Planta (1991)185:105-110
P l a n t a 9 Springer-Verlag1991
Over-expression of the oxygen-evolving enhancer 1 protein and its consequences on photosystem II accumulation Stephen P. Mayfield Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037, USA Received 6 February; accepted 18 April 1991
Abstract. By transformation with a cloned wild-type oeel gene, which codes for the oxygen-evolving enhancer 1 (OEE1)protein, we have constructed a strain of Chlamydomonas reinhardtii containing multiple copies of this gene. A transformant ( R 1 - K - 5 0 ) containing four to five copies of the oeel gene accumulated oeel m R N A in approximately threefold excess of the wild type. The OEE1 protein accumulated in proportion to the oeelm R N A levels in these cells. These data indicate that no apparant feedback mechanism is operating to reduce either transcription or translation of the introduced oeel genes as a means to regulate OEEl-protein accumulation. The OEE1 protein in R 1 - K - 5 0 was all of mature size, indicating that the transit peptide had been completely removed, and that all o f the protein was located within the thylakoid lumen. Photosystem II reactioncenter proteins D1 and D2 accumulated to wild-type levels, but not greater, in these cells, while there was no effect on accumulation o f any of the PSII peripheral proteins such as OEE2 or LItCII. The OEE1 protein which accumulated in excess of wild-type levels was not bound to the thylakoid membranes, indicating that a limited number o f binding sites for OEE1 exist on the thylakoid membranes. No difference in photosynthetic oxygen evolution was observed between wild-type and R 1 - K - 5 0 strains. These data show that whatever mechanisms are used to determine stoichiometry within the PSII complex they are not perturbed by overexpression of the OEE1 protein.
Key words: Chlamydomonas (photosynthesis) - Nuclear transformation - Oxygen-evolving proteins - Photosystern II accumulation
Introduction The synthesis and assembly of photosynthetic complexes in higher plants and algae requires the close cooperation Abbreviations: bp=basepairs; LHCII=light-harvesting complex II; OEE1 = oxygen-evolving enhancer 1; SDS= sodium dodecyl sulfate
of both the chloroplast and nuclear genomes. Proteins which comprise these multisubunit complexes are encoded in both the nuclear and chloroplastic genomes. Expression of photosynthetic proteins is coordinately regulated so that proteins within any of these complexes are always at or near their correct stoichiometry. Clearly there are a number of mechanisms which affect the accumulation of photosynthetic proteins, including transcription (Tobin and Silverthorne 1985), m R N A stability (Gruissem 1989) and protein translation (Mullet 1989). Each of these mechanisms acts to regulate protein accumulation, but to date there is no evidence which indicates that any one of these mechanisms regulates protein accumulation to the degree required to achieve the precise stoichiometries that are observed in photosystem complexes. It seems likely that a combination of all of these plus other, as yet determined, factors control this precise protein accumulation. One step of the biosynthetic pathway which strongly influences the accumulation of the photosynthetic complexes involves the degradation of component proteins in the absence of stable accumulation of a complete complex. Examination of a number of photosynthetic mutants in higher plants and algae has shown that accumulation of a complex requires the accumulation of every member of that complex (Apel 1978; Bennett 1981 ; Metz and Miles 1982; Harpster et al. 1984). Characterization of PSII mutants in C. reinhardtii and maize has shown that the loss of a single PSII core protein results in the loss of the entire complex (Chua and Bennoun 1975; Metz and Miles 1982; Bennoun et al. 1986; Kuchka et al. 1988; Rochaix et al. 1989). The synthesis o f PSII core proteins in these mutants, other than the protein directly affected by the mutation, continues at normal levels, but the newly synthesized proteins are rapidly degraded and never accumulate to wild-type levels. Peripheral PSII proteins, such as those of the oxygen-evolving and lightharvesting complexes, accumulate normally in PSII core mutants. Characterization of mutants deficient in one or another of the peripheral oxygen-evolving proteins has shown that two of these, called the oxygen-evolving enhancer
106 p r o t e i n s 1 a n d 2 ( O E E 1 a n d O E E 2 ; M a y f i e l d et al. 1987a, b), are a b s o l u t e l y r e q u i r e d for h i g h levels o f p h o t o s y n t h e t i c o x y g e n e v o l u t i o n . A b s e n c e o f the O E E 2 p r o t e i n r e s u l t s i n r e d u c e d rates o f p h o t o s y n t h e t i c o x y g e n e v o l u t i o n , a l t h o u g h cells w h i c h l a c k this p e p t i d e a r e still a b l e to g r o w p h o t o s y n t h e t i c a l l y . P h o t o s y s t e m II core a n d o t h e r p e r i p h e r a l p r o t e i n s a c c u m u l a t e to n o r m a l levels i n O E E 2 - d e f i c i e n t s t r a i n s ( M a y f i e l d et al. 1987b). A b s e n c e o f the O E E 1 p r o t e i n r e s u l t s in t h e c o m p l e t e loss o f p h o t o s y n t h e t i c o x y g e n e v o l u t i o n a n d these cells are u n a b l e to g r o w p h o t o s y n t h e t i c a l l y . P h o t o s y s t e m II react i o n - c e n t e r p r o t e i n s a c c u m u l a t e to o n l y 20 % o f w i l d - t y p e levels i n a n O E E l - d e f i c i e n t s t r a i n ( M a y f i e l d et al. 1987a). T h i s r e d u c t i o n i n a c c u m u l a t i o n o f c o r e P S I I p r o t e i n s is d u e to a d e c r e a s e i n the s t a b i l i t y o f t h e P S I I c o r e particle, n o t to a r e d u c t i o n i n s y n t h e s i s o f a n y o f the c o r e p r o t e i n s . T h e O E E 2 a n d O E E 3 p r o t e i n s a c c u m u l a t e to w i l d - t y p e levels in the O E E l - d e f i c i e n t cells ( M a y f i e l d et al. 1987a). T h u s O E E 1 s t r o n g l y affects the s t o i c h i o m e t r y o f P S I I c o r e p r o t e i n s , a n d in vitro e v i d e n c e i n d i c a t e s t h a t O E E 1 a n d t h e P S I I r e a c t i o n - c e n t e r p r o t e i n s D1 a n d D 2 are physically associated (reviewed Ghanotakis and Yocum 1990). Since the a b s e n c e o f a n y single m e m b e r o f the P S I I c o m p l e x so d r a m a t i c a l l y affects the a c c u m u l a t i o n o f o t h e r m e m b e r s o f t h e P S I I c o m p l e x , we were i n t e r e s t e d to d e t e r m i n e w h a t effect the o v e r e x p r e s s i o n o f o n e m e m b e r o f t h e c o m p l e x w o u l d h a v e o n the a c c u m u l a t i o n o f other PSII proteins. We therefore constructed a strain of C. reinhardtii w h i c h c o n t a i n e d m u l t i p l e c o p i e s o f the oeel gene.
