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Expression of the nuclear gene encoding oxygen-evolving enhancer protein 2 is required for high levels of photosynthetic oxygen evolution in <i>Chlamydomonas reinhardtii</i>

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Expression of the nuclear gene encoding oxygen-evolving enhancer protein 2 is required for high levels of photosynthetic oxygen evolution

in Chlamydomonas reinhardtii

MAYFIELD, Stephen Patrick, et al.

Abstract

We have cloned a cDNA encoding a 20-kDa polypeptide, oxygen-evolving enhancer protein 2 (OEE2), in Chlamydomonas reinhardtii. This polypeptide has been implicated in photosynthetic oxygen evolution, and it is associated with the photosystem II complex, the site of oxygen evolution in all higher plants and algae. The sequence of OEE2 cDNA, the deduced amino acid sequence of the preprotein, the N-terminal protein sequence of mature OEE2 protein, and the coding regions of the single OEE2 gene are presented. The protein is synthesized with a 57-amino acid N-terminal transit peptide that directs the transport of this polypeptide across three cellular membranes. A nuclear mutant of C. reinhardtii, deficient in oxygen-evolving activity, is shown to be specifically missing OEE2 polypeptides. This mutation, which results in the complete absence of all OEE2 mRNA and protein, does not affect the accumulation of other photosystem II polypeptides or their mRNAs.

MAYFIELD, Stephen Patrick, et al. Expression of the nuclear gene encoding oxygen-evolving enhancer protein 2 is required for high levels of photosynthetic oxygen evolution in

Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences, 1987, vol. 84, no. 3, p. 749-753

DOI : 10.1073/pnas.84.3.749

Available at:

http://archive-ouverte.unige.ch/unige:149393

Disclaimer: layout of this document may differ from the published version.

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Proc. Natl. Acad. Sci. USA Vol. 84,pp.749-753, February 1987 Botany

Expression of the nuclear gene encoding oxygen-evolving enhancer protein 2 is required for high levels of photosynthetic oxygen

evolution in Chlamydomonas reinhardtii

(photosystemII/thylakoid membrane/transitpeptide)

STEPHEN P. MAYFIELD*, MICHtLE RAHIRE*, GERHARD

FRANKt,

HERBERTZUBERt, AND JEAN-DAVID RoCHAIX*

*Departments of Molecular Biology and Plant Biology, University of Geneva, CH 1211, Geneva, Switzerland; andtInstituteof Molecular Biology and Biophysics,Eidgenossische TechnischeHochschule,Honggerberg, CH8093,Zurich,Switzerland

CommunicatedbyDiter von Wettstein, October 13, 1986 (receivedforreview September 8, 1986) ABSTRACT We have clonedacDNA encodinga 20-kDa

polypeptide, oxygen-evolving enhancer protein2 (OEE2), in Chlamydomonas reinhardtii. Thispolypeptide has been impli- cated in photosynthetic oxygenevolution, andit is associated with the photosystem II complex, the siteof oxygen evolution inall higher plants and algae. The sequenceof OEE2 cDNA, the deduced amino acid sequence of the preprotein, the N-terminalproteinsequenceofmatureOEE2protein, and the coding regions of the single OEE2 gene are presented. The proteinissynthesized witha57-amino acid N-terminal transit peptide that directs the transport ofthis polypeptide across three cellular membranes. A nuclear mutant ofC.reinhardtii, deficientinoxygen-evolving activity,is shown to bespecifically missingOEE2 polypeptides.Thismutation, whichresultsinthe complete absence of all OEE2 mRNAand protein, does not affecttheaccumulationof otherphotosystemIIpolypeptidesor their mRNAs.

All aerobic organismsare dependentupon the evolution of oxygen from the photolysis ofwaterduring photosynthesis for the oxygen required in respiration. Photosynthesis, the conversion oflightenergytochemicalenergyinhigher plants and algae, requires the cooperation of two photosystem reactioncentersandanelectron transportchainthat connects them. Thephotochemicalreactionof photosystemII (PS II) induces a chargeseparation across thethylakoidmembrane, thereby providing the oxidizing power necessary for the formation ofmolecular oxygen and electrons fromwater.The electrons derived from this reaction are passed along the electron transportchaintophotosystemIandareultimately used toreduce NADP. Considering theimportance of pho- tosyntheticoxygenevolution tothebiosphere, verylittle is known about either the enzymatic mechanism or the en- zyme(s) involved in this fundamental reaction.

