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Expression of the nuclear encoded OEE1 protein is required for oxygen evolution and stability of photosystem II particles in Chlamydomonas reinhardtii.

Expression of the nuclear encoded OEE1 protein is required for oxygen evolution and stability of... The EMBO Journal vol.6 no.2 pp.313-318, of Expression the nuclear encoded OEE1 is protein required for oxygen evolution and of stability II photosystem particles in Chlamydomonas reinhardtii Stephen P.Mayfieldl'3, Pierre Bennoun2 and be removed Jean-David from the salt particle by washing (Compiled in In- Rochaix1 oue et The removal of these al., 1983). water soluble extrinsic polypeptides from the PS II complex results in the loss of ox- 'Departments of Molecular Biology and Plant Biology, University of which can Geneva, CH 1211 ygen evolving activity, be restored Geneva, Switzerland and partially by the 2Institut de Biologie Physico- Chimique, 13, Rue Pierre et Marie Curie, Paris re-addition of the extrinsic 75005, France to the PS I core polypeptides (review- ed Murata and 3Present address: by Miyao, 1985; Critchley, Department of Molecular Biology, 1985). Scripps Clinic and Research Here we an in vivo Foundation, 10666 North Torrey Pines of Road, La Jolla, CA 92037, report analysis polypeptides involved USA in photosynthetic evolution. Mutants of oxygen Chlamydomonas a unicellular reinhardtii, green alga utilizing the Communicated by J.-D. Rochaix same photosyn- thetic scheme as most were higher plants, selected which In Chlamydomonas reinhardtii the oxygen evolving enhancer fluorescence displayed high chlorophyll compared to wild-type protein 1 (OEE1), which is part of the oxygen evolving com- which is indicative of the absence of cells, active PS H centers. plex of photosystem H (PS I), is coded for by a single nuclear Several of these mutants were tested for photosynthetic oxygen gene (psbl). The nuclear mutant FuD44 specifically lacks the evolution. One mutant, which is FuD44, completely lacking OEE1 polypeptide and is completely deficient in photosyn- photosynthetic oxygen evolving activity, specifically lacks a 26 kd thetic oxygen evolution. In this mutant a 5 kb DNA inser- nuclear encoded PS II polypeptide (OEE1), known as the 33 kd tion into the 5' region of the psbl gene results in the complete of polypeptide spinach oxygen evolving particles (Kuwabara and absence of OEE1 mRNA and protein. A revertant, FuD44-R Murata, Mutant FuD44 and a 1979). revertant of this mutant con- 2, which is capable of30% of the photosynthetic oxygen evolu- taining 30% of the oxygen evolving activity of wild-type cells, tion of wild-type cells, has lost 4 kb of the 5 kb DNA insert, were characterized. FuD44-R2, and accumulates both OEE1 mRNA and protein, although at levels somewhat less than those of wild-type cells. Absence Results of the OEE1 protein in the FuD44 mutant does not affect the Nuclear mutant FuD44 specifically lacks a 26 kd accumulation of other nuclear II nuclear encoded encoded PS peripheral poly- polypeptide associated with PS II peptides. OEE1 absence does, however, result in a more rapid turnover of the II chloroplast encoded PS core To polypeptides, determine the role of nuclear encoded PS polypeptides 11 in thus in a resulting substantial of II deficiency PS core photosynthetic oxygen evolution we isolated nuclear mutants poly- defi- peptides in FuD44 cells. II These PS core proteins again ac- cient in oxygen evolving activities. One such mutant FuD44, and cumulate in revertant FuD44-R2 cells. a revertant of this mutant containing 30% of the oxygen evolv- Key words: H Chlamydomonas reinhardtii/photosystem particles/ ing activity of wild-type cells, FuD44-R2, were characterized. stability/oxygen evolution/OEEl protein Water requirement soluble were proteins isolated from wild-type, FuD44, and FuD44-R2 cells, and separated on 7.5-15% polyacrylamide gels % SDS. containing 0.1 Following electrophoresis the gels were Introduction either stained with Coomassie Blue or (Figure IA) transferred to CNBr activated paper (Figure IB). The protein blots Photosynthetic oxygen evolution the were requires interaction of with antisera several hybridized raised against the three extrinsic different yet closely biochemical PS II coupled reactions. The polypeptides, light OEEl, OEE2 and OEE3, which have capturing, charge separating capacities of been im- photosystem H plicated in oxygen evolution. As must work in shown in Figure 1B only close cooperation with an one oxygen evolving com- of these polypeptides, is OEEl, lacking in mutant FuD44. This plex capable of utilizing this oxidizing power to split water into kd polypeptide is its (OEE1) again present in revertant molecular and Fu.D44-R2 species, oxygen hydrogen. Electrons stripped cells, although at of from only 50% the wild-type levels (Figure water during this reaction are funnelled back into iB, photo- wt X It is to note that 1/2). interesting while the growth rate of chemical reaction center and then II transported the elec- through wild-type and FuD44-R2 cells is about equal on acetate contain- tron transport chain to photosystem I, to be used for eventually ing media, the growth rate of the FuD44-R2 on minimal reduction of NADP. media, therefore relying 11 exclusively on photosynthesis for Photosystem and the are growth, is oxygen evolving complex physically about only one half that of wild-type cells. The two other linked. Isolation of oxygen a evolving particles yields complex peripheral PS polypeptides, OEE2 and OEE3, are present in containing a and chlorophylls b, carotenoids, quinones, lipids each of the different strains (Figure 1B), the although level of and an of at least and array eight polypeptides (Murata Miyao, OEE2 is somewhat reduced in both FuD44 and in FuD44-R2 1985). Five of known cells. these, Chlamydomonas reinhardtii as are encoded Dl, P6 and FuD44 specifically lacks OEEI D2, P5, C-b559, mRNAs chloroplast (Rochaix, 1981) and localized within the PS reaction center core (Inoue To determine if the absence of OEE1 protein in FuD44 cells was et Erickson et Associated with this al., core 1983; al., 1986). due to a mutation affecting expression of the gene encoding it are three extrinsic nuclear encoded PS particle and not polypeptides, due to a effect of a (psbl), pleiotropic mutation at another enhancer 2 and which we cloned oxygen can site, cDNAs the three extrinsic evolving proteins (OEE 1, encoding 3), polypeptides © IRL Press Limited, Oxford, 313 England S.P.Mayfield, P.Bennoun and J.-D.Rochaix Ai ..... '.. ij .. .... .