Materials and methods Construction of plasmid containing the oeel gene. Chlamydomonas reinhardtii genomic clones encoding the OEE1 protein were identified from a ;~-EMBL3 library (Mayfield et al. 1989). An 8-kb fragment, containing the entire oeel gene as well as 3 kb of DNA 5' and 2 kb of DNA 3', was subcloned into plasmid pUC19 at the unique Eco R1 and Kpn 1 sites to form plasmid pSB101 (Mayfield and Kindle 1990). Electroporation of Fud44 cells with pSBIO1 DNA. The Chlamydomonas reinhardtii OEE1- and cell-wall-deficient strain Fud44-cwl 5 was grown in complete medium (Gorman and Levine 1965) to a density of 1" 106 cells-ml -~ (log phase). Cells were pelleted by centrifugation at 6000 9g in a Beckman (Beckmann, Palo Alto, Cal., USA) SJ12 rotor at 4 ~ C for 5 min. The pellet was resuspended in water to a density of 1' 106 cells-ml -~ and pelleted again by centrifugation. The pellet was then resuspended in minimal medium (Sueoka 1960) at a density of 5 9 107 cells 9m l - 1 Fifty lal of cells in minimal media were pipetted into 500 lal microcentrifuge tubes and 1-5 lal of pSBI01 plasmid DNA (0.5 lag) was added directly to the cell suspension. The tubes containing the cell/DNA mixture were placed on ice prior to electroporation. The entire content of the tube (50 lal) was pipetted between two 1-cm-wide stainless-steel electrodes which were 0.5 mm apart (p/n 474 electrode; BTX Co. San Diego, Cal., USA). A single pulse was delivered to the cell mixture from a 500-laF capacitor (Transfector 300; BTX Co.) which was discharged through a voltage booster (Power Plus; BTX Co.). Immediately following the electrical discharge the cells were pipetted into 3 ml of liquid minimal medium, and then spread onto minimal-medium plates. The electrodes, electroporation apparatus, and liquid minimal medium were all maintained at room tem-
S.P. Mayfield: OEEI over-expression perature. The minimal plates containing the Fud44 cells were placed in a growth room at 25 ~ C with continuous light from fluorescent tubes (approx. 160 W - m - Z ) . After 10-14 d, small green colonies appeared which were subcloned onto minimal-medium plates. Colonies which grew on the second selective plate were characterized for the presence of oeel sequences and for the expression of oeel mRNA and protein. Isolation of DNA, RNA and protein from transformants. For DNA isolation, cells were grown in complete liquid media to a density of 1-107 cells" m1-1. Cells were pelleted, resuspended in an equal volume of 100 mM Tris pH 8.0, 400 mM NaCI, 40 mM EDTA and 2% sodium dodecyl sulfate (SDS). Pronase (protease XIV; Sigma, St. Louis, Mo., USA) was added to a final concentration of 4 lag - ml- x. Following incubation at 60 ~ C for 1 h the mixture was extracted twice with an equal volume of phenol/chloroform (1 : 1, v/v), and precipitated with 2.5 volumes of ethanol. The nucleic acid was pelleted by centrifugation at 10 000"9 for 10 min, air-dried, resuspended in a small volume of TE (10 mM Tris pH 7.5, 1 mM EDTA), and RNase A was added to a final concentration of 20 Ixg - m l - x. The mixture was incubated at 60~ C for 20 min prior to digestion at 37 ~ C with an appropriate restriction enzyme and buffer. Following digestion, samples were separated on 1% agarose gels and blotted to nylon membrane (Hybond-N; Amersham, Arlington Heights, Ill., USA) in 10x SSC (0.15 M NaC1, 15mM Sodium Citrate, pH 7.0). The DNA was fixed to the nylon membrane by UV irradiation (10 min on 300-nm UV light box). The filters were prehybridized in: 50% formamide, 5 x SSPE (0.18 M NaC1, 10mM NaHzPO4"2H20, l mM EDTA, pH7.0), 0.1% SDS, 0.5% non-fat dry milk and 10 lag " ml- 1 salmon-sperm DNA, at 42 ~ C for 1 h, and then hybridized in the same solution at 42 ~ C overnight with the addition of a denatured 32p_labele d DNA probe. The filters were washed (3 x 30 min in 1 x SSPE and 0.1% SDS at 60 ~ C, and then exposed to film (XAR; Kodak, Rochester, N. Y., USA) with an intensifying screen (Cronex, Dupont, Wilmington, DE., USA) at - 70 ~ C. For RNA and protein isolation the cells were grown to a density of 5" 106 cells" ml -a. Isolation of RNA, gel electrophoresis and blotting were essentially as described by Rochaix et al. (1987). Following fractionation on denaturing agarose gels, the RNA was electroblotted to nylon membrane and fixed by UV irradiation (Khadjian 1986). The filters were prehybridized, hybridized, washed and exposed to film exactly as for the DNA blots. For protein isolation, cells were resuspended in 750 mM Tris pH 8.0, 15% sucrose and 100 mM [3-mercaptoethanol (I3ME). The cells were disrupted by sonication with a microtip (Sonic 2000; Braun) for 15 s at 80% maximum power. The membrane fraction was pelleted at 1 0 0 0 0 ' 9 for 15 min at 4 ~ C. The supernatant (soluble fraction) was pipetted off, and the pellet (membrane fraction) resuspended in the above buffer. For separation of membrane and soluble proteins under conditions in which the OEE1 protein remains bound to the thylakoid membranes, cells were resuspended in 25 mM Tris pH 7.5, 100 mM NaC1, 5 mM MgCIz, 100 mM 13ME and 15 % sucrose. All other treatments to the cells were exactly as described above. The soluble proteins were made 2 % SDS, 2 % I3ME and separated by electrophoresis on 7.5-15 % acrylamide gels containing 0.1% SDS. For staining, the gels were treated with 50% methanol, 10 % acetic acid, 0.25 % Coomassie blue, and destained in 40 % methanol, 10 % acetic acid. For blotting the gels were incubated in 25mM Tris, 190mM glycine, 0.1% SDS for 20min at room temperature, and then electroblotted to nitrocellulose in the same buffer at 20 V for 2 h in a transblot apparatus. Following electroblotting the nitrocellulose filters were incubated in TBS (50 mM Tris pH 7.5, 150 mM NaC1, 0.1% Tween 20) containing 5% nonfat dry milk for 1 h at room temperature. Rabbit polyclonal antisera was added and the incubation continued for an additional 6 h. The filter was washed twice in TBS and then incubated in TBS, 5% nonfat milk containing goat-antirabbit IgG conjugated with alkaline phosphatase for 3 h at room temperature. The filter was washed three times in TBS and then developed with alkaline-phosphataseactivity staining.
S.P. Mayfield: OEE1 over-expression
107
Fig. 1. Southern analysis of oeel genes. DNAs were digested with Pst 1 and hybridized with a a2p-labeled 700-bp oeel genomic fragment. The oeel gene in the mutant Chlamydomonas strain Fud44 is 5 kb larger than in the wild type due to the insertion of a transposable element. A dilution of R1-K-50 shows that the wild-type oeel fragment in R1-K-50 is approximately threefold more abundant than in the wild type
Fig. 2A, B. Analysis ofoeel mRNA in R1-K-50. Total RNA from wild-type, Fud44 and R1-K-50 Chlamydomonascells was separated on denaturing agarose gels, blotted to nylon membrane and hybridized with a a2p-labelled oeel cDNA probe A. An identical filter was hybridized with a2p-labeled cDNA encoding the small subunit (SSu) of ribulose-l,5-bisphosphate carboxylase/oxygenase B
Results
fragments of 2.5 kb and 3.5 kb, which could also contain functional oeel genes, were also observed in the R I - K - 5 0 D N A . A fragment which hybridizes with the oeel gene but is too small to contain a full-length copy of the gene, and the endogenous Fud44 oeel fragment (7 kb), which is nonfunctional, are also observed in R 1 - K - 5 0 . C o m p a r i s o n o f a 1/2 and 1/5 dilution o f the R 1 - K - 5 0 D N A with the wild-type lane shows that the there are approximately three copies of the 2.2-kb oeel gene in the R 1 - K - 5 0 strain. Thus t r a n s f o r m a n t R 1 - K - 5 0 has potentially four or five functional copies of the oeel gene, as well as the Fud44 copy and a smaller partial copy of the gene.
