq u i l ib r a t ed wi th b u f f e r A co n ta in in g 0 .1 5 M NaC 1 . Ac t iv ef r ac t io n s a re a d j u s t ed t o 10 m M M g C 12 a n d e l u t e d t h r o u g h a c o l u m n(1 5 cm ) o f Re ac t ive R ed 120 agarose (S igma, S t. Lou is , M O ) equ i l i -b r a t e d w i t h b uf f er A c o n t a i n i n g 0 .1 5 M N a C1 a n d 1 0 m M M s C I 2 . U n a d -so rb ed i so p en ten y l P P i so merase i s ad ju s t ed to 5 0 % (v /v ) g ly ce ro l an ds to red a t - 20 .Properties. H o m o g e n e o u s i s o p e n te n y l P P i s o m e r a se i s a 3 3 - k D a m o n o -m e r . T h e p H o p t i m u m is a b o u t 7 .5 .(5 ) T h e K m v a l u e fo r is o p e n t e n y l P P is6 g M . T h e e n z y m e r e q u ir e s M n 2+ or Mg 2+ fo r max ima l ac t iv i ty . T h ea c ti v it y a s m e a s u r e d b y t h e c o n v e r s i o n o f i s o p e n te n y l P P t o d i m e t h y la l ly lP P i s m o r e t h a n 9 0% i n h i b i t e d b y 2 0 # M d i m e t h y l a ll y l P P . T h e e n z y m e issens i tive to th io l d i rec ted reagen ts .Characterization o f Geranylgeranyl Pyrophosphate Synthase

En zym e Assay Procedure. T h e a ssay m ix tu re (0 .1 ml ) co n ta in s 5 0 m MT ris -H C l , p H 7.6, 5 m M M g C 1 2 , 1 m M M n C I , 2 m M d i t h io t h r e it o l , 5 / ~ /[1-14C]isopen tenyl PP , 20 g M d im ethy la l ly l PP , geran y l PP , o r fa rnesy l PP ,an d th e en zy me ex t r ac t . Af t e r i n cu b a t io n fo r 2 0 r a in th e r eac t io n i ss t o p p e d a n d t h e m i x t u r e p r o c e ss e d a s d e s c ri b ed a b o v e i n t h e i s o m e r a s esec t ion .Purification. S o l id NaC 1 i s ad d ed (6 0 g /ml ) t o f r ac tio n I I en r i ch e d wi thg e ran y lg e ran y l P P sy n th ase ac tiv ity . T h e r e su l t in g so lu t io n i s ap p f i ed to aco lu m n o f P h en y l -S ep h a ro se (2 .5 X 1 5 e r a ) eq u i l ib r a ted wi th b u f f e r Ac o n t a i n i n g 1 M N a C I . T h e c o l u m n is w a s h e d b y a 1 a n d 0 .7 5 M N a C1 s t e pg rad ien t i n b u f fe r A fo l lo wed b y a 0 . 5 -0 M NaC 1 l in ea r g rad ien t i n b u f f e rA . Ac t iv e fr ac t io n s a re d i lu t ed th ree t imes an d ad so rb ed to a h y d ro x y ap a -r it e co lu m n (2.5 15 cm ) (p rep a red a s d e sc r ib ed ab o v e in th e i so merasesec tio n ). Ge ran y lg e ran y l P P sy n th ase is e lu t ed wi th a 1 0 m M to 0 . 3 MN a2 H P O 4 l i n ea r g rad ien t i n b u f f e r A . T h e p o o led p eak f r ac tio n s a re d i -l u t e d 10 t im e s , a n d t h e s o l u t i o n is a d s o rb e d o n t o a Q - S e p h ar o s e c o l u m n(2.5 15 e ra ) p ree qu i l ib ra te d wi th bu f fe r A. A ct ive f rac t ions a re e lu ted7 C. K. Mathews, F. Brown, and S. S. Cohen, J. Biol. C hem . 239, 2957 (1964).