-Il..1 il .,- : .... C.." --r 3, !.'' I.i. .:" LL k j-j AL b,.. .. b - 6 " OEE'- _ 4U i_. Fig. 1. of water soluble isolated from mutant Polyacrylamide gel electrophoresis and revertant FuD44-R2 cells. proteins FuD44, wild-type (wt), Following the were either stained with Coomassie Blue electrophoresis gels or transferred to CNBr activated (A), (B). The location of electrophoretically paper the OEEI is marked on the stained The protein (-) blots were with antisera to three extrinsic gel. protein associated with PS hybridized specific polypeptides II; OEE1, OEE2 and OEE3. A of wt sample lane 50% the loaded into the lane X is shown for of containing protein levels (wt 1/2) comparison OEE1 protein in the FuD44-R2 after sample. were visualized with 1251-labeled Protein-antibody complexes by autoradiography Staph-A protein. labeling To confirm (-4 pPll-12.4. that encoded the pPll-12.4 OEEl poly- the peptide cDNA insert was ,j- sequenced. Comparison of the .'). a deduced amino acid of sequence pPll-12.4 with the N-terminal 1u- ,a - 3m v > -i amino acid U L L.i a determined from sequence isolated mature OEEl pro- tein confirmed that encodes pPll-12.4 the OEEl polypeptide (se- to be quence reported elsewhere). Total RNA was separated on denaturing agarose gels, blotted to nylon membranes and stained with methylene blue to verify equal and even transfer of the loading RNA (Khandjian, 1986). The membranes were hybridized with nick translated cDNA in- OEEI-- serts from As -DEE2 plasmid pPII-12.4. shown in Figure 2 this cDNA 46 4b 40- -OEE3 hybridized to a 1.6 kb mRNA in wild-type cells which is com- so* pletely missing from mutant FuD44, but is again present in the revertant FuD44-R2. From dilutions of wild-type mRNA we estimate that the revertant contains only about 25% of the OEEl mRNA of not wild-type cells (data shown). Hybridization of iden- tical filters with cDNA encoding OEE2 (Mayfield et al., 1986) and OEE3 (cloning and sequence to be reported elsewhere) show that both of these mRNAs are present in equal quantities in wild- FuD44 and type, FuD44-R2 cells (Figure 2). FuD44 contains a 5 kb DNA insert in the 5' region of the single 2. OEE] Fig. Northern analysis of mRNA isolated from FuD44 gene wild-type (wt), and FuD44-R2 cells. Total RNA was on separated denaturing formaldehyde To characterize the mutation resulting in the specific absence of and electroblotted to membranes. The filters were gels nylon wth hybridized mRNA OEEl and protein we examined the chromosomal nick translated cloned cDNAs the PS II region encoding peripheral proteins OEE1, OEE2 and OEE3. containing the psbl gene by Southern analysis. DNA was isolated from wild-type, FuD44 and FuD44-R2 cells, digested with associated with oxygen evolving particles, and restriction measured the ac- endonucleases, separated on agarose gels, and blot- cumulation of their ted corresponding mRNAs. A C. to nitrocellulose reinhardtii membranes. The DNA blots were then cDNA library was constructed in 1 a Xgtl expression hybridized with nick translated vector and cloned cDNA insert. OEE1 As the expressed fusion proteins were shown identified by in Figure 3B the cDNA hybridization hybridized to a single fragment with antisera specific for the in three extrinsic polypeptides both the EcoRI and Hindlll digests in each of the different (Mayfield et al., 1986). Several DNA plaques were identified using samples. However, in mutant FuD44 the DNA fragment OEEI antisera, and individual phage were isolated. One of the containing the psbl gene is approximately 5 kb larger than the recombinant phage contained a 1.6 kb cDNA insert which was fragment in wild-type cells. In the revertant FuD44-R2 the frag- subcloned into the EcoRI site of plasmid pUC 19 ment to form plasmid containing psbl is approximately 4 kb smaller than the frag- 314 OEE1 protein: oxygen evolution and stability of PSH 1 wild-type counterpart. imately 5 kb and kb larger than their A B This analysis identified the site of insertion of foreign DNA into kb the psbl gene to a 220 bp Hindm-PstI fragment that is located at the 5' end of the psbl gene (see map Figure 3C). Sequencing 21.0- di a of the 220 bp wild-type PstI-HindIH genomic fragment show- a.- ed it to contain 60 nucleotides of an exon, and 160 nucleotides 65- 9 of an intron, located at the 5' end of the psbl gene (data not 5.0 - shown). The exact location of the 5 kb insert within this 220 bp 0m. fragment in FuD44 has not yet been determined. 3.5 - Ihe absence ofOEEJ results in the reduction ofcore PS IIpoly- 2.0- v peptides, but not in a deficiency of their mRNAs To assess what effect the absence of OEE1 had on PS H core polypeptides, membrane proteins were isolated from wild-type, FuD44 and FuD44-R2 cells, and separated on 7.5-15 % poly- 3 - acrylamide gels. Following electrophoresis the gels were either stained with Coomassie Blue (Figure 4A) or transferred to CNBr 0.8 - activated paper (Figure 4B). The protein blots were then hybridiz- ed with antisera specific to the PS II core polypeptides D2, DI, PS and P6, and to the PS II antenna chlorophyll binding protein, Ps t I - - I EcoRI PstI Sal HinIE IL LHC II. As shown in Figure SB all of the PS H core polypep- tides accumulate in FuD44 cells, but only to approximately 10-15% of wild-type levels. These PS H polypeptides ac- Cp P H S A HPt cumulate to a much greater extent in the revertant FuD44-R2 :. I P=71 i * VII, "-11V,'1111,4 cells, but are still reduced to - 50% of the wild-type levels. The LHC H polypeptides, like most of the membrane proteins as visualized by the stained protein gels, accumulate to similar levels in all three strains (Figure 4B). 3. Southern analysis of FuD44 and FuD44-R2 genomic Fig. wild-type, To determine if the reduction in PS H core proteins was a con- DNA. DNA was with restriction endonuclease as indicated on the digested sequence of reduced mRNA levels we measured the accumula- figure, separated by agarose gel electrophoresis, and blotted to nitrocellulose. For each digestion indicated the sample lanes are left to right; tion of chloroplastic mRNAs encoding the PS H core proteins. wild type, FuD44 and FuD44-R2. (A) The blot was hybridized with a nick Equal amounts of RNA from wild-type, FuD44 and FuD44-R2 translated 750 bp HindIII-HindIII cloned wild-type genomic fragment cells were separated on denaturing gels and electroblotted to nylon at the 5' end of the on located psbl gene (marked genomic map C). (B) membranes. The filters were then hybridized with nick translated After removal of this the filter was with a cDNA probe hybridized encoding this cDNA lies 3' to the PstI site within the cloned chloroplastic genomic fragments encoding the PS II core OEEI, completely coding chromosomal the region. (C) Genomic map of the fragment containing psbl polypeptides Dl, D2, PS and P6 (Rochaix, 1981). As shown in sites for PstI HindIll Sall and Aval are gene. Restriction (P), (H), (S) (A) Figure 5 all of the mRNAs encoding these PS II core polypep- are the 5' and 3' ends of the the site of the shown, as coding region (U), tides accumulate in FuD44 and revertant FuD44-R2 cells to levels location of the used as the 5' DNA insert (O), the genomic fragment probe similar to those of wild-type cells. the 5' limit of the cDNA (1z), and probe ( ). Deficiency ofcore PSIlpolypeptides in FuD44 is due to instability of the core complex kb than the ment in but still about 1 larger wild-type FuD44, To determine if the deficiency of PS H core proteins in FuD44 fragment (Figure 3A and B). cells was due to an increased instability of the complex or due Genomic clones containing the psbl gene were isolated from to a reduction in synthesis of the core polypeptides, wild-type, a EMBL3 - C. reinhardtii genomic library, and mapped using FuD44 and FuD44-R2 cells were pulse-labeled with [14C]acetate. common restriction endonucleases. A restriction map of the As shown in Figure 6A the pattern of proteins from cells label- chromosomal the wild-type psbl gene is segment containing ed for 10 with is identical in both presented in Figure 3C. Wild-type, FuD44 and FuD44-R2 DNA min [14C]acetate wild-type and FuD44 cells. Because this pattern of labeled proteins is com- was restricted with endonucleases Sail and HindH, all of PstI, plex we also labeled cells in the presence of cycloheximide, an which cut the into two or more fragments. The DNAs psbl gene inhibitor of cytoplasmic protein synthesis. As shown in Figure were on blotted to nitrocellulose filters separated agarose gels, 6C labeling of membrane proteins in this way allows for the easy and with a 750 bp HindRIl-HindRI cloned wild-type hybridized visualization of the PS II core proteins. The upper panel in Figure which is located at the extreme 5' end of the genomic fragment, 6C is an autoradiograph of proteins labeled for 45 min