Identification of a strain containing multiple copies of the oeel 9ene. The OEEl-deficient m u t a n t Fud44 was transformed by electroporation of cells in the presence o f plasmid D N A (pSB101) containing a cloned copy o f the wild-type oeel gene. T r a n s f o r m a n t s were selected for photosynthetic growth by plating onto minimal m e d i u m (Mayfield and Kindle 1990). Only cells which express the OEE1 protein (transformants) should be able to grow photosynthetically. Several dozen photosynthetic transformants were analyzed by Southern analysis and three were identified as containing multiple copies o f the oeel gene. One of these, R 1 - K - 5 0 , appeared to contain several complete copies o f the oeel gene. This transformant, which was obtained f r o m a transformation with pSB101 plasmid digested with EcoR1 and K p n I, was used for further characterization. T o assess the copy n u m b e r o f oeel genes in R 1 - K - 5 0 , D N A s were isolated f r o m wild type, Fud44 and transform a n t R 1 - K - 5 0 . The D N A s were digested with Pst I, separated on an agarose gel and blotted to nylon m e m brane. As shown in Fig. 1, R 1 - K - 5 0 has multiple copies of the oeel fragment which migrate at the same position as the wild-type fragment (2.2 kb). Additional
Multiple copies of the oee l gene results in accumulation of oeel mRNA in excess of wild type. T o determine if the presence of multiple copies o f the oeel gene results in accumulation of oeel m R N A in excess o f wild-type levels, oeel m R N A was measured in R 1 - K - 5 0 cells. Total R N A , 3 ~g/lane, f r o m wild-type, Fud44 and R 1 - K - 5 0 strains was separated on denaturing formaldehyde gels, blotted to nylon m e m b r a n e and fixed by U V irradiation. The filters were hybridized with a 32p-labeled oeel c D N A (Mayfield et al. 1989), and washed and exposed to film as described. As shown in Fig. 2A, oeel
108
S.P. Mayfield: OEE1 over-expression
oeel m R N A is approximately threefold more abundant in R 1 - K - 5 0 than in the wild type (Fig. 2A). As previously shown (Mayfield et al. 1987a), Fud44 fails to accumulate any detectable a m o u n t of oeel m R N A . A second identical filter was probed with a 32p-labeled c D N A encoding the small subunit (SSu) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Goldschmidt-Clermont 1986). As shown in Fig. 2B each on the lanes contains approximately equal amounts of the SSu m R N A .
Fig. 3. Western blot analysis of OEEl-protein accumulation in R l-K-50. Proteins were isolated, under conditions in which OEE 1 protein is soluble, from wild-type, Fud44 and R1-K-50 strains of Chlamydomonas, separated on SDS polyacrylamide gels and either stained with Coomassie blue (left panel) or transferred to nitrocellulose membrane and hybridized with either OEE1 or OEE2 antisera (right panels). The over-accumulation of OEE 1 can be observed in the stained gel (arrow)
m R N A , which runs as a distinct doublet in this gel system, accumulates in R 1 - K - 5 0 cells to a greater degree then in the wild-type strain. C o m p a r i s o n of a dilution of R I - K - 5 0 R N A with that o f the wild type shows that the
Overexpression of oeel m R N A leads to a proportional accumulation of OEE1 protein. To determine what affect the accumulation of excess oeel m R N A had on OEE1protein accumulation, we measured OEE1 protein in wild-type, Fud44 and R 1 - K - 5 0 strains. Total cellular proteins were separated into crude soluble and membrane fractions under conditions (750 m M Tris p H 8) in which the OEE1 proteins are soluble. Equal amounts of soluble proteins were loaded onto gradient polyacrylamide gels containing SDS. Following electrophoresis the gels were either stained with Coomassie blue or transferred to nitrocellulose membrane. The nitrocellulose membranes were reacted with antisera against the OEE1 or OEE2 protein. As shown in Fig. 3 (left panel) most of the Coomassie-staining bands are c o m m o n to the wildtype, Fud44 and R 1 - K - 5 0 strains. Reaction of OEE1 antisera with blotted proteins reveals that the OEE1 protein accumulates to a greater degree in the R 1 - K - 5 0 cells than in the wild type (Fig. 3, central panel). Comparison of a dilution of R 1 - K - 5 0 with that of the wild type shows that OEE1 accumulates in R 1 - K - 5 0 to levels approximately threefold greater than in the wild type. The over-accumulation of OEE1 in R 1 - K - 5 0 cells can also be seen in the Coomassie-stained gel (arrow, left panel). As previously shown, no OEE1 protein accumulates in the Fud44 strain (Mayfield et al. 1987a). Measurement of OEE2 in a similar blot (Fig. 3, right
Fig. 4. Western blot analysis of membrane-protein accumulation in R1-K 50. Proteins were isolated from wild-type, Fud44 and R1-K-50 strains of Chlamydomonas separated on SDS polyacrylamide gels and either stained with Coomassie blue (left panel) or transferred to nitrocellulose membrane and decorated with either D1, D2 or LHCII antisera (right panels)
S.P. Mayfield: OEE1 over-expression
Fig. 5. Representative western blot analysis of OEE 1 protein association with thylakoids in R1-K-50. Proteins were isolated, under conditions in which OEE1 protein remains bound to the thylakoid membranes (mere), and separated in membrane and soluble (sol) fractions. Both soluble and membrane proteins from wild-type, Fud44 and RI-K-50 Chlamydomonas were separated on SDS polyacrylamide gels and either stained with Coomassie blue (left panel) or transferred to nitrocellulose membrane and hybridized with either OEE1 or OEE2 antisera (right panels). The accumulation of OEE1 in the soluble fraction of R1-K-50 can be observed in the stained gel (arrow) panel), shows that OEE2 accumulates to equal amounts in each of the strains.
Photosystem H reaction-center proteins accumulate to wild-type levels in R1-K-50 cells. As the absence of OEE1 protein results in a substantial reduction of PSII reaction-center proteins we were interested to see if overexpression of the OEE1 protein would result in an increased accumulation of PSII reaction-center proteins. To measure PSII reaction-center proteins, equal amounts of membrane proteins from the wild type, Fud44 and R 1 - K - 5 0 were separated on gradient polyacrylamide gels containing SDS. Following electrophoresis the gels were either stained with Coomassie blue (Fig. 4, left panel) or transferred to nitrocellulose filters for antibody hybridization. As shown in Fig. 4 (central panels) both the D1 and D2 reaction-center proteins accumulate to wild-type levels, but not more, in the R 1 - K - 5 0 strain. As previously shown D1 and D2 accumulate to only 20% of wild-type levels in the OEEl-deficient strain Fud44 (Mayfield et al. 1987a), while the L H C I I proteins accumulate to similar levels in each of the strains (right panel). Attachment of OEE1 protein to photosynthetic membranes is similar in wild-type and R1-K-50 strains. To determine
109 if OEE1 protein which accumulates in excess of the amount normally associated with the PSII core was bound to the membranes or was free in solution we measured OEE proteins under conditions in which the OEE proteins are normally retained on the thylakoid membranes. Wild-type and R 1 - K - 5 0 cells were pelleted, resuspended in a low-salt buffer and briefly sonicated to disrupt the cells. The membrane fraction, which should retain the OEE proteins if they are bound to the thylakoid, was pelleted and the supernatant (soluble fraction) saved. Soluble and membrane fractions were separated on SDS-polyacrylamide gels and either stained with Coomassie blue or transfered to nitrocellulose for antibody hybridization. As shown in Fig. 5 (left panel) the proteinstaining pattern is similar for both wild-type and R 1 - K - 5 0 cells. Western analysis using OEE1 antisera shows that wild-type and R l - K - 5 0 membrane fractions contain approx, equal amounts of the OEE1 protein (Fig. 5, central panel). N o OEE1 is detected in the soluble fraction of wild-type cells, while OEE1 is detected in the soluble fraction of the R l - K - 5 0 cells (central panel). A similar' filter probed with the OEE2 antisera shows that equal amounts o f OEE2 are found in the membrane fraction of both the wild type and R 1 - K - 5 0 while no OEE2 is found in either of the soluble protein fractions.