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358 MOLECULAR AND CELL BIOLOGY [32]

with a 0 -0 .4 M NaC1 l inear gradient in buffer A. Af ter d i lu t ion (5 t imes) ,the pooled p eak f rac t ions previous ly adjus ted to 10 m M MgC I2 are ad-sorbed onto a R eact ive Re d 120 agarose colum n (2.5 15 cm ) preequil i-bra ted w i th buffer A co nta in ing 10 m M MgC12. Th e co lum n is e lu ted wi tha 0 .1 -0 .5 M NaC1 l inear gradient in the sam e buffer . I f impu r i t ies rema in ,the ac t ive f rac t ions are d i lu ted (4 t imes) and adsorbed onto a smal l Q-Sepharose colu m n (1.5 5 cm) . T he co lum n is washed w i th buffer Adevoid of 2-mercaptoethanol before e lu t ing geranylgeranyl PP us ing thesame buffer adjusted to 0.3 M NaC1. Final ly, the enzyme solut ion is ap-pl ied on to a sm all (1.5 X 3 cm ) co lum n o f Afl i-Gel 501 (Bio-Rad, Rich -m ond , C A) washed wi th buffer A con ta in ing 0.3 M NaC 1 bu t devo id o f2-m ercap toethan ol . Gera nylge ranyl PP synthase is eluted specif ically w ithbuffer A conta in ing 10 m M di th io thre i to l. Pooled f rac t ions are conc en-trated a gainst dry po lyehty lene glycol 20,000 an d dialyzed against buffer Acon ta in ing 0 .5 M NaC I be fo re sto ring a t - 2 0 .Properties. Gerany lgeranyl PP synthases f rom al l prepara t ions thus farexam ined a re d im er i c p ro te ins com posed o f apparen t ly iden t i ca l subun it so f a p p ro x i m a te ly 3 6 - 3 7 k D a a n d h a v e a p H o p t i m u m o f a b o u t 7 - 7 . 6 .Th e Km values are as follows: 3 pA for isop enten yl PP; 0.95 # M fordime thyla l ly pp; 1 # M for geranyl PP; an d 1 .2 p M for farnesyl pp .5 Th een zy m e is sensi tive to thiol-directed reagents .Characterization of Phytoene Syn thase

Enzyme Assay Procedure. Two m ethods a re used accord ing to thepresence o r the absence o f i sopen teny l PP i som erase and ge rany lge rany l PPsynthase . In the p resence of these com pa nio n enzym es , the reac t ion mix-ture (0 .2 m l fina l volum e) conta ins 50 m M Tr is -HC l buffer , pH 7 .6 , 5 m MMgCI2, 2 .5 m M MnC12, 2 m M di th io thre i to l, 1 0 p M [1-~*C]isopentenylPP, an d enz ym e extrac t. Af ter 20 ra in to 1 hr o f incu bat ion a t 30* thereact ion is s topped by adding 0 .5 m l o f chlo rofo rm /m etha nol (2 : 1, v /v).Af ter addi t ion of unlabeled s tandards (geranylgeranio l an d phytoene ) , la -beled phytoen e in the ch loroform ph ase is pur i fied by th in- layer chrom a-tography on s ilica ge l p la tes developed wi th be nzen e/e thyl ace ta te (90: 10,v /v ) o r by H PL C us ing a C t s/~B ondapak co lum n (W ate rs , M i lfo rd , M A )an d m ethan ol /cycloh exan e/hex ane (80: 10: 10 , v /v) as the m obi le phase . 5Th e incorpo ra ted radioact iv i ty is de term ined by l iquid sc in ti l la t ion coun t -ing . In the second m ethod , t he sam e incuba t ion m ix tu re i s adop ted excep tth at [l-~4C]isopentenyl PP is rep lace d by 2 / t M [1,5,9-~4C]geranylgeranylPP enzyrnat iea l ly synthes ized f rom dimethyla l ly l P P an d [ 1-~4C]isopentenyl PP using the gerany lgeranyl PP synthase pu rif ied above.