in the line in 3C). As shown in single psbl gene (crossed Figure Figure on presence of 8 p4g/ml cycloheximide prior to separation poly- this to a fragment in the PstI and 3A probe hybridized single M and shows that the DI and acrylamide gels containing 8 urea, HindIll and to two in the Sail Both the digests fragments digest. D2 are in amounts in proteins synthesized approximately equal PstI and the 1.8 kb Sail are than their wild- fragments larger The lower in 6C is an autoradio- all three strains. panel Figure in FuD44 and while the 750 Hin- type counterpart FuD44-R2, bp of the same labeled on non-urea con- graph proteins separated and the 3.5 kb Sail are the same in all dIl fragment fragment of P4 which allows for the was removed from the filters taining gels, separation polypeptides three DNA This samples. probe and P6 are and shows that both PS with a cDNA which lies and PS, polypeptide synthesiz- and the blot was rehybridized probe ed in three all strains. in 3C. As shown in 3' to the PstI site as indicated Figure Figure without the addition of were chased are the same while labeled 3B the PstI of each size, Cells cycloheximide fragments sample of cold acetate the 10 for 90 in the min of FuD44 and FuD44-R2 are min 3' presence following the HindmI fragments approx- 315 P.Bennoun S.P.Mayfield, and J.-D.Rochaix A4 A4 .. .; a& a..... Fig. 4. of membrane isolated from FuD44 and FuD44-R2 Polyacrylamide gel electrophoresis proteins cells. the wild-type (wt), Following electrophoresis gels Blue or transferred to CNBr activated The blots were were either stained with Coomassie (A), with antisera to the PS paper (B). protein hybridized specific II P5 and to the PS antenna core proteins DI, D2, and P6, II LHC The were visualized chlorophyll binding protein II. proteins by autoradiography after of with 1251-labeled labelling the antigen-antibody complexes Staph-A protein. ! A 4~~~. p(f em - P5 - a,a I--- - ._ _- - P4pz --_ t a&er Fig. 5. Northern analysis of mRNA isolated from FuD44 wild-type (wt), FuD44-R2 and cells. Total RNA was on separated denaturing formaldehyde gels and electroblotted to membranes. The filters were nylon hybridized with nick translated cloned the PS chloroplastic genomic fragments encoding II core proteins D1, D2, P5 and P6. PL pulse. As shown in 6B the of Figure patterns labeled proteins in wild-type and FuD44 cells show some differences. This dif- ference is most easily observed in the Dl and P5 which proteins Fig. 6. Autoradiograph of in vivo labeled membrane proteins from wild-type are in clearly underrepresented the FuD44 cells after the 90 min (wt), FuD44 and FuD44-R2 cells. (A) Cells labeled for 10 min with chase (Figure 6B). Polypeptides P6 and D2 co-migrate with other and [14C]acetate, (B) chased for 90 min in the presence of non-radioactive labeled proteins on these acetate. The site of gels and are thus not as easily observ- migration of polypeptides P5, P6, and D2 is DI marked. (C) Cells labeled for 40 min in the presence of cycloheximide. ed in the autoradiograph (Figure 6B). The loss of P5 and Dl Upper panel shows the proteins separated on polyacrylamide gels containing protein, and we assume P6 and D2 as well, in FuD44 cells must 8 M urea, and the lower panel shows a section of the autoradiograph with therefore be due to a more rapid turnover of the in protein FuD44 similar samples separated on non-urea gels which allows for the separation cells compared to wild-type cells. of polypeptides P4 and PS. 316 oxygen evolution and stability of PSI1 OEE1 protein: exception all of them have identical patterns on Southern analysis, Discussion indicating that the reversion of this mutant is probably not a ran- Several groups have isolated oxygen evolving particles which dom event. It is also interesting to note that the revertant, although contain the PSII reaction center core proteins and three extrin- able to accumulate both OEEl mRNA and protein, does not ac- sic nuclear encoded proteins (reviewed by Critchley, 1985; cumulate either to wild-type levels. This data suggest that this Murata and Miyao, 1985). There is, however, some debate as DNA insert may be a transposable element, but proof of this must to which of the extrinsic proteins are really necessary for await the cloning and analysis of the mutant OEEl locus and photosynthetic 02 evolution. Several groups have reported that its revertants. in vitro the PSII core and OEEl comprise the minimal particle capable of oxygen evolution (Ono and Inoue, 1984; Ikeuchi et Materials and methods al., 1985; Preston and Critchley, 1985; Ghanotakis et al., 1984), others have reported that the PSII core andOEE2 are sufficient Isolation of mutant FuD44, and cell culture conditions for 02 evolution in vitro (Moller and Hoj, 1983; Henry et al., Wild-type cells 137c were treated with 5-fluorodeoxyuridine and metronidazole 1982), while still others (Tang and Satoh, 1985) have concluded as described (Bennoun et al., 1978; Schmidt et al., 1977). Mutant FuD44 was isolated as a high chlorophyll fluorescent colony which was back crossed to wild- that 02 evolution is associated with the PS II core alone. type cells and the mutation shown to be inherited in a Mendelian (nuclear) fashion. Recently we have shown that in vitroOEE2 is required for Complementation analysis in young zygotes (Bennoun et al., 1980) with mutant high levels of 02 evolution, but that in its complete absence there BF25, a nuclear mutant which affects the expression of the OEE2 polypeptide is still some 02 evolution (5% of wild-type levels, Mayfield et (Mayfield et al., 1986), showed that the two mutations are in different genes al., 1986). Here we show that in vivoOEEl is absolutely re- (data not shown). Revertants of mutant FuD44 were selected by plating the mu- tant on agar plates containing minimal media in the light, thus selecting for quired for any photosynthetic oxygen evolution, and that cells photosynthetic growth. Revertants were observed at a frequency of -1 x 10-6 deficient for OEEI, unlike those deficient for OEE2, are in- to l0-7 cells, and 12 colonies, capable of growth on minimal media, were capable of photoautotrophic growth, thus OEE1 is absolutely re- isolated. With one exception all of these revertants showed similar patterns on quired for photosynthesis. Southern analysis so only one revertant, FuD44-R2, was used for further characterization. Oxygen evolution was measured on whole cells with a Clark- Two interesting observations can be made from the Western type oxygen electrode (Rank Brothers, England). analysis of PS II polypeptides. The first is that the absence of Wild-type C. reinhardtii strain 137c, nuclear mutant FuD44 and revertant OEE1 does not result in the loss of eitherOEE2 or OEE3. Thus FuD44-R2 were grown in liquid Tris-acetate-phospha,te media pH 7.0 (Gor- OEE1 is not required for the stability of the other OEE polypep- man and Levine, 1965) under dim fluorescent lighting to a density of 1-2 x 106 cells/ml. The cells were harvested by centrifugation at 8000 g for 10 min tides. We have not, however, attempted to localize OEE2 or resuspended in 1/20 volume fresh media and pelleted again at 8000 for 10 min. OEE3 within the chloroplast and thus we can not say whether Protein isolation, gel electrophoresis, electroblotting and polyacrylamide antbody these polypeptides remain anchored to the membrane in the hybridization absence of OEE1, only that they accumulate to near wild-type Protein isolation and sample preparation were described previously (Mayfield levels and are the correct, mature, size. The second observation et al., 1986). Electrophoresis of proteins on urea containing gels was described is that the absence ofOEE1 causes