Discussion
Analysis of mutants deficient in single PSII proteins has shown that the absence of a single protein results in the loss of the entire PSII reaction-center complex. The loss of these proteins is not due to a decrease in synthesis or assembly, but rather to a more rapid degradation of the proteins after insertion into the thylakoid membranes (Erickson et al. 1986; Mayfield et al. 1987a; Kuchka et al. 1988). Since deficiencies of single proteins have such profound effects on PSII-complex accumulation we were interested to determine if PSII-protein accumulation would be influenced by overexpression of one member (OEE1 protein) of the PSII complex. As a means of overexpressing the OEE1 protein a strain of C. reinhardtii was engineered which contained multiple copies of the oeel gene. Although a number of factors are likely to influence the expression o f introduced oeel genes, the end result was that multiple copies of the oeel gene resulted in m R N A and protein accumulation in excess of wild-type levels. Examination of a number of oeel transformants showed that m R N A accumulation was roughly proportional to the copy number of the introduced gene, and that OEEl-protein accumulation was proportional to the level of oeel m R N A . Thus no apparant feedback mechanism is operating to reduce either the transcription or translation of oeel as a means to regulate OEE 1-protein accumulation. All of the OEE1 protein observed in the R 1 - K - 5 0 strain is mature protein, showing that the transit peptide has been completely removed and indicating that all of the protein is found within the lumen of the thylakoid
110 m e m b r a n e . There was no difference in p h o t o s y n t h e t i c oxygen evolution between wild-type and R 1 - K - 5 0 cells, which is n o t too surprising as the PSII reaction-center complexes a p p e a r identical in R 1 - K - 5 0 and wild-type strains. This also indicates that the excess OEE1 is not c o m p e t i n g for some factor required for photosynthetic oxygen evolution a n d thereby reducing oxygen evolution in R 1 - K - 5 0 cells. E x a m i n a t i o n o f the PSII reaction-center proteins D 1 and D 2 showed that b o t h a c c u m u l a t e d to wild-type levels, but n o t greater, in the R 1 - K - 5 0 strain. There was also no affect on the a c c u m u l a t i o n o f either the O E E 2 or the L H C I ! proteins. T h u s when OEE1 is limiting, the reaction-center proteins are susceptable to degradation, but when O E E 1 is in excess a c c u m u l a t i o n o f reactioncenter protein is n o t directly affected. This clearly shows that OEE1 is n o t a prime determinant o f PSII protein stoichiometry, and also indicates that protein stoichiom e t r y is n o t determined simply by degrading any protein not associated with other P S I I proteins or with the thylakoid m e m b r a n e s (as excess O E E 1 accumulates freely in the soluble phase). The a m o u n t o f O E E 1 b o u n d to the thylakoid m e m brane is the same in R 1 - K 50 and the wild type, indicating that O E E l - b i n d i n g sites on the thylakoid m e m b r a n e are limited. This w o u l d be expected if one o f the reactioncenter proteins, like D1 or D 2 or a c o m b i n a t i o n o f the two, contained the binding site for O E E 1 . It is also interesting to note that all o f the O E E 2 f o u n d in the R 1 - K - 5 0 strain is b o u n d to the thylakoid membrane. I f O E E 1 contained the binding site o f O E E 2 , as some authors have suggested, it w o u l d be expected that some o f the O E E 2 w o u l d be associated with the u n b o u n d OEE1 and thus a p p e a r in the soluble protein fraction o f R 1 - K - 5 0 , which it does not. Thus it is unlikely that the binding site for O E E 2 resides on O E E 1 , a l t h o u g h it is possible that O E E 2 does bind to OEE1 but only when OEE1 is b o u n d to the m e m b r a n e . The d a t a presented here show that a l t h o u g h stoichiometries o f proteins within the P S I I reaction centers are greatly influenced by the absence o f OEE1 they are not p e r t u r b e d by its overexpression. W h e t h e r this would also be the case in overexpression o f one o f the core proteins such as D 1 or D 2 c a n n o t be determined f r o m these data. W h a t e v e r m e c h a n i s m is used to determine stoichiometry within the P S I I c o m p l e x it is clearly not a case o f simple d e g r a d a t i o n o f all proteins not associated with a complex. These d a t a also indicate that if a m e c h a n i s m exists which regulates P S I I - p r o t e i n stoichiometry by m o n i t o r ing P S I I - p r o t e i n c o n t e n t it is operating independent o f OEE1.
The author wishes to thank Karen Freriks for excellent technical assistance in every aspect of this project. I also thank Mich Hein (Scripps Clinic, La Jolla, Cal., USA) for helpful discussions throughout the project an Avi Danon (Scripps Clinic) and Laurel Spear-Bernstein (Scripps Clinic) for a critical reading of the manuscript. This work was supported by grant GM41353 from the National Institutes of Health, USA.