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[ 3 2 ] P L A N T P H Y T OE N E S Y N T H AS E C O M P L EX 3 5 9/Purification. F r a c t i o n I II c o n t a i n i n g t h e b u l k o f p h y t o e n e s y n t h as e ist r ea t ed a s d e sc r ib ed fo r t h e p u r i f i c a tio n o f g e ran y lg e ran y l P P sy n th aseex cep t t h a t d u r in g th e R eac t iv e R ed 1 20 ag aro se ch ro m a to g rap h y a 0 .1 -

1 .5 M N aC 1 l in ea r g rad ien t i n b u f f e r A co n ta in in g 1 0 m M Mg C 12 i s u sed toe lu t e th e en z y m e . I f imp u r i t i e s r ema in , t h e ac t iv e f r ac tio n s ar e d i lu t ed (1 0t im e s ) a n d c h r o m a t o g r a p h d r a p id l y o n t o a s m a l l c o l u m n o f Q - S e p ha r o sean d A ffi-Gel 501 as descr ibed fo r gerany lgerany l PP syn thase .Properties. Purified p h y t o e n e s y n th a s es f r o m a ll p r e p a r a t io n s t h u s f a re x a m i n e d a re m o n o m e r i c p ro t e in s o f a p p ro x im a t e ly . T h e K m v a lu es fo rg e ran y lg e ran y l P P an d p rep h y to en e P P axe 0 . 3 0 an d 0 . 2 7 # M , r e sp ec tiv ely .T h e a c ti v it y o f t h e e n z y m e i s s tr ic tl y d e p e n d e n t o n M n 2+. T h e e n z y m e i ssens i tive to a rg in ine-d i rec ted reagen ts .~

I m m u n o c h e m i s t r y o f P h y t o e n e S y n th a se C o m p l e x C o m p o n e n tE n z y m e sT h e d if fe re n t c o m p o n e n t e n z y m e s o f t h e p h y t o e n e s y n th a se c o m p l e xa re u sed to e l i c it im m u n e r e sp o n ses in r ab b i t s a cco rd in g to s t an d a rdpro toco ls .8 T h e d i f fe r en t p o ly c lo n a l an t ib o d ie s o b ta in ed u n d e r th e se co n -d i t ion s a re n o t ac t ive s i te d i rec ted , s ince they fai l to inh ib i t d i rec t ly in vitrothe enz ym at ic ac t iv ity . The i r spec i fic ity i s eva lua ted as descr ibed be low.

l mm u n o i n h i b i t i o n As s a y Pr o ce du reS amp le s o f so lu b le f r ac t io n s (2 0 0 /z l ) a r e in cu b a ted wi th an t ib o d ie sag a in s t iso p en ten y l P P i so merase , g e ran y lg eran y l P P sy n th ase , o r p h y to e n es y n th a s e ( 5 - 2 0 #1 ) f o r 3 0 m i n a t r o o m t e m p e r a t u r e , t h e n f o r 2 h r t oo v e r n i g h t at 4 . T h e r e a f te r t w o k i n d s o f i m m u n o p r e c i p i t a t i o n s c a n b eu sed . In th e f i r s t me th o d , p ro te in A-S ep h a ro se b ead s (S ig ma ; tw ice th ev o l u m e o f p r e i m m u n e s e r u m o r a n t ib o d ie s ) a re a d d e d t o t h e p r o t e inso lu t io n , an d th e su sp en s io n i s i n cu b a ted a t 4 fo r 1 h r wh i l e swi r lin g tod is pe rs e a n y c l u m p s t h a t m a y f o r m . T h e p r o t e in A - S e p h a r o s e - a n t i b o d y -an t ig en co m p lex is p e l le t ed b y cen t r i fu g a t io n a t 1 7,0 00 rp m fo r 2 0 m in inJ A 1 8 .1 r o t o r ( B e c k m a n ) . T h e r e su l ti n g s u p e r n a t a n t i s u s e d f o r e n z y m ea ssa y. I n t h e s e c o n d m e t h o d , t h e a n t i b o d y - a n t i g e n c o m p l e x i s p r e c i p it a t edw i t h o u t t h e a d d i t io n o f p r o t e in A - S e p h ar o se b e a ds . D e p e n d i n g o n t h ec o n c e n t r a t i o n o f a n ti b o d i e s u s e d , p r e p a r at i o n s o b t a i n e d i n t h e se m a n n e r su su a l ly h av e le ss t h an 5 0 to 9 0 % ac t iv i ty co m p a red to co n t ro l s .