a substantial reduction of core in Piccioni et al. (1981). Polyacrylamide gel electrophoresis of non-urea con- PS II polypeptides and active PS H centers, but not a reduction taining gels, protein blotting, antibody hybridization, and autoradiography were as described by Mayfield and Taylor (1984). Following autoradiography the filters in other polypeptides closely associated with PS II, like the LHC (CNBr activated paper) were stripped of the primary antibodies by washing the II polypeptides. The in vivo labelling experiments clearly show filters for 30 in 7.5 M guanidinium hydrochloride, 5% (3-mercaptoethanol min that the deficiency of the PS H core proteins in FuD44 cells is and then for 15 min in 0.2 M Glycine-HCl pH 2.8. The filters were then wash- not due to a decrease in synthesis of these proteins, but rather ed for 30 min in hybridization buffer before being rehybridized with a new an- due to a more rapid turnover of the newly synthesized polypep- tisera. As long as the filters were never allowed to completely dry they could be reused several times without apparent loss of signal. tides in the absence ofOEEl protein. This suggests that OEEI, Isolation of cDNA and genomic clones, and RNA and DNA isolation, elec- or perhaps a portion of it, is required for the stable formation trophoresis and blotting of the PS II core particle, perhaps even having some structural Isolation of cDNA clones was described by Mayfield et al. (1986), but basically role within the complex. An alternative explanation could be that involved identification of OEE1 protein expressed in a Xgtl 1 expression vector the loss ofOEEl exposes the PS H core or one of its proteins (Young and Davis, 1985) using the same rabbit polyclonal antisera used for the in such a way that the particle becomes susceptible to proteolytic Western blots. Genomic clones were then isolated from a C. reinhardtii EMBL3 genomic library 1986) by the method of Benton and Davis degradation. Recently we have shown that another 02 evolving (Goldschmidt-Clermont, (1977) using nick translated OEE1 cDNA inserts from the Xgtl 1 clones. deficient mutant of C. reinhardtii, which is missing the OEE2 RNA was isolated with guanidinium hydrochloride as described (Nelson et al., polypeptide (Mayfield et al., 1986), accumulates almost wild- 1984). Total RNA, 10 per sample lane, was separated on denaturing for- jig type levels of all of the core PS H proteins, thus the instability maldehyde agarose gels and then electroblotted to nylon membrane (genescreen) of the PS H core proteins in FuD44 cells must be due to the in 25 mM phosphate pH 6.5. Following electroblotting the RNA was fixed to the filters by u.v. irradiation, and then stained with methylene blue to verify even absence of OEE1 and not due to some pleiotropic effect of the loading and transfer of RNA (Khandjian, 1986). The filters were then prehybridized loss Of 02 evolving activities. and hybridized as described (Johnson et al., 1984). The mutation which results in the absence of OEE1 mRNA Isolation of C. reinhardtii DNA, digestion with restriction endonucleasees, in FuD44 cells is a large, 5 kb, DNA insertion into the 5 prime and blotting to nitrocellulose membranes was as previously describ- electrophoresis ed (Rochaix, 1978). region of the single psbl gene. Revertants of the original mu- Pulse labeling of with [14C]acetate tant, which are again capable of oxygen evolution, have lost 4 kb proteins of this DNA insert. These revertants, although still harboring Cells were grown in complete media to a cell density of 1-2 x 106 cells/ml, then transferred to media lacking acetate for 1 h prior to labelling. The cyclohex- 1 kb DNA insert in the psbl gene, again accumulate OEE1 imide treated cells were made 8 final concentration 10 min prior to the mRNA. We assume from this that the DNA insert must be in ltg/IL addition of label, and were labeled for 45 min with 2 PCi/mi [14C]acetate (56 the non-coding region of the 220 bp genomic fragment which was added to 40 mi of cells which had not been 1Ci of [ 14C]acetate jACi/iM). contains the insert, as the RNA is still correctly spliced to yield and allowed to incorporate for 10 min. The media with cycloheximide treated 50 mM acetate by the addition of 2 M non-radioactive acetate, mature OEE1 mRNA of the correct size. Analysis of then made functional was harvested by centrifugation for 2 min at 10 000 g. Half cells quickly and the of FuD44 (data not shown) shows that with one 12 revertants 317 S.P.Mayfield, P.Bennoun and J.-D.Rochaix of the cells were quick frozen in dry ice ethanol and the other half returned to complete media containing non-radioactive acetate and allowed to grow for an additional 90 min, then harvested and frozen as above. Protein samples were prepared and electrophoresed exactly as the samples used for the stained gels except that following destaining the gels were soaked for 20 min in Enlighting (New England Nuclear), dried onto filter paper under vacuum, and then expos- to film at -70°C. ed Acknowledgements to thank O.Jenni and Y.DeLotto for help in preparing the figures. We We wish also thank Yves Pierre for technical assistance, and P.Shaw for helpful discus- sions and a critical reading of the manuscript. This work was supported by grant 3.587.084 from the Swiss National Foundation to J.D.R., CNRS grant ATP Biologie Moleculaire Vegetale to P.B., and NIH postdoctoral fellowship GM 10246-02 to S.P.M. References Masson,P. and Delosme,M. (1980) Genetics, 95, 39-47. Bennoun,P., and Davis,R.W. (1977) Science, 1%, 180-182. Benton,W.D. Biochim. Biophys. Acta, 811, 33-46. Critchley,C. (1985) Erickson,J.E., Rahire,M., Malnoe,P., Girard-Bascou,J., Bennoun,P. and Rochaix,J.D. (1986) EMBO J., 5, 1745-1754. Ghanotakis,D.F., Babcock,G.T. and Yocum,C.F. (1984) Biochim. Biophys. Acta, 765, 388-398. Goldschmidt-Clermont,M. and Rahire,M. (1986) J. Mol. Biol., 191, 421-432. Gorman,D.S. and Levine,R.P. (1965) Proc. Natl. Acad. Sci. USA, 54, 1665-1669. Andersson,B. and Akerlund,H.-E. (1982) Carls. Henry,L.E.A., Moller,B.L., Res. Commun., 47, 187-198. Ikeuchi,M., Yuasa,M. and Inoue,Y. (1985) FEBS Lett., 185, 316-322. Inoue,Y., Crofts,A.R:, Govindjee, Murata,N., Benger,G. and Satoh,K. (1983) 7he Oxygen Evolving System of Photosynthesis. Academic Press, Tokyo. Johnson,D.A., Gantsch,J.W., Sportman,J.R. and Elder,J.H. (1984) Gene Anal. Techn., 1, 3-8. (1986) Mol. Biol. Rep., 11, 107-115. Khandjian,E.W. Kuwabara,T. and Murata,N. (1979) Biochim. Biophys. Acta, 581, 228-215. Rahire,M., Frank,G., Zuber,H., Rochaix,J.D. (1986) Proc. Natl. Mayfield,S.P., Acad. Sci. USA, in press. and Taylor,W.C. (1984) Planta, 161, 481-480. Mayfield,S.P. and (1983) Carls. Res. Conunn., 48, 161-185. Moller,B.L. Hoj,P.B. (1985) Trends Biochem. Sci., 10, 122-124. Murata,N. and Miyao,M. Mayfield,S.P. and Taylor,W.C. (1984) J. Cell Biol., Nelson,T., Harpster,M.H., 558-564. 98, and FEBS Len., 166, 381-384. Ono,T.-A. Inoue,Y. (1984) Bennoun,P. and Chua,N.H. (1981) Eur. J. Biochem., 117, 93-102. Piccioni,P., and Critchley,C. (1985) FEBS Lea., 184, 318-322. Preston,C. Rochaix,J.-D. (1978) J. Mol. Biol., 126, 597-617. Experientia, 37, 323-332. Rochaix,J.-D. (1981) and Chua,N.H. (1977) Proc. Natl. Acad. Sci. USA, Schmidt,G.W., Matlin,K.S. 610-614. 74, (1985) FEBS Lea., 179, 60-64. Tang,X.-S. and Satoh,K. and (1985) In Setlow,J.K. and Hollaender,A. (eds), Young,R.A. Davis,R.W. and Methods. Plenum Press, NY, Vol. 7, Genetic Engineering: Principles 29-41. pp. Received on 20 November 1986 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Expression of the nuclear encoded OEE1 protein is required for oxygen evolution and stability of photosystem II particles in Chlamydomonas reinhardtii.