S.P. Mayfield: OEE1 over-expression
References Apel, K. (1979) Phytochrome induced appearance ofm-RNA activity for the apoprotein of the light-harvesting chlorophyll a/b protein of barley. Eur. J. Biochem. 97, 183-188 Bennett, J. (1981) Biosynthesis of the light-harvesting chlorophyll a/b protein: polypeptide turn-over in darkness. Eur. J. Biochem. 118, 61-70 Bennoun, P., Spierer-Herz, M., Erickson, J., Girard-Bascou, J., Pierre, Y., Delosme, M., Rochaix, J.-D. (1986) Characterization of photosystem II mutants of Chlamydomonas reinhardtii lacking the psbA gene. Plant Mol. Biol. 6, 151-160 Chua, N.H., Bennoun, P. (1975) Thylakoid membrane polypeptides of Chlamydomonas reinhardtii: wild type and mutant strains deficient in photosystem II reaction centers. Proc. Natl. Acad. Sci. USA. 72, 2175-2179 Erickson, J.M., Rahire, M., Malnoe, P., Girard-Bascou, J., Pierre, Y. Bennoun, P., Rochaix, J.-D. (1986) Lack of the D2 protein in a Chlamydomonas reinhardtii psbD mutant affects photosystern II stability and D1 expression. EMBO J. fi, 1745-1754 Ghanotakis, D.F., Yocum, C.F. (1990) Photosystem I1 and the oxygen-evolving complex. Annu. Rev. Plant Physiol. 41, 255-276 Goldschmidt-Clermont, M. (1986) The two genes for the small subunit of RuBP carboxylase/oxygenase are closely linked in Chlamydomonas reinhardtii Plant Mol. Biol. 6, 13-21 Gorman, D.S., Levine, R.P. (1965) Cytochrome f and plastocyanin : their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 54, 1665-1669 G ruissem, W. (1989) Chloroplast gene expression: How plants turn their plastids on. Cell. 56, 161 170 Harpster, M.H., Mayfield, S.P., Taylor, W.C. (1984) Effects of pigment deficient mutants on the accumulation of photosynthetic proteins of maize. Plant Mol. Biol. 3, 59-71 Khandjian, E.W. (1986) U.V. crosslinking of RNA to nylon membrane enhances signal. Mol. Biol. Rep. 11, 107-115 Kuchka, M.R., Mayfield, S.P., Rochaix, J.-D. (1988) Nuclear mutations specifically affect the synthesis and/or degradation of the chloroplast-encoded D2 polypeptide of Photosystem II in Chlamydomonas reinhardtii. EMBO J. 7, 319-324 Mayfield, S.P., Kindle, K. (1990) Stable nuclear transformation of Chlamydomonas reinhardtii using a C. reinhardtii gene as the selectable marker. Proc. Natl. Acad. Sci. USA 87, 2087-2091 Mayfield, S.P., Bennoun, P., Rochaix, J.-D. (1987a) Expression of the nuclear encoded OEE 1 protein is required for oxygen evolution and stability of photosystem I1 particles in Chlamydomonas reinhardtii. EMBO J. 6, 313-318 Mayfield, S.P., Rahire, M., Frank, G., Zuber, H., and Rochaix J.-D.. (1987b) Expression of the nuclear OEE2 gene is required "for high levels of photosynthetic oxygen evolution in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA. 84, 749-753 Mayfield, S.P., Rahire-Schirmer, M., Frank, G., Zuber, H., Rochaix, J.-D. (1989) Analysis of the genes of the OEE1 and OEE3 proteins of the Photosystem II complex from Chlamydomonas reinhardtii. Plant Mol. Biol. 12, 683-693 Metz, J.G., Miles, D. (1982) Use of nuclear mutations of maize to identify components ofphotosystem II. Biochem. Biophys. Acta 681, 95-102 Mullet, J. (1988) Chloroplast development and gene expression. Annu. Rev. Plant Physiol. 39, 475-502 Rochaix, J.-D., Kuchka, M. R., Mayfield, S.P., Schirmer-Rahire, M., Girard-Bascou, J., Bennoun, P. (1989) Nuclear and chloroplast mutations affect the synthesis or stability of the chloroplast psbC gene product in Chlamydomonas reinhardtii. EMBO J. 8, 1013 1021 Sueoka, N. (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardtii. Proc. Natl. Sci. USA. 46, 83-91 Tobin, E.M., Silverthorne, J. (1985) Light regulation of gene expression in higher plants. Annu. Rev. Plant Physiol. 36, 569-593
P l a n t a 9 Springer-Verlag1991
Over-expression of the oxygen-evolving enhancer 1 protein and its consequences on photosystem II accumulation Stephen P. Mayfield Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037, USA Received 6 February; accepted 18 April 1991
Abstract. By transformation with a cloned wild-type oeel gene, which codes for the oxygen-evolving enhancer 1 (OEE1)protein, we have constructed a strain of Chlamydomonas reinhardtii containing multiple copies of this gene. A transformant ( R 1 - K - 5 0 ) containing four to five copies of the oeel gene accumulated oeel m R N A in approximately threefold excess of the wild type. The OEE1 protein accumulated in proportion to the oeelm R N A levels in these cells. These data indicate that no apparant feedback mechanism is operating to reduce either transcription or translation of the introduced oeel genes as a means to regulate OEEl-protein accumulation. The OEE1 protein in R 1 - K - 5 0 was all of mature size, indicating that the transit peptide had been completely removed, and that all o f the protein was located within the thylakoid lumen. Photosystem II reactioncenter proteins D1 and D2 accumulated to wild-type levels, but not greater, in these cells, while there was no effect on accumulation o f any of the PSII peripheral proteins such as OEE2 or LItCII. The OEE1 protein which accumulated in excess of wild-type levels was not bound to the thylakoid membranes, indicating that a limited number o f binding sites for OEE1 exist on the thylakoid membranes. No difference in photosynthetic oxygen evolution was observed between wild-type and R 1 - K - 5 0 strains. These data show that whatever mechanisms are used to determine stoichiometry within the PSII complex they are not perturbed by overexpression of the OEE1 protein.
Key words: Chlamydomonas (photosynthesis) - Nuclear transformation - Oxygen-evolving proteins - Photosystern II accumulation
Introduction The synthesis and assembly of photosynthetic complexes in higher plants and algae requires the close cooperation Abbreviations: bp=basepairs; LHCII=light-harvesting complex II; OEE1 = oxygen-evolving enhancer 1; SDS= sodium dodecyl sulfate
of both the chloroplast and nuclear genomes. Proteins which comprise these multisubunit complexes are encoded in both the nuclear and chloroplastic genomes. Expression of photosynthetic proteins is coordinately regulated so that proteins within any of these complexes are always at or near their correct stoichiometry. Clearly there are a number of mechanisms which affect the accumulation of photosynthetic proteins, including transcription (Tobin and Silverthorne 1985), m R N A stability (Gruissem 1989) and protein translation (Mullet 1989). Each of these mechanisms acts to regulate protein accumulation, but to date there is no evidence which indicates that any one of these mechanisms regulates protein accumulation to the degree required to achieve the precise stoichiometries that are observed in photosystem complexes. It seems likely that a combination of all of these plus other, as yet determined, factors control this precise protein accumulation. One step of the biosynthetic pathway which strongly influences the accumulation of the photosynthetic complexes involves the degradation of component proteins in the absence of stable accumulation of a complete complex. Examination of a number of photosynthetic mutants in higher plants and algae has shown that accumulation of a complex requires the accumulation of every member of that complex (Apel 1978; Bennett 1981 ; Metz and Miles 1982; Harpster et al. 1984). Characterization of PSII mutants in C. reinhardtii and maize has shown that the loss of a single PSII core protein results in the loss of the entire complex (Chua and Bennoun 1975; Metz and Miles 1982; Bennoun et al. 1986; Kuchka et al. 1988; Rochaix et al. 1989). The synthesis o f PSII core proteins in these mutants, other than the protein directly affected by the mutation, continues at normal levels, but the newly synthesized proteins are rapidly degraded and never accumulate to wild-type levels. Peripheral PSII proteins, such as those of the oxygen-evolving and lightharvesting complexes, accumulate normally in PSII core mutants. Characterization of mutants deficient in one or another of the peripheral oxygen-evolving proteins has shown that two of these, called the oxygen-evolving enhancer
106 p r o t e i n s 1 a n d 2 ( O E E 1 a n d O E E 2 ; M a y f i e l d et al. 1987a, b), are a b s o l u t e l y r e q u i r e d for h i g h levels o f p h o t o s y n t h e t i c o x y g e n e v o l u t i o n . A b s e n c e o f the O E E 2 p r o t e i n r e s u l t s i n r e d u c e d rates o f p h o t o s y n t h e t i c o x y g e n e v o l u t i o n , a l t h o u g h cells w h i c h l a c k this p e p t i d e a r e still a b l e to g r o w p h o t o s y n t h e t i c a l l y . P h o t o s y s t e m II core a n d o t h e r p e r i p h e r a l p r o t e i n s a c c u m u l a t e to n o r m a l levels i n O E E 2 - d e f i c i e n t s t r a i n s ( M a y f i e l d et al. 1987b). A b s e n c e o f the O E E 1 p r o t e i n r e s u l t s in t h e c o m p l e t e loss o f p h o t o s y n t h e t i c o x y g e n e v o l u t i o n a n d these cells are u n a b l e to g r o w p h o t o s y n t h e t i c a l l y . P h o t o s y s t e m II react i o n - c e n t e r p r o t e i n s a c c u m u l a t e to o n l y 20 % o f w i l d - t y p e levels i n a n O E E l - d e f i c i e n t s t r a i n ( M a y f i e l d et al. 1987a). T h i s r e d u c t i o n i n a c c u m u l a t i o n o f c o r e P S I I p r o t e i n s is d u e to a d e c r e a s e i n the s t a b i l i t y o f t h e P S I I c o r e particle, n o t to a r e d u c t i o n i n s y n t h e s i s o f a n y o f the c o r e p r o t e i n s . T h e O E E 2 a n d O E E 3 p r o t e i n s a c c u m u l a t e to w i l d - t y p e levels in the O E E l - d e f i c i e n t cells ( M a y f i e l d et al. 1987a). T h u s O E E 1 s t r o n g l y affects the s t o i c h i o m e t r y o f P S I I c o r e p r o t e i n s , a n d in vitro e v i d e n c e i n d i c a t e s t h a t O E E 1 a n d t h e P S I I r e a c t i o n - c e n t e r p r o t e i n s D1 a n d D 2 are physically associated (reviewed Ghanotakis and Yocum 1990). Since the a b s e n c e o f a n y single m e m b e r o f the P S I I c o m p l e x so d r a m a t i c a l l y affects the a c c u m u l a t i o n o f o t h e r m e m b e r s o f t h e P S I I c o m p l e x , we were i n t e r e s t e d to d e t e r m i n e w h a t effect the o v e r e x p r e s s i o n o f o n e m e m b e r o f t h e c o m p l e x w o u l d h a v e o n the a c c u m u l a t i o n o f other PSII proteins. We therefore constructed a strain of C. reinhardtii w h i c h c o n t a i n e d m u l t i p l e c o p i e s o f the oeel gene.