8 N . H a r b o e a n d A . I n g i ld , in " Q uan t i t a t i ve I m m unoe l ec t r ophor es i s " ( N . H . A xe l s en , J .K rol l , and B . Weeks , eds. ), p. 161. Blackwel l, Ox ford, 1977.


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[3 2 ] PLANT PHYTOENESYNTHASECOMPLEX 361W e s t e r n B l o t t i n g

P r o t e i n s a r e s e p a r a t e d b y o n e - d i m e n s i o n a l s o d i u m d o d e c y l s u l f a t e( S D S ) - 1 0% a c r y l a m i d e g e l e l e ct r o p h o r e si s . 9 T h e t r a n s f e r o f p r o t e i n s f r o mt h e g e l t o n i t ro c e ll u lo s e o r I m m o b i l o n - P i s p e r f o r m e d i n a c o l d r o o m a t 4 u s i ng a T r a n s p h o r ( H o e f e r S ci en ti fi c I n s t ru m e n t s ) a n d b u f f e r ed ( p H 8 .3 )t r a n s f e r m e d i u m c o n t a i n i n g 0 . 3 % ( w / v ) T r is , 0 .1 5 % ( w / v ) g ly c in e , a n d 2 0 %( v /v ) m e t h a n o l . A l t e rn a t iv e l y a N o v a b l o t ( L K B ) a p p a r a t u s i s u s e d w i t h at r a n s f e r m e d i u m c o n t a i n i n g 0 . 3 % ( w / v ) g l y c i n e , 0 . 6 % ( w / v ) T r i s , 0 . 0 5 %( w / v ) s o d i u m d o d e c y l s u l f a t e , a n d 2 0 % ( v / v ) e t h a n o l . T h e b l o t t i n g i sc a r r ie d o u t f o r 2 t o 4 h r . T h e p r o t e i n s a r e v i s u a li z e d u s in g s p e c if ic a n t i b o d -i e s ( 1 / 1 0 0 0 d i l u t i o n ) a g a i n s t t h e i n d i v i d u a l e n z y m e s a c c o r d i n g t o t h epro ced ure de sc r ibed . ~ A ty p ica l r e su l t i s p re sen ted in F ig . 5 , show ing tha ti s o p e n t e n y l P P i s o m e r a s e , g e r a n y l g e r a n y l P P s y n t h a s e , a n d p h y t o e n es y n t h a s e a r e q u a n t i t a t i v e l y r e c o v e r e d i n t h e s o l u b l e s t r o m a l f r a c t i o n o fc h l o r o p l a s t s a n d c h r o m o p l a s t s . F u r t h e r m o r e , t h e d a t a p r e s e n t e d d e m o n -s tr a te t h a t t h e se e n z y m e s a r e m o r e h i g hl y ex p r e ss e d in c h r o m o p l a s t s c o m -p a r e d t o c h l o r o p la s t s.I m m u n o c y t o c h e m i s t r y