Mayfield, S.P.; Bennoun, P.; Rochaix, J.D.
The EMBO Journal , Volume 6 (2) – Feb 1, 1987
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Springer Journals
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Copyright © European Molecular Biology Organization 1987
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1002/j.1460-2075.1987.tb04756.x
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The EMBO Journal vol.6 no.2 pp.313-318, of Expression the nuclear encoded OEE1 is protein required for oxygen evolution and of stability II photosystem particles in Chlamydomonas reinhardtii Stephen P.Mayfieldl'3, Pierre Bennoun2 and be removed Jean-David from the salt particle by washing (Compiled in In- Rochaix1 oue et The removal of these al., 1983). water soluble extrinsic polypeptides from the PS II complex results in the loss of ox- 'Departments of Molecular Biology and Plant Biology, University of which can Geneva, CH 1211 ygen evolving activity, be restored Geneva, Switzerland and partially by the 2Institut de Biologie Physico- Chimique, 13, Rue Pierre et Marie Curie, Paris re-addition of the extrinsic 75005, France to the PS I core polypeptides (review- ed Murata and 3Present address: by Miyao, 1985; Critchley, Department of Molecular Biology, 1985). Scripps Clinic and Research Here we an in vivo Foundation, 10666 North Torrey Pines of Road, La Jolla, CA 92037, report analysis polypeptides involved USA in photosynthetic evolution. Mutants of oxygen Chlamydomonas a unicellular reinhardtii, green alga utilizing the Communicated by J.-D. Rochaix same photosyn- thetic scheme as most were higher plants, selected which In Chlamydomonas reinhardtii the oxygen evolving enhancer fluorescence displayed high chlorophyll compared to wild-type protein 1 (OEE1), which is part of the oxygen evolving com- which is indicative of the absence of cells, active PS H centers. plex of photosystem H (PS I), is coded for by a single nuclear Several of these mutants were tested for photosynthetic oxygen gene (psbl). The nuclear mutant FuD44 specifically lacks the evolution. One mutant, which is FuD44, completely lacking OEE1 polypeptide and is completely deficient in photosyn- photosynthetic oxygen evolving activity, specifically lacks a 26 kd thetic oxygen evolution. In this mutant a 5 kb DNA inser- nuclear encoded PS II polypeptide (OEE1), known as the 33 kd tion into the 5' region of the psbl gene results in the complete of polypeptide spinach oxygen evolving particles (Kuwabara and absence of OEE1 mRNA and protein. A revertant, FuD44-R Murata, Mutant FuD44 and a 1979). revertant of this mutant con- 2, which is capable of30% of the photosynthetic oxygen evolu- taining 30% of the oxygen evolving activity of wild-type cells, tion of wild-type cells, has lost 4 kb of the 5 kb DNA insert, were characterized. FuD44-R2, and accumulates both OEE1 mRNA and protein, although at levels somewhat less than those of wild-type cells. Absence Results of the OEE1 protein in the FuD44 mutant does not affect the Nuclear mutant FuD44 specifically lacks a 26 kd accumulation of other nuclear II nuclear encoded encoded PS peripheral poly- polypeptide associated with PS II peptides. OEE1 absence does, however, result in a more rapid turnover of the II chloroplast encoded PS core To polypeptides, determine the role of nuclear encoded PS polypeptides 11 in thus in a resulting substantial of II deficiency PS core photosynthetic oxygen evolution we isolated nuclear mutants poly- defi- peptides in FuD44 cells. II These PS core proteins again ac- cient in oxygen evolving activities. One such mutant FuD44, and cumulate in revertant FuD44-R2 cells. a revertant of this mutant containing 30% of the oxygen evolv- Key words: H Chlamydomonas reinhardtii/photosystem particles/ ing activity of wild-type cells, FuD44-R2, were characterized. stability/oxygen evolution/OEEl protein Water requirement soluble were proteins isolated from wild-type, FuD44, and FuD44-R2 cells, and separated on 7.5-15% polyacrylamide gels % SDS. containing 0.1 Following electrophoresis the gels were Introduction either stained with Coomassie Blue or (Figure IA) transferred to CNBr activated paper (Figure IB). The protein blots Photosynthetic oxygen evolution the were requires interaction of with antisera several hybridized raised against the three extrinsic different yet closely biochemical PS II coupled reactions. The polypeptides, light OEEl, OEE2 and OEE3, which have capturing, charge separating capacities of been im- photosystem H plicated in oxygen evolution. As must work in shown in Figure 1B only close cooperation with an one oxygen evolving com- of these polypeptides, is OEEl, lacking in mutant FuD44. This plex capable of utilizing this oxidizing power to split water into kd polypeptide is its (OEE1) again present in revertant molecular and Fu.D44-R2 species, oxygen hydrogen. Electrons stripped cells, although at of from only 50% the wild-type levels (Figure water during this reaction are funnelled back into iB, photo- wt X It is to note that 1/2). interesting while the growth rate of chemical reaction center and then II transported the elec- through wild-type and FuD44-R2 cells is about equal on acetate contain- tron transport chain to photosystem I, to be used for eventually ing media, the growth rate of the FuD44-R2 on minimal reduction of NADP. media, therefore relying 11 exclusively on photosynthesis for Photosystem and the are growth, is oxygen evolving complex physically about only one half that of wild-type cells. The two other linked. Isolation of oxygen a evolving particles yields complex peripheral PS polypeptides, OEE2 and OEE3, are present in containing a and chlorophylls b, carotenoids, quinones, lipids each of the different strains (Figure 1B), the although level of and an of at least and array eight polypeptides (Murata Miyao, OEE2 is somewhat reduced in both FuD44 and in FuD44-R2 1985). Five of known cells. these, Chlamydomonas reinhardtii as are encoded Dl, P6 and FuD44 specifically lacks OEEI D2, P5, C-b559, mRNAs chloroplast (Rochaix, 1981) and localized within the PS reaction center core (Inoue To determine if the absence of OEE1 protein in FuD44 cells was et Erickson et Associated with this al., core 1983; al., 1986). due to a mutation affecting expression of the gene encoding it are three extrinsic nuclear encoded PS particle and not polypeptides, due to a effect of a (psbl), pleiotropic mutation at another enhancer 2 and which we cloned oxygen can site, cDNAs the three extrinsic evolving proteins (OEE 1, encoding 3), polypeptides © IRL Press Limited, Oxford, 313 England S.P.Mayfield, P.Bennoun and J.-D.Rochaix Ai ..... '.. ij .. .... .