The photochemical reaction center of PS II and the oxygen-evolving complex are biochemically closely linked andconsistofatleastfive chloroplast-encoded polypeptides, which are members of the core PS II particle, and three nucleus-encoded extrinsic polypeptides, which bind to the lumen side of PS II(forreviewseeref. 1). With theexception of the proteins responsible for chlorophyll binding, it has proven difficult to assign particular functions to specific polypeptides within the PS II complex. Recently several groups have isolatedphotosynthetic membranes or submem- braneparticles withhigh specificactivities of oxygen evolu- tion in vitro (reports compiled in ref. 2). These particles generally contain the fivecorepolypeptides associated with the reaction center of PS II as well as the three extrinsic

polypeptides. The extrinsic polypeptides can be removed from the oxygen-evolving particles by salt washes. The removal ofthese polypeptides results in the lossofoxygen- evolving activities of the particle. Restoration of theactivity canbeaccomplished by the readdition of thesepolypeptides (forreview see ref. 3) or to a limited degreeby the addition ofnonphysiological levels of calcium or chloride ions (4).

Anin vivoapproach to thequestion of whatpolypeptides might be involved in oxygen evolution can be taken by analyzing mutantsof Chlamydomonasreinhardtii, aunicel- lular green algautilizingthesamephotosynthetic schemeas higher plants, that are deficient in oxygenevolution. These mutants canbe separated into two classes; those that lack all PS II activities, including oxygenevolution, and those that aredeficientonly foroxygen-evolvingactivities. The former mutants lack polypeptidesassociated with the reaction center of PS 11 (5, 6), while the latter mutants retainatleastsomeof the core PS IIpolypeptides(7).

Here we report that a C. reinhardtii nuclear mutant deficient inoxygen-evolvingactivity (7), butcontainingother PS IIactivities, specificallylacks both a 20-kDaextrinsicPS IIpolypeptide(oxygen-evolving enhancerprotein2, OEE2) andthemRNAencoding it. All other PS IIpolypeptidesand their mRNAs accumulate to normal levels in thesecells.We also report thecloning and nucleotide sequenceofcDNAand genomic fragments encoding the OEE2 polypeptide, the predicted aminoacid sequenceof the entirepreprotein, the coding regions of thesingleOEE2 gene, and theN-terminal amino acid sequence of purified mature OEE2protein.

MATERIALS AND METHODS

Cell Culture.Wild-typeChlamydomonas reinhardtii strain 137 and lowchlorophyll fluorescence nuclearmutant BF25 (7) were grown inliquidTris/acetate/phosphatemedium, pH 7.0 (8), underfluorescentlightingto a densityof 2-3 x 106 cells per ml. The cells wereharvested by centrifugation at 8000 x gfor10min,resuspendedin½iovol freshmedium,and pelleted againat8000 x gfor 10 min.

ProteinIsolation, Polyacrylamide Gel Electrophoresis,Elec- troblotting,andAntibodyHybridization. Forprotein isolation thepelleted cells were resuspended in 0.8 M TrisHCl, pH 8.3/0.4Msucrose/1%2-mercaptoethanol, quicklyfrozen in adry ice/ethanol bath, allowed to thaw onice, andbriefly sonicated(three10-sectreatments) withamicrotip tobreak thecells. The lysed cellswerepelletedat 15,000 x g for 15 min, thesupernatant(solublefraction) was removed, and the pellet (membrane fraction) was resuspended in the above buffer. Under theseconditions the three extrinsicpolypep- tides associated with PS II are released into the soluble

Abbreviations:OEE1, OEE2,andOEE3, oxygen-evolvingenhancer proteins 1, 2, and 3; PS I and II, photosystems I andII.

749 Thepublicationcostsofthis articleweredefrayedinpartbypagecharge payment. This article must therefore be hereby marked "advertisement"

in accordancewith 18U.S.C.§1734 solelytoindicate this fact.

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Proc.Natl. Acad. Sci. USA 84 (1987) faction and can thus easily be separated from other PS II

polypeptides. Polyacrylamide gel electrophoresis, protein blotting, antibodyhybridization, andautoradiographywere aspreviouslydescribed (9).

Mature OEE2 polypeptides were isolated from C. rein- hardtii mutant F54-14(10)whichlacksboth the PS I and the chloroplastATPasecomplexes.Useofthismutantfacilitated the isolation of PS IIparticles freeofcontaminating poly- peptides. PS II particles wereprepared accordingto Diner and Wollman (11).ThePSIIparticleswereremovedfromthe sucrosegradient, adjustedto5% trifluoroaceticacid,and loaded ontoaHPLCreverse-phase C8 column(Whatman,300-A pore size). Theproteinswereelutedfrom thematrix witha0-100%o gradient of acetonitrile. The samples were lyophilized to re- moveacetonitrile andtheaminoacidsequencewasdetermined byusinganAppliedBiosystemsmodel 470Aproteinsequenator accordingtostandardprocedures.

Isolation of cDNAs andGenomic Clones Encoding OEE2. A C. reinhardtii cDNA library was constructedin XgtlO (14).