Materials and methods Construction of plasmid containing the oeel gene. Chlamydomonas reinhardtii genomic clones encoding the OEE1 protein were identified from a ;~-EMBL3 library (Mayfield et al. 1989). An 8-kb fragment, containing the entire oeel gene as well as 3 kb of DNA 5' and 2 kb of DNA 3', was subcloned into plasmid pUC19 at the unique Eco R1 and Kpn 1 sites to form plasmid pSB101 (Mayfield and Kindle 1990). Electroporation of Fud44 cells with pSBIO1 DNA. The Chlamydomonas reinhardtii OEE1- and cell-wall-deficient strain Fud44-cwl 5 was grown in complete medium (Gorman and Levine 1965) to a density of 1" 106 cells-ml -~ (log phase). Cells were pelleted by centrifugation at 6000 9g in a Beckman (Beckmann, Palo Alto, Cal., USA) SJ12 rotor at 4 ~ C for 5 min. The pellet was resuspended in water to a density of 1' 106 cells-ml -~ and pelleted again by centrifugation. The pellet was then resuspended in minimal medium (Sueoka 1960) at a density of 5 9 107 cells 9m l - 1 Fifty lal of cells in minimal media were pipetted into 500 lal microcentrifuge tubes and 1-5 lal of pSBI01 plasmid DNA (0.5 lag) was added directly to the cell suspension. The tubes containing the cell/DNA mixture were placed on ice prior to electroporation. The entire content of the tube (50 lal) was pipetted between two 1-cm-wide stainless-steel electrodes which were 0.5 mm apart (p/n 474 electrode; BTX Co. San Diego, Cal., USA). A single pulse was delivered to the cell mixture from a 500-laF capacitor (Transfector 300; BTX Co.) which was discharged through a voltage booster (Power Plus; BTX Co.). Immediately following the electrical discharge the cells were pipetted into 3 ml of liquid minimal medium, and then spread onto minimal-medium plates. The electrodes, electroporation apparatus, and liquid minimal medium were all maintained at room tem-
S.P. Mayfield: OEEI over-expression perature. The minimal plates containing the Fud44 cells were placed in a growth room at 25 ~ C with continuous light from fluorescent tubes (approx. 160 W - m - Z ) . After 10-14 d, small green colonies appeared which were subcloned onto minimal-medium plates. Colonies which grew on the second selective plate were characterized for the presence of oeel sequences and for the expression of oeel mRNA and protein. Isolation of DNA, RNA and protein from transformants. For DNA isolation, cells were grown in complete liquid media to a density of 1-107 cells" m1-1. Cells were pelleted, resuspended in an equal volume of 100 mM Tris pH 8.0, 400 mM NaCI, 40 mM EDTA and 2% sodium dodecyl sulfate (SDS). Pronase (protease XIV; Sigma, St. Louis, Mo., USA) was added to a final concentration of 4 lag - ml- x. Following incubation at 60 ~ C for 1 h the mixture was extracted twice with an equal volume of phenol/chloroform (1 : 1, v/v), and precipitated with 2.5 volumes of ethanol. The nucleic acid was pelleted by centrifugation at 10 000"9 for 10 min, air-dried, resuspended in a small volume of TE (10 mM Tris pH 7.5, 1 mM EDTA), and RNase A was added to a final concentration of 20 Ixg - m l - x. The mixture was incubated at 60~ C for 20 min prior to digestion at 37 ~ C with an appropriate restriction enzyme and buffer. Following digestion, samples were separated on 1% agarose gels and blotted to nylon membrane (Hybond-N; Amersham, Arlington Heights, Ill., USA) in 10x SSC (0.15 M NaC1, 15mM Sodium Citrate, pH 7.0). The DNA was fixed to the nylon membrane by UV irradiation (10 min on 300-nm UV light box). The filters were prehybridized in: 50% formamide, 5 x SSPE (0.18 M NaC1, 10mM NaHzPO4"2H20, l mM EDTA, pH7.0), 0.1% SDS, 0.5% non-fat dry milk and 10 lag " ml- 1 salmon-sperm DNA, at 42 ~ C for 1 h, and then hybridized in the same solution at 42 ~ C overnight with the addition of a denatured 32p_labele d DNA probe. The filters were washed (3 x 30 min in 1 x SSPE and 0.1% SDS at 60 ~ C, and then exposed to film (XAR; Kodak, Rochester, N. Y., USA) with an intensifying screen (Cronex, Dupont, Wilmington, DE., USA) at - 70 ~ C. For RNA and protein isolation the cells were grown to a density of 5" 106 cells" ml -a. Isolation of RNA, gel electrophoresis and blotting were essentially as described by Rochaix et al. (1987). Following fractionation on denaturing agarose gels, the RNA was electroblotted to nylon membrane and fixed by UV irradiation (Khadjian 1986). The filters were prehybridized, hybridized, washed and exposed to film exactly as for the DNA blots. For protein isolation, cells were resuspended in 750 mM Tris pH 8.0, 15% sucrose and 100 mM [3-mercaptoethanol (I3ME). The cells were disrupted by sonication with a microtip (Sonic 2000; Braun) for 15 s at 80% maximum power. The membrane fraction was pelleted at 1 0 0 0 0 ' 9 for 15 min at 4 ~ C. The supernatant (soluble fraction) was pipetted off, and the pellet (membrane fraction) resuspended in the above buffer. For separation of membrane and soluble proteins under conditions in which the OEE1 protein remains bound to the thylakoid membranes, cells were resuspended in 25 mM Tris pH 7.5, 100 mM NaC1, 5 mM MgCIz, 100 mM 13ME and 15 % sucrose. All other treatments to the cells were exactly as described above. The soluble proteins were made 2 % SDS, 2 % I3ME and separated by electrophoresis on 7.5-15 % acrylamide gels containing 0.1% SDS. For staining, the gels were treated with 50% methanol, 10 % acetic acid, 0.25 % Coomassie blue, and destained in 40 % methanol, 10 % acetic acid. For blotting the gels were incubated in 25mM Tris, 190mM glycine, 0.1% SDS for 20min at room temperature, and then electroblotted to nitrocellulose in the same buffer at 20 V for 2 h in a transblot apparatus. Following electroblotting the nitrocellulose filters were incubated in TBS (50 mM Tris pH 7.5, 150 mM NaC1, 0.1% Tween 20) containing 5% nonfat dry milk for 1 h at room temperature. Rabbit polyclonal antisera was added and the incubation continued for an additional 6 h. The filter was washed twice in TBS and then incubated in TBS, 5% nonfat milk containing goat-antirabbit IgG conjugated with alkaline phosphatase for 3 h at room temperature. The filter was washed three times in TBS and then developed with alkaline-phosphataseactivity staining.