-Il..1 il .,- : .... C.." --r 3, !.'' I.i. .:" LL k j-j AL b,.. .. b - 6 " OEE'- _ 4U i_. Fig. 1. of water soluble isolated from mutant Polyacrylamide gel electrophoresis and revertant FuD44-R2 cells. proteins FuD44, wild-type (wt), Following the were either stained with Coomassie Blue electrophoresis gels or transferred to CNBr activated (A), (B). The location of electrophoretically paper the OEEI is marked on the stained The protein (-) blots were with antisera to three extrinsic gel. protein associated with PS hybridized specific polypeptides II; OEE1, OEE2 and OEE3. A of wt sample lane 50% the loaded into the lane X is shown for of containing protein levels (wt 1/2) comparison OEE1 protein in the FuD44-R2 after sample. were visualized with 1251-labeled Protein-antibody complexes by autoradiography Staph-A protein. labeling To confirm (-4 pPll-12.4. that encoded the pPll-12.4 OEEl poly- the peptide cDNA insert was ,j- sequenced. Comparison of the .'). a deduced amino acid of sequence pPll-12.4 with the N-terminal 1u- ,a - 3m v > -i amino acid U L L.i a determined from sequence isolated mature OEEl pro- tein confirmed that encodes pPll-12.4 the OEEl polypeptide (se- to be quence reported elsewhere). Total RNA was separated on denaturing agarose gels, blotted to nylon membranes and stained with methylene blue to verify equal and even transfer of the loading RNA (Khandjian, 1986). The membranes were hybridized with nick translated cDNA in- OEEI-- serts from As -DEE2 plasmid pPII-12.4. shown in Figure 2 this cDNA 46 4b 40- -OEE3 hybridized to a 1.6 kb mRNA in wild-type cells which is com- so* pletely missing from mutant FuD44, but is again present in the revertant FuD44-R2. From dilutions of wild-type mRNA we estimate that the revertant contains only about 25% of the OEEl mRNA of not wild-type cells (data shown). Hybridization of iden- tical filters with cDNA encoding OEE2 (Mayfield et al., 1986) and OEE3 (cloning and sequence to be reported elsewhere) show that both of these mRNAs are present in equal quantities in wild- FuD44 and type, FuD44-R2 cells (Figure 2). FuD44 contains a 5 kb DNA insert in the 5' region of the single 2. OEE] Fig. Northern analysis of mRNA isolated from FuD44 gene wild-type (wt), and FuD44-R2 cells. Total RNA was on separated denaturing formaldehyde To characterize the mutation resulting in the specific absence of and electroblotted to membranes. The filters were gels nylon wth hybridized mRNA OEEl and protein we examined the chromosomal nick translated cloned cDNAs the PS II region encoding peripheral proteins OEE1, OEE2 and OEE3. containing the psbl gene by Southern analysis. DNA was isolated from wild-type, FuD44 and FuD44-R2 cells, digested with associated with oxygen evolving particles, and restriction measured the ac- endonucleases, separated on agarose gels, and blot- cumulation of their ted corresponding mRNAs. A C. to nitrocellulose reinhardtii membranes. The DNA blots were then cDNA library was constructed in 1 a Xgtl expression hybridized with nick translated vector and cloned cDNA insert. OEE1 As the expressed fusion proteins were shown identified by in Figure 3B the cDNA hybridization hybridized to a single fragment with antisera specific for the in three extrinsic polypeptides both the EcoRI and Hindlll digests in each of the different (Mayfield et al., 1986). Several DNA plaques were identified using samples. However, in mutant FuD44 the DNA fragment OEEI antisera, and individual phage were isolated. One of the containing the psbl gene is approximately 5 kb larger than the recombinant phage contained a 1.6 kb cDNA insert which was fragment in wild-type cells. In the revertant FuD44-R2 the frag- subcloned into the EcoRI site of plasmid pUC 19 ment to form plasmid containing psbl is approximately 4 kb smaller than the frag- 314 OEE1 protein: oxygen evolution and stability of PSH 1 wild-type counterpart. imately 5 kb and kb larger than their A B This analysis identified the site of insertion of foreign DNA into kb the psbl gene to a 220 bp Hindm-PstI fragment that is located at the 5' end of the psbl gene (see map Figure 3C). Sequencing 21.0- di a of the 220 bp wild-type PstI-HindIH genomic fragment show- a.- ed it to contain 60 nucleotides of an exon, and 160 nucleotides 65- 9 of an intron, located at the 5' end of the psbl gene (data not 5.0 - shown). The exact location of the 5 kb insert within this 220 bp 0m. fragment in FuD44 has not yet been determined. 3.5 - Ihe absence ofOEEJ results in the reduction ofcore PS IIpoly- 2.0- v peptides, but not in a deficiency of their mRNAs To assess what effect the absence of OEE1 had on PS H core polypeptides, membrane proteins were isolated from wild-type, FuD44 and FuD44-R2 cells, and separated on 7.5-15 % poly- 3 - acrylamide gels. Following electrophoresis the gels were either stained with Coomassie Blue (Figure 4A) or transferred to CNBr 0.8 - activated paper (Figure 4B). The protein blots were then hybridiz- ed with antisera specific to the PS II core polypeptides D2, DI, PS and P6, and to the PS II antenna chlorophyll binding protein, Ps t I - - I EcoRI PstI Sal HinIE IL LHC II. As shown in Figure SB all of the PS H core polypep- tides accumulate in FuD44 cells, but only to approximately 10-15% of wild-type levels. These PS H polypeptides ac- Cp P H S A HPt cumulate to a much greater extent in the revertant FuD44-R2 :. I P=71 i * VII, "-11V,'1111,4 cells, but are still reduced to - 50% of the wild-type levels. The LHC H polypeptides, like most of the membrane proteins as visualized by the stained protein gels, accumulate to similar levels in all three strains (Figure 4B). 3. Southern analysis of FuD44 and FuD44-R2 genomic Fig. wild-type, To determine if the reduction in PS H core proteins was a con- DNA. DNA was with restriction endonuclease as indicated on the digested sequence of reduced mRNA levels we measured the accumula- figure, separated by agarose gel electrophoresis, and blotted to nitrocellulose. For each digestion indicated the sample lanes are left to right; tion of chloroplastic mRNAs encoding the PS H core proteins. wild type, FuD44 and FuD44-R2. (A) The blot was hybridized with a nick Equal amounts of RNA from wild-type, FuD44 and FuD44-R2 translated 750 bp HindIII-HindIII cloned wild-type genomic fragment cells were separated on denaturing gels and electroblotted to nylon at the 5' end of the on located psbl gene (marked genomic map C). (B) membranes. The filters were then hybridized with nick translated After removal of this the filter was with a cDNA probe hybridized encoding this cDNA lies 3' to the PstI site within the cloned chloroplastic genomic fragments encoding the PS II core OEEI, completely coding chromosomal the region. (C) Genomic map of the fragment containing psbl polypeptides Dl, D2, PS and P6 (Rochaix, 1981). As shown in sites for PstI HindIll Sall and Aval are gene. Restriction (P), (H), (S) (A) Figure 5 all of the mRNAs encoding these PS II core polypep- are the 5' and 3' ends of the the site of the shown, as coding region (U), tides accumulate in FuD44 and revertant FuD44-R2 cells to levels location of the used as the 5' DNA insert (O), the genomic fragment probe similar to those of wild-type cells. the 5' limit of the cDNA (1z), and probe ( ). Deficiency ofcore PSIlpolypeptides in FuD44 is due to instability of the core complex kb than the ment in but still about 1 larger wild-type FuD44, To determine if the deficiency of PS H core proteins in FuD44 fragment (Figure 3A and B). cells was due to an increased instability of the complex or due Genomic clones containing the psbl gene were isolated from to a reduction in synthesis of the core polypeptides, wild-type, a EMBL3 - C. reinhardtii genomic library, and mapped using FuD44 and FuD44-R2 cells were pulse-labeled with [14C]acetate. common restriction endonucleases. A restriction map of the As shown in Figure 6A the pattern of proteins from cells label- chromosomal the wild-type psbl gene is segment containing ed for 10 with is identical in both presented in Figure 3C. Wild-type, FuD44 and FuD44-R2 DNA min [14C]acetate wild-type and FuD44 cells. Because this pattern of labeled proteins is com- was restricted with endonucleases Sail and HindH, all of PstI, plex we also labeled cells in the presence of cycloheximide, an which cut the into two or more fragments. The DNAs psbl gene inhibitor of cytoplasmic protein synthesis. As shown in Figure were on blotted to nitrocellulose filters separated agarose gels, 6C labeling of membrane proteins in this way allows for the easy and with a 750 bp HindRIl-HindRI cloned wild-type hybridized visualization of the PS II core proteins. The upper panel in Figure which is located at the extreme 5' end of the