The XgtlO DNA was digested with EcoRI, and the cDNA inserts were purified by agarose gel electrophoresis and inserted intotheuniqueEcoRIsiteofXgtll (PromegaBiotec, Madison, WI). Immunoscreening ofthe Xgtll recombinant phageswas asdescribedby Young and Davis(12),usingthe sameantiseraasusedfortheproteinblothybridizations. A single positive plaquewasdetectedwith antiseraspecificto OEE2,and the cDNAinsertwassubcloned in the EcoRIsite in plasmid pUC19. The resultant plasmid, pPII-19.1, con- taineda 1.4-kilobase (kb)cDNAinsert. To isolate genomic clones encoding OEE2 the cDNA insert ofpPII-19.1 was nick-translatedandhybridized bythemethod ofBenton and Davis (13) toa C. reinhardtiigenomiclibrary (14) cloned in X EMBL3. Six overlapping clones were isolated and a restriction map was compiled (Fig. 1). One ofthe clones, E19-2, containedtheentirecodingregion of OEE2 andwas usedfor subcloninginplasmidpUC19.

500 bp

4- 3 E19-2

500bp

LI

PPel9-1

FIG.1 Retito. ma an seunigshm fcN ln

1-so*o PI- * I ar

X . ~E

FIG. 1. Restriction map and sequencing scheme of cDNA clone pPII-19.1 andgenomic clone E19-2. bp, Base pairs. The restriction sites for EcoRI (o), Pst I(o~),Sal I(.),AvaI(u),and Pvu I(tx)are shown forgenomiccloneE19-2,and the restriction sites forEcoRI (o), Hinfl (e),BstNI(v), TaqI(o), Sau96I(v), andAvaI(n)are shownfor cDNA clonepPII-19.1.Theheavylineongenomicclone E19-2indicates thecodingregionsofthesingleOEE2 gene.Thethree AvaI(m) sites within thegenomicclonearealsofound in the cDNA.

Forsequencingthe 1.4-kb cDNAclonewasdigestedwith Hinfl(A, B), BstNI(C), TaqI(D),AvaI(E),and Sau96I(F).Genomic clone E19-2wasdigestedwithAvaI.Fragmentswerelabeledateither their 5'(I)ortheir 3'(x)ends and the sequencewasdetermined for the length indicatedbythearrow.

Sequencing of pPll-19.1 and Genomic Clone E19-2. The 1.4-kbcDNA insert ofplasmid pPII-19.1 was digested with restriction endonucleases and labeled either 5' or 3' as diagrammedinFig. 1. Genomicfragments from clone E19-2 were subclonedin plasmid pUC19, digested withrestriction endonuclease Ava I, and labeled at the 5' and 3' ends as diagrammedinFig. 1. Thelabeledfragments weredenatured, separatedon strand-separating acrylamidegels, eluted, and thenchemicallycleaved,and the sequences were determined asdescribed by Maxamand Gilbert (15).

RNA and DNA Isolation, Electrophoresis, and Blotting.

RNA was isolated with guanidinium hydrochloride as de- scribed (16). Total RNA, 10 ug per sample lane, was separatedondenaturingformaldehyde/agarosegelsand then electroblotted to nylon membrane (GeneScreen; New En- gland Nuclear) in 25 mM sodiumphosphate, pH 6.5. After electroblotting thefilters were baked in a vacuum oven at 80'Cfor1 hrandstained withmethyleneblue (17) to check forevenloadingandtransferofRNA. Thefilterswerethen prehybridized and hybridizedas described (18).

IsolationofC. reinhardtiiDNA,digestion withrestriction endonucleases, electrophoresis, and blotting to nitrocellu- lose membranes were as previously described (19).

RESULTS

Cloningof OEE2 cDNA and Characterization of the Corre- sponding Single Nuclear Gene in C.

reinhardiji.

Indirect evidence suggestedthat allthreeoftheextrinsicpolypeptides associatedwith oxygenevolutionareencodedby thenucleus in C. reinhardtii (6) as they are in higher plants (20). We therefore constructed aXgtll expression vector library con- tainingC.reinhardtiicDNAinserts and identified theexpressed fusionprotein products by reactionwith antisera specific for eachofthe threepolypeptides. cDNAclones wereobtained for each of the three nucleus-encoded polypeptides. A single plaque containing a phage with a 1.4-kb cDNA insert was obtainedby using antiseraspecificforOEE2. This cDNA clone, which hybridizedto a1.5-kb mRNA, wassubclonedinplasmid pUC19 and sequenced by themethod ofMaxamandGilbert (15). Arestrictionmap and the sequencing scheme of cDNA clonepPII-19.1 areshowninFig. 1.