S.P. Mayfield: OEE1 over-expression
107
Fig. 1. Southern analysis of oeel genes. DNAs were digested with Pst 1 and hybridized with a a2p-labeled 700-bp oeel genomic fragment. The oeel gene in the mutant Chlamydomonas strain Fud44 is 5 kb larger than in the wild type due to the insertion of a transposable element. A dilution of R1-K-50 shows that the wild-type oeel fragment in R1-K-50 is approximately threefold more abundant than in the wild type
Fig. 2A, B. Analysis ofoeel mRNA in R1-K-50. Total RNA from wild-type, Fud44 and R1-K-50 Chlamydomonascells was separated on denaturing agarose gels, blotted to nylon membrane and hybridized with a a2p-labelled oeel cDNA probe A. An identical filter was hybridized with a2p-labeled cDNA encoding the small subunit (SSu) of ribulose-l,5-bisphosphate carboxylase/oxygenase B
Results
fragments of 2.5 kb and 3.5 kb, which could also contain functional oeel genes, were also observed in the R I - K - 5 0 D N A . A fragment which hybridizes with the oeel gene but is too small to contain a full-length copy of the gene, and the endogenous Fud44 oeel fragment (7 kb), which is nonfunctional, are also observed in R 1 - K - 5 0 . C o m p a r i s o n o f a 1/2 and 1/5 dilution o f the R 1 - K - 5 0 D N A with the wild-type lane shows that the there are approximately three copies of the 2.2-kb oeel gene in the R 1 - K - 5 0 strain. Thus t r a n s f o r m a n t R 1 - K - 5 0 has potentially four or five functional copies of the oeel gene, as well as the Fud44 copy and a smaller partial copy of the gene.
Identification of a strain containing multiple copies of the oeel 9ene. The OEEl-deficient m u t a n t Fud44 was transformed by electroporation of cells in the presence o f plasmid D N A (pSB101) containing a cloned copy o f the wild-type oeel gene. T r a n s f o r m a n t s were selected for photosynthetic growth by plating onto minimal m e d i u m (Mayfield and Kindle 1990). Only cells which express the OEE1 protein (transformants) should be able to grow photosynthetically. Several dozen photosynthetic transformants were analyzed by Southern analysis and three were identified as containing multiple copies o f the oeel gene. One of these, R 1 - K - 5 0 , appeared to contain several complete copies o f the oeel gene. This transformant, which was obtained f r o m a transformation with pSB101 plasmid digested with EcoR1 and K p n I, was used for further characterization. T o assess the copy n u m b e r o f oeel genes in R 1 - K - 5 0 , D N A s were isolated f r o m wild type, Fud44 and transform a n t R 1 - K - 5 0 . The D N A s were digested with Pst I, separated on an agarose gel and blotted to nylon m e m brane. As shown in Fig. 1, R 1 - K - 5 0 has multiple copies of the oeel fragment which migrate at the same position as the wild-type fragment (2.2 kb). Additional
Multiple copies of the oee l gene results in accumulation of oeel mRNA in excess of wild type. T o determine if the presence of multiple copies o f the oeel gene results in accumulation of oeel m R N A in excess o f wild-type levels, oeel m R N A was measured in R 1 - K - 5 0 cells. Total R N A , 3 ~g/lane, f r o m wild-type, Fud44 and R 1 - K - 5 0 strains was separated on denaturing formaldehyde gels, blotted to nylon m e m b r a n e and fixed by U V irradiation. The filters were hybridized with a 32p-labeled oeel c D N A (Mayfield et al. 1989), and washed and exposed to film as described. As shown in Fig. 2A, oeel
108
S.P. Mayfield: OEE1 over-expression
oeel m R N A is approximately threefold more abundant in R 1 - K - 5 0 than in the wild type (Fig. 2A). As previously shown (Mayfield et al. 1987a), Fud44 fails to accumulate any detectable a m o u n t of oeel m R N A . A second identical filter was probed with a 32p-labeled c D N A encoding the small subunit (SSu) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Goldschmidt-Clermont 1986). As shown in Fig. 2B each on the lanes contains approximately equal amounts of the SSu m R N A .
Fig. 3. Western blot analysis of OEEl-protein accumulation in R l-K-50. Proteins were isolated, under conditions in which OEE 1 protein is soluble, from wild-type, Fud44 and R1-K-50 strains of Chlamydomonas, separated on SDS polyacrylamide gels and either stained with Coomassie blue (left panel) or transferred to nitrocellulose membrane and hybridized with either OEE1 or OEE2 antisera (right panels). The over-accumulation of OEE 1 can be observed in the stained gel (arrow)
m R N A , which runs as a distinct doublet in this gel system, accumulates in R 1 - K - 5 0 cells to a greater degree then in the wild-type strain. C o m p a r i s o n of a dilution of R I - K - 5 0 R N A with that o f the wild type shows that the
Overexpression of oeel m R N A leads to a proportional accumulation of OEE1 protein. To determine what affect the accumulation of excess oeel m R N A had on OEE1protein accumulation, we measured OEE1 protein in wild-type, Fud44 and R 1 - K - 5 0 strains. Total cellular proteins were separated into crude soluble and membrane fractions under conditions (750 m M Tris p H 8) in which the OEE1 proteins are soluble. Equal amounts of soluble proteins were loaded onto gradient polyacrylamide gels containing SDS. Following electrophoresis the gels were either stained with Coomassie blue or transferred to nitrocellulose membrane. The nitrocellulose membranes were reacted with antisera against the OEE1 or OEE2 protein. As shown in Fig. 3 (left panel) most of the Coomassie-staining bands are c o m m o n to the wildtype, Fud44 and R 1 - K - 5 0 strains. Reaction of OEE1 antisera with blotted proteins reveals that the OEE1 protein accumulates to a greater degree in the R 1 - K - 5 0 cells than in the wild type (Fig. 3, central panel). Comparison of a dilution of R 1 - K - 5 0 with that of the wild type shows that OEE1 accumulates in R 1 - K - 5 0 to levels approximately threefold greater than in the wild type. The over-accumulation of OEE1 in R 1 - K - 5 0 cells can also be seen in the Coomassie-stained gel (arrow, left panel). As previously shown, no OEE1 protein accumulates in the Fud44 strain (Mayfield et al. 1987a). Measurement of OEE2 in a similar blot (Fig. 3, right
Fig. 4. Western blot analysis of membrane-protein accumulation in R1-K 50. Proteins were isolated from wild-type, Fud44 and R1-K-50 strains of Chlamydomonas separated on SDS polyacrylamide gels and either stained with Coomassie blue (left panel) or transferred to nitrocellulose membrane and decorated with either D1, D2 or LHCII antisera (right panels)
S.P. Mayfield: OEE1 over-expression
Fig. 5. Representative western blot analysis of OEE 1 protein association with thylakoids in R1-K-50. Proteins were isolated, under conditions in which OEE1 protein remains bound to the thylakoid membranes (mere), and separated in membrane and soluble (sol) fractions. Both soluble and membrane proteins from wild-type, Fud44 and RI-K-50 Chlamydomonas were separated on SDS polyacrylamide gels and either stained with Coomassie blue (left panel) or transferred to nitrocellulose membrane and hybridized with either OEE1 or OEE2 antisera (right panels). The accumulation of OEE1 in the soluble fraction of R1-K-50 can be observed in the stained gel (arrow) panel), shows that OEE2 accumulates to equal amounts in each of the strains.