genomic fragment, 6C is an autoradiograph of proteins labeled for 45 min in the line in 3C). As shown in single psbl gene (crossed Figure Figure on presence of 8 p4g/ml cycloheximide prior to separation poly- this to a fragment in the PstI and 3A probe hybridized single M and shows that the DI and acrylamide gels containing 8 urea, HindIll and to two in the Sail Both the digests fragments digest. D2 are in amounts in proteins synthesized approximately equal PstI and the 1.8 kb Sail are than their wild- fragments larger The lower in 6C is an autoradio- all three strains. panel Figure in FuD44 and while the 750 Hin- type counterpart FuD44-R2, bp of the same labeled on non-urea con- graph proteins separated and the 3.5 kb Sail are the same in all dIl fragment fragment of P4 which allows for the was removed from the filters taining gels, separation polypeptides three DNA This samples. probe and P6 are and shows that both PS with a cDNA which lies and PS, polypeptide synthesiz- and the blot was rehybridized probe ed in three all strains. in 3C. As shown in 3' to the PstI site as indicated Figure Figure without the addition of were chased are the same while labeled 3B the PstI of each size, Cells cycloheximide fragments sample of cold acetate the 10 for 90 in the min of FuD44 and FuD44-R2 are min 3' presence following the HindmI fragments approx- 315 P.Bennoun S.P.Mayfield, and J.-D.Rochaix A4 A4 .. .; a& a..... Fig. 4. of membrane isolated from FuD44 and FuD44-R2 Polyacrylamide gel electrophoresis proteins cells. the wild-type (wt), Following electrophoresis gels Blue or transferred to CNBr activated The blots were were either stained with Coomassie (A), with antisera to the PS paper (B). protein hybridized specific II P5 and to the PS antenna core proteins DI, D2, and P6, II LHC The were visualized chlorophyll binding protein II. proteins by autoradiography after of with 1251-labeled labelling the antigen-antibody complexes Staph-A protein. ! A 4~~~. p(f em - P5 - a,a I--- - ._ _- - P4pz --_ t a&er Fig. 5. Northern analysis of mRNA isolated from FuD44 wild-type (wt), FuD44-R2 and cells. Total RNA was on separated denaturing formaldehyde gels and electroblotted to membranes. The filters were nylon hybridized with nick translated cloned the PS chloroplastic genomic fragments encoding II core proteins D1, D2, P5 and P6. PL pulse. As shown in 6B the of Figure patterns labeled proteins in wild-type and FuD44 cells show some differences. This dif- ference is most easily observed in the Dl and P5 which proteins Fig. 6. Autoradiograph of in vivo labeled membrane proteins from wild-type are in clearly underrepresented the FuD44 cells after the 90 min (wt), FuD44 and FuD44-R2 cells. (A) Cells labeled for 10 min with chase (Figure 6B). Polypeptides P6 and D2 co-migrate with other and [14C]acetate, (B) chased for 90 min in the presence of non-radioactive labeled proteins on these acetate. The site of gels and are thus not as easily observ- migration of polypeptides P5, P6, and D2 is DI marked. (C) Cells labeled for 40 min in the presence of cycloheximide. ed in the autoradiograph (Figure 6B). The loss of P5 and Dl Upper panel shows the proteins separated on polyacrylamide gels containing protein, and we assume P6 and D2 as well, in FuD44 cells must 8 M urea, and the lower panel shows a section of the autoradiograph with therefore be due to a more rapid turnover of the in protein FuD44 similar samples separated on non-urea gels which allows for the separation cells compared to wild-type cells. of polypeptides P4 and PS. 316 oxygen evolution and stability of PSI1 OEE1 protein: exception all of them have identical patterns on Southern analysis, Discussion indicating that the reversion of this mutant is probably not a ran- Several groups have isolated oxygen evolving particles which dom event. It is also interesting to note that the revertant, although contain the PSII reaction center core proteins and three extrin- able to accumulate both OEEl mRNA and protein, does not ac- sic nuclear encoded proteins (reviewed by Critchley, 1985; cumulate either to wild-type levels. This data suggest that this Murata and Miyao, 1985). There is, however, some debate as DNA insert may be a transposable element, but proof of this must to which of the extrinsic proteins are really necessary for await the cloning and analysis of the mutant OEEl locus and photosynthetic 02 evolution. Several groups have reported that its revertants. in vitro the PSII core and OEEl comprise the minimal particle capable of oxygen evolution (Ono and Inoue, 1984; Ikeuchi et Materials and methods al., 1985; Preston and Critchley, 1985; Ghanotakis et al., 1984), others have reported that the PSII core andOEE2 are sufficient Isolation of mutant FuD44, and cell culture conditions for 02 evolution in vitro (Moller and Hoj, 1983; Henry et al., Wild-type cells 137c were treated with 5-fluorodeoxyuridine and metronidazole 1982), while still others (Tang and Satoh, 1985) have concluded as described (Bennoun et al., 1978; Schmidt et al., 1977). Mutant FuD44 was isolated as a high chlorophyll fluorescent colony which was back crossed to wild- that 02 evolution is associated with the PS II core alone. type cells and the mutation shown to be inherited in a Mendelian (nuclear) fashion. Recently we have shown that in vitroOEE2 is required for Complementation analysis in young zygotes (Bennoun et al., 1980) with mutant high levels of 02 evolution, but that in its complete absence there BF25, a nuclear mutant which affects the expression of the OEE2 polypeptide is still some 02 evolution (5% of wild-type levels, Mayfield et (Mayfield et al., 1986), showed that the two mutations are in different genes al., 1986). Here we show that in vivoOEEl is absolutely re- (data not shown). Revertants of mutant FuD44 were selected by plating the mu- tant on agar plates containing minimal media in the light, thus selecting for quired for any photosynthetic oxygen evolution, and that cells photosynthetic growth. Revertants were observed at a frequency of -1 x 10-6 deficient for OEEI, unlike those deficient for OEE2, are in- to l0-7 cells, and 12 colonies, capable of growth on minimal media, were capable of photoautotrophic growth, thus OEE1 is absolutely re- isolated. With one exception all of these revertants showed similar patterns on quired for photosynthesis. Southern analysis so only one revertant, FuD44-R2, was used for further characterization. Oxygen evolution was measured on whole cells with a Clark- Two interesting observations can be made from the Western type oxygen electrode (Rank Brothers, England). analysis of PS II polypeptides. The first is that the absence of Wild-type C. reinhardtii strain 137c, nuclear mutant FuD44 and revertant OEE1 does not result in the loss of eitherOEE2 or OEE3. Thus FuD44-R2 were grown in liquid Tris-acetate-phospha,te media pH 7.0 (Gor- OEE1 is not required for the stability of the other OEE polypep- man and Levine, 1965) under dim fluorescent lighting to a density of 1-2 x 106 cells/ml. The cells were harvested by centrifugation at 8000 g for 10 min tides. We have not, however, attempted to localize OEE2 or resuspended in 1/20 volume fresh media and pelleted again at 8000 for 10 min. OEE3 within the chloroplast and thus we can not say whether Protein isolation, gel electrophoresis, electroblotting and polyacrylamide antbody these polypeptides remain anchored to the membrane in the hybridization absence of OEE1, only that they accumulate to near wild-type Protein isolation and sample preparation were described previously (Mayfield levels and are the correct, mature, size. The second observation et al., 1986). Electrophoresis of proteins on urea containing