The1.4-kb cDNA clonepPII-19.1wasnick-translated and hybridizedto genomicDNA blots. As shownin Fig. 2, the OEE2 cDNA hybridized to a single or small set ofDNA fragments, depending on the restriction enzyme used. Ge- nomic clones encoding OEE2 were isolated from a C.

reinhardtii library.Severaloverlappingclones wereobtained

kb

19.6- 9* 9

9.4-

5.9- 3.9-

0.5-

FIG.2. Southernanalysis ofwild-type C. reinhardtii genomic DNA. DNA was

digestedwitharestrictionendonucleaseas

* indicated on thefigure, separated by agar- ose gel electrophoresis, and blotted to nitrocellulose. The blot was hybridized witha nick-translated cDNA insert from plasmidpPII-19.1.

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and a restriction map of the chromosomal segment was compiled by using commonrestriction endonucleases (Fig.

1). Note thatinthe Pst Idigest ofgenomicDNAshown inFig.

2 there are only two hybridization signals and that the restriction map of the cloned genomic segment (Fig. 1) predicts 2 fragments ofapproximately 900 bp(strongband) and 1 fragmentof1.7 kb (weak band). Comparisonof other restriction fragments from thegenomiccloneswith Southern blot analysis of genomic DNA confirmed that OEE2 is a single-copy gene (data notshown). Genomicclones contain- ing the entire OEE2 gene weresubclonedinplasmid pUC19, digested with theappropriate restrictionendonuclease, and labeled at either their 5' or 3' ends. The labeled fragments were then hybridizedwithanexcess ofC. reinhardtiiRNA and thecodingregionsweremapped by using S1exonuclease

Proc.Natl. Acad. Sci. USA 84 (1987) 751 (21).The four codingregionsand the 5' and 3'boundariesof thesingle OEE2gene areshown in the upper map ofFig. 1.

OEE2 Is Encoded as a 245-AminoAcid Precursor Polypep- tide That Is Cleaved to a Mature Proteinof188 AminoAcids.

Toverify that pPII-19.1 encoded OEE2 and to determine the aminoacid sequence of the OEE2 protein, the OEE2 cDNA was sequenced. OEE2 cDNA was digested and labeled at either the 5' or 3' end as diagrammed in Fig. 1. The labeled fragments were then sequenced by the chemical cleavage method of Maxam and Gilbert (15). The cDNA pPII-19.1 containedthe 3'poly(A) tail but was truncatedatthe 5' end.

To obtain the 5' coding sequence of OEE2, subclones of genomic fragments containing the 5' end of the gene were digested with Ava I, labeledatboth the 5' and 3' ends, and sequenced as before (see map in Fig. 1). The composite