Photosystem H reaction-center proteins accumulate to wild-type levels in R1-K-50 cells. As the absence of OEE1 protein results in a substantial reduction of PSII reaction-center proteins we were interested to see if overexpression of the OEE1 protein would result in an increased accumulation of PSII reaction-center proteins. To measure PSII reaction-center proteins, equal amounts of membrane proteins from the wild type, Fud44 and R 1 - K - 5 0 were separated on gradient polyacrylamide gels containing SDS. Following electrophoresis the gels were either stained with Coomassie blue (Fig. 4, left panel) or transferred to nitrocellulose filters for antibody hybridization. As shown in Fig. 4 (central panels) both the D1 and D2 reaction-center proteins accumulate to wild-type levels, but not more, in the R 1 - K - 5 0 strain. As previously shown D1 and D2 accumulate to only 20% of wild-type levels in the OEEl-deficient strain Fud44 (Mayfield et al. 1987a), while the L H C I I proteins accumulate to similar levels in each of the strains (right panel). Attachment of OEE1 protein to photosynthetic membranes is similar in wild-type and R1-K-50 strains. To determine
109 if OEE1 protein which accumulates in excess of the amount normally associated with the PSII core was bound to the membranes or was free in solution we measured OEE proteins under conditions in which the OEE proteins are normally retained on the thylakoid membranes. Wild-type and R 1 - K - 5 0 cells were pelleted, resuspended in a low-salt buffer and briefly sonicated to disrupt the cells. The membrane fraction, which should retain the OEE proteins if they are bound to the thylakoid, was pelleted and the supernatant (soluble fraction) saved. Soluble and membrane fractions were separated on SDS-polyacrylamide gels and either stained with Coomassie blue or transfered to nitrocellulose for antibody hybridization. As shown in Fig. 5 (left panel) the proteinstaining pattern is similar for both wild-type and R 1 - K - 5 0 cells. Western analysis using OEE1 antisera shows that wild-type and R l - K - 5 0 membrane fractions contain approx, equal amounts of the OEE1 protein (Fig. 5, central panel). N o OEE1 is detected in the soluble fraction of wild-type cells, while OEE1 is detected in the soluble fraction of the R l - K - 5 0 cells (central panel). A similar' filter probed with the OEE2 antisera shows that equal amounts o f OEE2 are found in the membrane fraction of both the wild type and R 1 - K - 5 0 while no OEE2 is found in either of the soluble protein fractions.
Discussion
Analysis of mutants deficient in single PSII proteins has shown that the absence of a single protein results in the loss of the entire PSII reaction-center complex. The loss of these proteins is not due to a decrease in synthesis or assembly, but rather to a more rapid degradation of the proteins after insertion into the thylakoid membranes (Erickson et al. 1986; Mayfield et al. 1987a; Kuchka et al. 1988). Since deficiencies of single proteins have such profound effects on PSII-complex accumulation we were interested to determine if PSII-protein accumulation would be influenced by overexpression of one member (OEE1 protein) of the PSII complex. As a means of overexpressing the OEE1 protein a strain of C. reinhardtii was engineered which contained multiple copies of the oeel gene. Although a number of factors are likely to influence the expression o f introduced oeel genes, the end result was that multiple copies of the oeel gene resulted in m R N A and protein accumulation in excess of wild-type levels. Examination of a number of oeel transformants showed that m R N A accumulation was roughly proportional to the copy number of the introduced gene, and that OEEl-protein accumulation was proportional to the level of oeel m R N A . Thus no apparant feedback mechanism is operating to reduce either the transcription or translation of oeel as a means to regulate OEE 1-protein accumulation. All of the OEE1 protein observed in the R 1 - K - 5 0 strain is mature protein, showing that the transit peptide has been completely removed and indicating that all of the protein is found within the lumen of the thylakoid
110 m e m b r a n e . There was no difference in p h o t o s y n t h e t i c oxygen evolution between wild-type and R 1 - K - 5 0 cells, which is n o t too surprising as the PSII reaction-center complexes a p p e a r identical in R 1 - K - 5 0 and wild-type strains. This also indicates that the excess OEE1 is not c o m p e t i n g for some factor required for photosynthetic oxygen evolution a n d thereby reducing oxygen evolution in R 1 - K - 5 0 cells. E x a m i n a t i o n o f the PSII reaction-center proteins D 1 and D 2 showed that b o t h a c c u m u l a t e d to wild-type levels, but n o t greater, in the R 1 - K - 5 0 strain. There was also no affect on the a c c u m u l a t i o n o f either the O E E 2 or the L H C I ! proteins. T h u s when OEE1 is limiting, the reaction-center proteins are susceptable to degradation, but when O E E 1 is in excess a c c u m u l a t i o n o f reactioncenter protein is n o t directly affected. This clearly shows that OEE1 is n o t a prime determinant o f PSII protein stoichiometry, and also indicates that protein stoichiom e t r y is n o t determined simply by degrading any protein not associated with other P S I I proteins or with the thylakoid m e m b r a n e s (as excess O E E 1 accumulates freely in the soluble phase). The a m o u n t o f O E E 1 b o u n d to the thylakoid m e m brane is the same in R 1 - K 50 and the wild type, indicating that O E E l - b i n d i n g sites on the thylakoid m e m b r a n e are limited. This w o u l d be expected if one o f the reactioncenter proteins, like D1 or D 2 or a c o m b i n a t i o n o f the two, contained the binding site for O E E 1 . It is also interesting to note that all o f the O E E 2 f o u n d in the R 1 - K - 5 0 strain is b o u n d to the thylakoid membrane. I f O E E 1 contained the binding site o f O E E 2 , as some authors have suggested, it w o u l d be expected that some o f the O E E 2 w o u l d be associated with the u n b o u n d OEE1 and thus a p p e a r in the soluble protein fraction o f R 1 - K - 5 0 , which it does not. Thus it is unlikely that the binding site for O E E 2 resides on O E E 1 , a l t h o u g h it is possible that O E E 2 does bind to OEE1 but only when OEE1 is b o u n d to the m e m b r a n e . The d a t a presented here show that a l t h o u g h stoichiometries o f proteins within the P S I I reaction centers are greatly influenced by the absence o f OEE1 they are not p e r t u r b e d by its overexpression. W h e t h e r this would also be the case in overexpression o f one o f the core proteins such as D 1 or D 2 c a n n o t be determined f r o m these data. W h a t e v e r m e c h a n i s m is used to determine stoichiometry within the P S I I c o m p l e x it is clearly not a case o f simple d e g r a d a t i o n o f all proteins not associated with a complex. These d a t a also indicate that if a m e c h a n i s m exists which regulates P S I I - p r o t e i n stoichiometry by m o n i t o r ing P S I I - p r o t e i n c o n t e n t it is operating independent o f OEE1.
The author wishes to thank Karen Freriks for excellent technical assistance in every aspect of this project. I also thank Mich Hein (Scripps Clinic, La Jolla, Cal., USA) for helpful discussions throughout the project an Avi Danon (Scripps Clinic) and Laurel Spear-Bernstein (Scripps Clinic) for a critical reading of the manuscript. This work was supported by grant GM41353 from the National Institutes of Health, USA.
S.P. Mayfield: OEE1 over-expression
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