Per ica rp t i s sue f rom green a nd red f ru i t s a re f ixed a t 4 (2 t imes , 15 ra ine a c h) i n t h e p r e s e n c e o f 2 % p a r a f o r m a l d e h y d e a n d 1 % g l u t a ra l d e h y d ed is so lv e d i n a m e d i u m c o n ta in i ng 2 5 m M K H 2 P O 4 - N a 2 H P O 4 b u f fe r ( p H7 . 2 ) s u p p l e m e n t e d w i t h 0 . 1 M s u c r o s e . T h e t is s u e s a r e e m b e d d e d i nE p o n .l ~ P o l y m e r i z a t i o n i s c a r d e d o u t a t 6 0 f o r 2 t o 3 d a y s . S e c t io n s (1 t o1 . 5 / z m t h i c k ) a r e c u t a n d m o u n t e d o n g l a s s s l i d e s . T h e s e c t i o n s a r e o v e r -l a i d w i t h p h o s p h a t e - b u f f e r e d s a li n e ( P B S ) c o n t a i n i n g 1 % ( w / v ) b o v i n es e r u m a l b u m i n a n d 0 . 1 % ( w / v ) T r i t o n X - 1 0 0 ( B T - P B S ) a n d c o n t a i n i n gp r e i m m u n e s e r u m o r d i l u te d 0 / 1 0 0 f o r g r e en ti ss u es a n d 1 / 1 0 0 0 f o r r e dt i s s u e s ) a n t i g e r a n y l g e r a n y l P P s y n t h a s e o r a n t i p h y t o e n e s y n t h a s e a n t i b o d -9 B. D . H aines, in "G el Elcctrophoresis of Proteins: A Practical App roach" (B. D . Hainesand D. Rickwood,eds.), p. I. IRL Press, Ox ford, 1984.1o M. S. B lake, K. H. Russel-Jones,and E. C. Gotslich,Ana l. Biochem. 136 , 175 (1984).H j. H. Luft, J. Biochem. Cytol.9, 409 (1961).

FI~. 5. Electrophoretic analysis followed by imm unoblotting of the different plastidsubfractions. (A) S trom al (S) and mem brane proteins (M b) fro m chloroplasts (Ch l) andchromoplasts (Chr) were separated by SD S-polyacrylam idegel electrophoresis. Size stan-dards are indicated at left. (B) A fter electrophoreti transfer the po lypcptideswere visualizedwith antiisopentenyl PP isomerase antibodies mixe d w ith antigeranylgeranyl PP synthaseantibodies (I + G) or with antiphytoene synthase antibodies (P). A rrowheads indicate posi-tions of isopentenyl PP isomeras (I), geranylgeranyl PP synthase (G), and phytoen esynthase(P) subunits.


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362 MOLECULAR AND CELL BIOLOGY [32]ie s. F o l l o w i n g i n c u b a t i o n i n a m o i s t c h a m b e r a n d w a s h i n g 3 ti m e s ( o v e r a4 0 - m i n i n te r v a l) w i t h B T - P B S , t h e s li d e s a re d r a in e d a n d o v e r l ai d w i t h 1%( w / v ) f lu o r e s c e in i s o t h io c y a n a t e - c o u p l e d g o a t a n ti- ra b bit i m m u n o g l o b u l i nG ( S i g m a ) , f o r u p t o 2 h r i n t h e d a r k . T h e n t h e s l i d e s a r e w a s h e d w i t hB T - P B S f o l l o w e d b y p h o s p h a t e b u f f e r a n d f i n a l l y t r e a t e d w i t h 0 . 0 2 %E v a n ' s B l u e ( M e r c k , D a r m s t a d t , G e r m a n y ) . T h e s l i d e s a r e s u b s e q u e n t l y

1~. 6. Immunofluorescence microscopy of green and red pericarp tissue of Capsicumimmunolabeled with polyclonal antibodies to geranylgeranyl PP synthase or preimmuneserum (control). The fluorescene micrographs were taken with a Zeiss photomicroscope. (a,b) Green lL~sues treated with antigeranylgeranyl PP synthase antibodies and preimmuneserum. (c, d) Red tissues treated with antigeranylgeranyl PP synthase antibodies and prdm-mune serum. Ep, Epidermia; N, nucleus; clap, chloroplast; chr, chromoplast.