.-15o . . . . .-100

CCCGCGCTGCRTCGTGGCCRTTTTGGCGCGCGCGCCCRGRGRRTGCTGCRTGGGCTGCRRGRTCTTCCGTRCGG

.-50 Si

TTCRTCGCCRGCCCCCTTRRTTTCTTCCTTGCGCGRCCGRGCCRGCTTGTCRCCTRTTCRCGTTRGGCRGRTRCCRRRR

.30 . .60

RTG GCG RCC GCT CTG TGC RRC RRG GCC TTC GCT GCC GCC CCC GTG GCT CGC CCG GCT TCG

£. M R T R L C N K R F R R R P V AR P R S

pPHI-19-1 E19A

4 90 ..120

CGC CGC TCG GCT GTC GTC GTC CGC GCC TCG GGC TCG GRT GTG RGC CGC CGC GCT GCT CTG

21. R R S R V V V R R S G S 0 V S R R R R L

.I50

GCT GGC TTC GCT GGC GCT GCC GCT CTG GTG TCC TCG AGC

41. R G F R G R R R L V S S S

*.* .180

CCC GCC RRC GCT GCC TRC GGT

P R N R R Y G

R Y G

.210 . .240

GRC TCT GCC RRC GTG TTC GGC RRG GTG RCC RRC RRG TCC GGC TTC GTG CCC TRC GCC GGC

61. 0 S R N V F G K V T N K S G F V P Y R G

0 S R N V F G K V T N K S G F V P Y R G

.270 . . 300

GRT GGC TTC GCT CTG CTG CTG CCC GCC RRG TGG RRC CCC RGC RRG GRG RRC GRC TTC CCC

el. 0 G F R L L L P R K w N P S K E N 0 F P

- G F R MS L L P R K

GGC GTG RTC CTG CGC TRC GRG

101. G V I L R Y E

CRG GRC RCC GRC RRG RRG GCG

121. 0 0 T 0 K K R

.330 . . .360

GRC RRC TTC GRC GCC GTG RRC RRC CTG GTC GTC RTT GCC

0 N F 0 R V N N L V V I R

.390 . .420

RTC GCC GRC TTC GGC RGC CRG GRC RRG TTC CTG GRG TCC

I R 0 F G S a 0 K F L E S

GTC TCC TRC CTG CTG GGC RRG CRG GCC TRC TCC GGC.450

141. V S Y L L G K 0 R Y S G

.480

GRG RCC CRG TCG GRG GGT GGC TTC

E T 0 S E G G F

.510 . .540

GCC TCC CTG CTG GRC GTG TCG RCC RCG RCC GRC RRG RRG

R S L L 0 V S T T T 0 K K

.570

GGC RRG RCT TRC TRC RRG TAC GRG CTG CTG GTC CGC

181. G K T Y r K Y E L L V R

.630 CGC CRC CRG CTG RTC GGC GCC RCC GTT GGC TCC GRC

201. R H a L I G R T V G S 0

TCC GCC GRT GGC GRT GRG GGC GGC600

S R D G 0 E G G

ARC RRG CTG TRC RTC RTC RRG RTC.660

N K L Y I I K I

.690 . .720

CRG RTT GGC GRC RRG CGC TGG TTC RRG GGT GCC RRG RRG GRG GCC RTG GGC GCC TTC GRC

221. 0 I G 0 K R W F K G R K K E R M G R F 0

.750

TCC TTC RCC GTT GTG TRR GCGGCCRRCCTGGCTCGCGTRRGGRRCGGGTGGRRGGRRGRGRGCTCGGARRGCC 241. S F T V V

.800 . . . .950

GCRTCCTGGTGCGTCGGGCGTCTGCRGGCGCCCTCGCTCGGRGCTCGGRGCCCGGCGCRRGCTGCTGCTGCGGRCTTGR

.900 . . . .950

GTGCGRGRRCTCTGGGCTGCTGRRTGGRGRGCRGCGRGGRGTTGCGCGTGRGGGRRRGCRTGGRGCRGGGRTCGGCRTG .1000

RRTGGCGRTTTTCGGRTTCGCRGCGGTGTCRTTTTGTGTTRGCCCGGGCRTGCGCCTTGCGTGGRGCTGTGCCTRTTCG

.1050 . . . .1100

TGGGRGTGTGRCCGGGTGCGTGGCRGTCGGCGGCRTRGCCCCRGCGCCTTGTGGTGRGCCRGTGCGGTTGGCCTGRGGG .1150

TGGGGCRRTCTGRTGCCCCGRCCTGCTGTGCGTGTTTTGCGTGTCRTTTTGGRRCTTGTTCTTRCCGGTTCCGRCGGTG

.t200 . . . .1250

CCTRCCRTRGCGGTGGRGGTGGGTGCTGCRRTRTGGCGGRGTGTGTGTGGGCGGTGTCRRRGCGCCGCCRCRCRGCTRG

.1300

CRGRGGRCGRCTGCGGCCRTTTTGCRRRTCRCRGGCRRRRTGCCRRCGRRGTTTGGCRCCCRGTRCRGCGTGTTTCGTT

.1350 . . . w . .1400

CRTAGTCCCCTCTGCRRCCCTCTTGRGRRGRRRRCTGTARRCCCRTTTCCGTTRRRRRRRRARRRRRRRRRRRARRRRR RRRARRARR

FIG. 3. Nucleotide sequence of cDNApPII-19.1 (arrowpointing right) and genomic fragment E19-2 (arrow pointingleft), predictedamino acidse- quence ofOEE2, and N-terminal se- quenceofmatureOEE2protein. Ami- noacid residuesarerepresented bythe standard one-lettercode. The 5'termi- nus(Si)of the mRNA is 32 bases from the methionine initiation codon ATG (residue1). Note that A isneverused in the thirdpositionof any codon and that themethionine initiationcodon is pre- ceded by four A residues (=); this renders all three reading frames null just prior to the start codon. The N terminus ofthe matureOEE2proteinis 58 aminoacids(*)from theNterminus of the deducedpreprotein, and differs atonepoint(M1),whichisprobablydue to a misreading of themature protein sequence. A possible polyadenylyla- tion site(wavyoverline)hasbeen found in all C. reinhardtii nuclear genes se-

quencedtodate(refs.6and22;S.P.M.

unpublisheddata).Alsonotethatthe 3' untranslatedportionof the sequence is almost aslongasthatof thetranslated portion; the reason for this long 3' extension isnotclear.

GCC CCC RRC CGT GTG TCG GCT

161. R P N R V S R

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Proc. Natl.Acad. Sci. USA 84(1987) sequence derived from both the cDNA and genomic frag-

ments is presented in Fig. 3.