gels was described is that the absence ofOEE1 causes a substantial reduction of core in Piccioni et al. (1981). Polyacrylamide gel electrophoresis of non-urea con- PS II polypeptides and active PS H centers, but not a reduction taining gels, protein blotting, antibody hybridization, and autoradiography were as described by Mayfield and Taylor (1984). Following autoradiography the filters in other polypeptides closely associated with PS II, like the LHC (CNBr activated paper) were stripped of the primary antibodies by washing the II polypeptides. The in vivo labelling experiments clearly show filters for 30 in 7.5 M guanidinium hydrochloride, 5% (3-mercaptoethanol min that the deficiency of the PS H core proteins in FuD44 cells is and then for 15 min in 0.2 M Glycine-HCl pH 2.8. The filters were then wash- not due to a decrease in synthesis of these proteins, but rather ed for 30 min in hybridization buffer before being rehybridized with a new an- due to a more rapid turnover of the newly synthesized polypep- tisera. As long as the filters were never allowed to completely dry they could be reused several times without apparent loss of signal. tides in the absence ofOEEl protein. This suggests that OEEI, Isolation of cDNA and genomic clones, and RNA and DNA isolation, elec- or perhaps a portion of it, is required for the stable formation trophoresis and blotting of the PS II core particle, perhaps even having some structural Isolation of cDNA clones was described by Mayfield et al. (1986), but basically role within the complex. An alternative explanation could be that involved identification of OEE1 protein expressed in a Xgtl 1 expression vector the loss ofOEEl exposes the PS H core or one of its proteins (Young and Davis, 1985) using the same rabbit polyclonal antisera used for the in such a way that the particle becomes susceptible to proteolytic Western blots. Genomic clones were then isolated from a C. reinhardtii EMBL3 genomic library 1986) by the method of Benton and Davis degradation. Recently we have shown that another 02 evolving (Goldschmidt-Clermont, (1977) using nick translated OEE1 cDNA inserts from the Xgtl 1 clones. deficient mutant of C. reinhardtii, which is missing the OEE2 RNA was isolated with guanidinium hydrochloride as described (Nelson et al., polypeptide (Mayfield et al., 1986), accumulates almost wild- 1984). Total RNA, 10 per sample lane, was separated on denaturing for- jig type levels of all of the core PS H proteins, thus the instability maldehyde agarose gels and then electroblotted to nylon membrane (genescreen) of the PS H core proteins in FuD44 cells must be due to the in 25 mM phosphate pH 6.5. Following electroblotting the RNA was fixed to the filters by u.v. irradiation, and then stained with methylene blue to verify even absence of OEE1 and not due to some pleiotropic effect of the loading and transfer of RNA (Khandjian, 1986). The filters were then prehybridized loss Of 02 evolving activities. and hybridized as described (Johnson et al., 1984). The mutation which results in the absence of OEE1 mRNA Isolation of C. reinhardtii DNA, digestion with restriction endonucleasees, in FuD44 cells is a large, 5 kb, DNA insertion into the 5 prime and blotting to nitrocellulose membranes was as previously describ- electrophoresis ed (Rochaix, 1978). region of the single psbl gene. Revertants of the original mu- Pulse labeling of with [14C]acetate tant, which are again capable of oxygen evolution, have lost 4 kb proteins of this DNA insert. These revertants, although still harboring Cells were grown in complete media to a cell density of 1-2 x 106 cells/ml, then transferred to media lacking acetate for 1 h prior to labelling. The cyclohex- 1 kb DNA insert in the psbl gene, again accumulate OEE1 imide treated cells were made 8 final concentration 10 min prior to the mRNA. We assume from this that the DNA insert must be in ltg/IL addition of label, and were labeled for 45 min with 2 PCi/mi [14C]acetate (56 the non-coding region of the 220 bp genomic fragment which was added to 40 mi of cells which had not been 1Ci of [ 14C]acetate jACi/iM). contains the insert, as the RNA is still correctly spliced to yield and allowed to incorporate for 10 min. The media with cycloheximide treated 50 mM acetate by the addition of 2 M non-radioactive acetate, mature OEE1 mRNA of the correct size. Analysis of then made functional was harvested by centrifugation for 2 min at 10 000 g. Half cells quickly and the of FuD44 (data not shown) shows that with one 12 revertants 317 S.P.Mayfield, P.Bennoun and J.-D.Rochaix of the cells were quick frozen in dry ice ethanol and the other half returned to complete media containing non-radioactive acetate and allowed to grow for an additional 90 min, then harvested and frozen as above. Protein samples were prepared and electrophoresed exactly as the samples used for the stained gels except that following destaining the gels were soaked for 20 min in Enlighting (New England Nuclear), dried onto filter paper under vacuum, and then expos- to film at -70°C. ed Acknowledgements to thank O.Jenni and Y.DeLotto for help in preparing the figures. We We wish also thank Yves Pierre for technical assistance, and P.Shaw for helpful discus- sions and a critical reading of the manuscript. This work was supported by grant 3.587.084 from the Swiss National Foundation to J.D.R., CNRS grant ATP Biologie Moleculaire Vegetale to P.B., and NIH postdoctoral fellowship GM 10246-02 to S.P.M. References Masson,P. and Delosme,M. (1980) Genetics, 95, 39-47. Bennoun,P., and Davis,R.W. (1977) Science, 1%, 180-182. Benton,W.D. Biochim. Biophys. Acta, 811, 33-46. Critchley,C. (1985) Erickson,J.E., Rahire,M., Malnoe,P., Girard-Bascou,J., Bennoun,P. and Rochaix,J.D. (1986) EMBO J., 5, 1745-1754. Ghanotakis,D.F., Babcock,G.T. and Yocum,C.F. (1984) Biochim. Biophys. Acta, 765, 388-398. Goldschmidt-Clermont,M. and Rahire,M. (1986) J. Mol. Biol., 191, 421-432. Gorman,D.S. and Levine,R.P. (1965) Proc. Natl. Acad. Sci. USA, 54, 1665-1669. Andersson,B. and Akerlund,H.-E. (1982) Carls. Henry,L.E.A., Moller,B.L., Res. Commun., 47, 187-198. Ikeuchi,M., Yuasa,M. and Inoue,Y. (1985) FEBS Lett., 185, 316-322. Inoue,Y., Crofts,A.R:, Govindjee, Murata,N., Benger,G. and Satoh,K. (1983) 7he Oxygen Evolving System of Photosynthesis. Academic Press, Tokyo. Johnson,D.A., Gantsch,J.W., Sportman,J.R. and Elder,J.H. (1984) Gene Anal. Techn., 1, 3-8. (1986) Mol. Biol. Rep., 11, 107-115. Khandjian,E.W. Kuwabara,T. and Murata,N. (1979) Biochim. Biophys. Acta, 581, 228-215. Rahire,M., Frank,G., Zuber,H., Rochaix,J.D. (1986) Proc. Natl. Mayfield,S.P., Acad. Sci. USA, in press. and Taylor,W.C. (1984) Planta, 161, 481-480. Mayfield,S.P. and (1983) Carls. Res. Conunn., 48, 161-185. Moller,B.L. Hoj,P.B. (1985) Trends Biochem. Sci., 10, 122-124. Murata,N. and Miyao,M. Mayfield,S.P. and Taylor,W.C. (1984) J. Cell Biol., Nelson,T., Harpster,M.H., 558-564. 98, and FEBS Len., 166, 381-384. Ono,T.-A. Inoue,Y. (1984) Bennoun,P. and Chua,N.H. (1981) Eur. J. Biochem., 117, 93-102. Piccioni,P., and Critchley,C. (1985) FEBS Lea., 184, 318-322. Preston,C. Rochaix,J.-D. (1978) J. Mol. Biol., 126, 597-617. Experientia, 37, 323-332. Rochaix,J.-D. (1981) and Chua,N.H. (1977) Proc. Natl. Acad. Sci. USA, Schmidt,G.W., Matlin,K.S. 610-614. 74, (1985) FEBS Lea., 179, 60-64. Tang,X.-S. and Satoh,K. and (1985) In Setlow,J.K. and Hollaender,A. (eds), Young,R.A. Davis,R.W. and Methods. Plenum Press, NY, Vol. 7, Genetic Engineering: Principles 29-41. pp. Received on 20 November 1986

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The EMBO Journal – Springer Journals

Published: Feb 1, 1987

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