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[32] PLANT PHYTOENE SYNTHASE COMPLEX 36 3m o u nte d w i th Ci t i fluo r (Ag a r Aids , S ta nsted , Eng land) . Th e t is sues a reo b se rv ed a n d p h o t o g ra p h e d w i t h a Z e i ss p h o t o m i c r o s c o p e e q u i p p e d w i t hep i f luo rescence i l lumina t io n . Pho to micro g ra phs a re reco rded o n Ag -f a c h r o m e 2 0 0 R S f i l m . T h e r e s u l t s o b t a i n e d a t c o m p l e t i o n a r e s h o w n i nFigs . 6 and 7 , which c lear ly indicate that geranylgeranyl PP synthase andph y to en e sy ntha se a re ex c lus iv e ly lo ca l i zed in the p la s tid co m pa rtm ent .

F la . 7 . Im m un of luorescence m ic roscopy o f g reen and r ed pe r i ca rp t is sue o f Capsicumi m m u n o l a b e l e d w i th p o l y c lo n a l antibodies to phy toene synthase o r p re im m une se rum (con-t rol ) . (a , b) Green t i ssues treated with anf iphy toene syn thase antibodis and p r e i m m u n eserum. (c, d) Red t issues treated with an t iphy toene syn thase antibodies and p r e i m m u n eserum. Ep, Ep idermis ; N , nucleus , ch p, chlorop las t ; chr , chro mo plas t ; v , vacuole .


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3 6 4 MOLECULAR AND CELL BIOLOGY [ 3 2 ]1 2 3 4

M r x 1 0 -3

6 7 - -

4 3 - - ~ i

3 0 - -

2 0 - -

Fxo. 8. F luo rogram af te r e lec t rophores is o f t r ans la t ion produc ts d i rec ted by po ly(A) RN Aisolated f rom red p er icarp tissue o f Capsicum. Lan e 1, tota l translation produc ts ; lanes 2-4,polyp eptides im m uno prec ipita te d with an tiisopente nyl PP isomerase, anf igeranylgeranyl PPsynthase, an d an f iphyto ene synthase antibodies .

B i og en e si s o f P h y t o e n e S y n t h a se C o m p o n e n t E n z y m e sR N A s a re i so l at ed f ro m p e r i ca rp ti ssu es fro zen in l i q u id n i t ro g en ac-co rd in g to a p rev io u s ly d e sc r ib ed m e th o d , n P o ly (A)+ PaNAs a re i so la ted bya f f in i ty ch ro ma to g rap h y o n o l ig o (d T ) -ce l lu lo se .13 Poly(A)+ R N A s a r et rans la ted in vitro i n a r ab b i t r e t i cu lo cy te ly sa t e k i t (Amersh am) in th ep r e se n c e o f 2 0 m C i / m l [3 5S ]m e th io nin e, a s r e c o m m e n d e d b y t h e m a n u f a c -tu re r . Af t e r 9 0 m in a t 3 0 , t r an s l a t io n i s t e rm in a ted b y th e a d d i t io n o fso d iu m d o d ecy l su lf a te t o 1% (w/v ) fo l lo wed b y b o i l in g fo r I m in . A na l iq u o t (2 5 ]d ) o f t h e b o i l ed t r an s l a tio n m ix tu re i s d i lu t ed w i th 2 5 0 /1 1 o f th eim m u n o p rec i p i t a t io n b u f f e r ( IB ) (50 m M T ris -HC 1 b u f f er , p H 7 .6 , 0 .1 5 M

12 M . K untz, J . L. Evrard, A. d 'Harlin gue, J. H . W eil , and B. Cam ara, Mol. Gen. Genet. 216,156 (1989).13 H. Av iv an d P. Leder , Proc. Natl. Acad. Sci. U.S.A. 69, 1408 (1972).


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32 Plant Phytoene Synthase Complex