Toconfirm the identityof pPII-19.1 as the cDNAencoding OEE2,we isolated mature OEE2proteinfrom C.reinhardtii cells anddetermined theN-terminal aminoacidsequence.As shownin Fig. 3, the Nterminus of the mature OEE2protein does not correspond to the N terminus of the deduced preprotein, but rathercorresponds to a sectionofthe poly- peptide that is 57aminoacidsfromthe methionineinitiation amino acid. The 25-amino acid sequence derived from the mature protein differs atonly one point from the deduced aminoacid sequence (Fig. 3,aminoacid67). Wetake thisas proofthat pPII-19.1 and E19-2 encode OEE2.

A Nuclear Mutant of C. reinhardtii Deficient in Oxygen Evolution Specifically Lacks OEE2 Polypeptides. A nuclear mutant of C. reinhardtii (BF25) that has only 5% of the oxygen-evolving activity of wild-type cells yet contains normallevels of other activities associated withPS II (7) is lacking a 20-kDapolypeptide associated with PS IIparticles (Fig. 4A). Proteins were isolated from wild-type and BF25 cells,separated intosoluble and membraneprotein fractions, and electrophoretically separated on denaturing polyacryl- amidegels in the presence of0.1% sodium dodecyl sulfate.

Afterelectrophoresiseither theproteingels were stainedwith Coomassie blue or the proteins were transferred toCNBr- activated paper for reaction with specific antisera. Exami- nation of the stained gels reveals that a 20-kDa protein is missing from the soluble protein fraction ofmutant BF25, whilemost otherpolypeptides appear to be present inequal quantities in bothmembraneand solubleprotein fractions in both wild-type and mutantcells (Fig. 4 A and C). Reaction withantisera specific to polypeptides associatedwithPS II showsthatonly the 20-kDaextrinsic polypeptide (OEE2) is missing from the mutant (Fig. 4B). The 26-kDa (OEE1, referred to as the 33-kDa protein in spinach) and 17-kDa (OEE3) extrinsic polypeptides and all of the core PS II proteins accumulate to similar levels in both wild-type and BF25cells (Fig. 4 B andD).

Mutant BF25 Lacks Only the mRNA EncodingOEE2. To determineiftheloss of OEE2 proteinwas directly dueto a mutationin the gene encoding thispolypeptide and not to a pleiotropic effectof amutationatanothersite,weused the OEE2 cDNA toprobe RNA and DNAblotsofwild-type and BF25cells.Equal amounts of total RNA fromwild-type and BF25 cells werefractionatedondenaturingagarosegelsand electroblotted onto nylon membranes. The blotswerethen hybridized withnick-translated cDNAprobes encodingthe 26- and 17-kDapolypeptides associated withoxygen-evolv- ingactivity (OEE1 andOEE3;cloning and identificationtobe reportedelsewhere)and with nick-translatedpPII-19.1.RNA blots were also probed withnick-translated cloned chloro- plasticgenomicfragmentsspecificfor mRNAsencodingthe corePS IIpolypeptides D1, D2,P5, and P6(23). Asshown in Fig.5, all of thechloroplast mRNAsencodingcorePS II polypeptides accumulate to similar levels in bothwild-type andBF25 cells. The two nuclearmRNAsencodingOEE1 and OEE3also accumulate towild-typelevelsin BF25 cells. The only mRNAencodingaPSIIprotein that failedto accumu- latein BF25 cells was OEE2 mRNA. Southern blotanalysis of genomic DNA from wild-type and BF25 cells failed to revealany differences between the two (datanotshown).

DISCUSSION

We have shown that a nuclear mutantofC. reinhardtii that has only5% of the oxygen-evolvingactivity ofwild-typecells lacks a single 20-kDa water-soluble protein(OEE2) associ- atedwith PSII.The lossof OEE2polypeptide is duetothe complete failure of OEE2 mRNA accumulation. Thus, the mutation ofBF25ismostlikelywithin thesingleOEE2gene, but itmayalsobe in another genethatsomehow affects the

A

H LL

kDa

B C14LLL-)

~1dUc

C04LL

3: CD

92.5- _

66- 4

W..

31- 9

OEE1-U.j.

-.0 EP -OEE2

22.5- 14.5-

c CN

kDa 92.5- #

EE3

-ililSb

D Ln

LL

,:,T

Ln LO Ln

F- LL LL H 11

3o co 1co

66- i4

5- P6-

31-

-D2

.4 -2

21.5 14.5-

FIG. 4. Polyacrylamide gelelectrophoresis ofwater-soluble (A

andB) and membrane(C andD) proteins isolatedfrom wild-type (WT)andmutantBF25cells.Afterelectrophoresistheproteinswere

either stained with Coomassie blue (A and C) or transferred to

CNBr-activatedpaper(BandD).Theproteinblots in Bwereallowed

to react with antisera specific for three extrinsic polypeptides

associated with PS II: OEE1,OEE2, and OEE3. Theproteinblots

inDwereallowedto reactwithantiseraspecificfor the PS IIcore proteins D1, D2, P5, and P6. The proteins were visualized by autoradiography after labeling of the antigen-antibody complexes

with 125I-labeled StaphylococcusaureusproteinA.

stable accumulation of OEE2 mRNA. The loss of OEE2 polypeptide does notaffecttheaccumulation of other PS II

polypeptides ortheirmRNAs.

Sequence analysis

ofmature OEE2 protein and of cloned OEE2 cDNA and

genomic

fragments showed that the

polypeptide

is

synthesized

as a

245-aminoacid precursorpolypeptide, which is cleavedtoa

188-amino acidmatureproteinbythe time it reaches its final destination within the chloroplast.

Several groups have isolatedchloroplast membrane

parti-

cles thatarecapableofhighratesofoxygenevolutioninvitro.

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Proc. Natl.Acad. Sci. USA 84 (1987) 753

A In L Ln , B a a

w u LU LL LL u1

i: m c:o i m mcomBco

P6- *

OEE1-@

P5-4* * -D2

V-Di

FIG. 5. Blot hybridization analysis of mRNA is wild-type (WT) and mutant BF25 cells. Total RNA was denaturing formaldehyde gels and electroblotted to branes. The filters were hybridized with nick-transl cDNAs encoding OEE1, OEE2, or OEE3 (B) and chloroplastgenomic fragments encodingthe PS II core D2, P5,orP6(A).

Analysesof theseparticleshave shown them to reaction center proteins of PS II and in most extrinsicpolypeptides, referred to here as OEE1, OEE3, which could be removed fromthe corePi by salt washing. Removal of these proteins fron coreparticleresulted in the loss ofoxygen-evolvi which could be restoredtovarious degrees by the of the extrinsicpolypeptides. Thegeneral pictur from this in vitro work is that OEE2 and OEE 17-kDapolypeptides ofspinach)mayplay some binding or concentrating of Ca2' and Cl-, w

(33-kDapolypeptideofspinach)isnecessaryfor ofMn2+ (for reviewseerefs. 1 and3).

There is some debate on which of the perip]

polypeptides are absolutely necessary for 02 Several groups have reported that the PS II cc OEE1proteinaresufficient for02evolution (4, 24 others havereported thatthe PSIIcoreandOEE theminimaloxygenevolving complex (31-33).He that, in vivo, OEE2 is absolutely requiredforhis oxygen evolution. However, it is important t(

mutant BF25, although deficient in 02 evolut completely devoid of02evolution and in fact is photosynthetic growth (approximately 5% ofwi els). Although OEE2 is required for 02 evo apparent from the protein blotting data that 0 necessaryfor theaccumulation of thecore PS I]

for the accumulation of eitherOEE1 orOEE3.

The57-aminoacid transitpeptideof OEE2mu- polypeptideacrossthechloroplastouterandinni membranes and thenacrossthethylakoidmembr for thepolypeptidetoreachitsbindingsiteonthe of the PS II complex. Examination of the trar shows ittocontainmostlyhydrophobicand unchE acids, inparticularalanine (33%), serine (14%), (10%).Thechargedresidues, mainly arginine,are

evenly throughouttheN-terminal half of the se(

are missing in the C-terminal portion. Asimilar region has alsobeen found in the C terminus ol peptideofplastocyanin,aproteinthat,likeOEE:

on the lumen side of the thylakoid membrane ( features are also foundinthetransit sequence ofI

encodedyeast mitochondrialcytochromecl,apr boundtotheinnermitochondrial membrane and(

HL.L the intermembranespace.The N-terminal half ofthis transit sequence is abletotarget theproteininto the mitochondrial matrix, while it appears that the C terminus of the transit peptideacts asastop transfersignalfor the innermembrane

(35).Itmay be thatasimilarmechanism is used for thesorting ofOEE2 withinC. reinhardtii.

-OEE2 We thank P. F. Spahr for help with the HPLC isolation ofproteins,

**-OEE3 0. Jenni for help in preparing the figures, N. H. Chua and L.

McIntoshforproviding antisera,M.Goldschmidt-Clermontand A.

Shawforconstructing the XgtlOcDNA library,and A. Dayfor a

criticalreadingof themanuscript.WeareespeciallythankfultoP.

Shawforhelpfuldiscussionthroughouttheseexperimentsandfora

criticalreadingofthemanuscript.This workwassupported byGrant 3.587.084from the Swiss NationalFoundationtoJ.-D.R.,andNational Institutes of Health PostdoctoralFellowshipGM10246-02toS.P.M.

1. Critchley,C. (1985)Biochim.Biophys.Acta811,33-46.

2. Inoue, Y.,Crofts,A. R.,Govindjee, Murata, N., Benger,G. &

Satoh,K.,eds.(1983)TheOxygenEvolvingSystemofPhoto- olated from synthesis (Academic, Tokyo).

separatedon 3. Murata, N. & Miyao, M. (1985) Trends Biochem. Sci. 10, nylon mem- 122-124.

lated cloned 4. Preston,C. &Critchley,C.(1985)FEBSLett. 184,318-322.

with cloned 5. Chua,N.-H. &Bennoun,P.(1975)Proc.Natl.Acad. Sci. USA proteinsD1, 72,2175-2179.

6. Delepelaire,P.(1984)EMBO J.3,701-706.

7. Bennoun, P., Diner, B.A., Wollman, F.-A., Schmidt, G. &

contain the Chua,N.-H.(1981)inPhotosynthesis III,ed.Akoyunoglou,G.

cases three (Balaban,Philadelphia),pp.839-849.

OEE2,and 8. Gorman, D. S. & Levine, R. P.(1965)Proc.Natl.Acad. Sci.

SII particle USA54,1665-1669.

n the PS II 9.

Mayfield,

S. P. & Taylor, W. C. (1984) Planta 161, 481-480.

Ing activity, 10. Chua, N.-H.,Matlin,K. & Bennoun, P. (1975) J. Cell Biol. 67,

-readdition

11. Diner, B. A. &Woilman, F.-A. (1980) Eur. J. Biochem. 110, re emerging 521-526.

_3 (23- and 12. Young, R. A.&Davis, R. W. (1985) in Genetic Engineering:

role inthe PrinciplesandMethods,eds.Setlow,J. K. &Hollaender,A.

rhile OEE1 (Plenum, NewYork),Vol. 7.pp. 29-41.

thebinding 13. Benton, W. D. & Davis, R. W. (1977) Science196,180-182.

14. Goldschmidt-Clermont, M. & Rahire, M. (1986) J. Mol. Biol.

heral PS II 191,421-432.

evolution. 15. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65,

:)reand

the499-560.

3re

and the 16. Nelson, T., Harpster, M. H.,Mayfield,S. P.& Taylor, W. C.

1-30),while (1984) J. Cell Biol. 98, 558-564.

2 constitute 17. Khandjian, E. W. (1986) Mol. Biol. Rep. 11,107-115.

,re weshow 18. Johnson, D.A., Gantsch, J.W., Sportman, J. R. & Elder, ghlevels of J. H.(1984)Gene Anal. Tech.1,3-8.

) note that 19. Rochaix, J.-D. (1978) J. Mol. Biol. 126, 597-617.

Lion, is not 20. Westhoff, P., Jansson, C., Klein-Hitpab, L., Berzborn, R., capable of Larsson, C. & Bartlett, S. G.(1985)Plant Mol. Biol.4, 137-146.

id-type

levo

21. Berk,A.J.&Sharp,P. A. (1977)Cell12,721-732.

id-type 1ev- 22. Silflow, C.D., Chisolm, R. L., Conner, T. W. & Ranum,

lution it i L. P. W. (1985)Mol. Cell.Biol. 5, 2389-2398.

tEE2 iS not 23. Rochaix, J.-D. (1981) Experientia 37, 323-332.

[particleor 24. Ono, T.-A.&Inoue, Y. (1983)FEBS Lett. 164, 255-260.

25. Ono,T.-A.&Inoue,Y. (1984)FEBSLett. 166,381-384.

stdirect the 26. Ikeuchi, M., Yuasa, M. &Inoue, Y. (1985)FEBSLett. 185, erenvelope 316-322.

aneinorder 27. Tang, X.-S. & Satoh, K. (1985)FEBSLett. 179, 60-64.

-lumen side 28. Satoh, K., Ohno, T. & Katoh, S. (1985)FEBSLett. 180, 326-330.

nsitpptide 29. Ghanotakis, D. F., Babcock, G. T. & Yocum, C. F. (1985) nsit peptide FEBSLett. 192, 1-3.

arged

amino 30. Ghanotakis, D. F., Babcock, G. T. & Yocum, C. F. (1984)

,(and valine Biochim. Biophys. Acta 765, 388-398.

distributed 31. Henry, L. E. A. & Moller,13. L. (1981) Carlsberg Res. Com- quence, but mun.46,227-242.

runcharged 32. Henry, L. E.A., Moller, B.L., Andersson,B. &Akerlund, f the transit H.-E. (1982)CarlsbergRes. Commun. 47, 187-198.

2, is located 33. Moller, B. L. &Hoj,P. B. (1983) Carlsberg Res. Commun. 48,

'34).34). SimilarSimilar ~34. 1115Smeekens, S., de Groot,M.,von Binsberger, J. & Weisbeck, thenucleus- P. (1985) Nature (London) 317, 456-458.

*otein that is 35. van Loon, A. P., Braendli, A. W. & Schatz, G. (1986) Cell44, extendsinto 801-812.

Botany: Mayfield

etal.

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