Article
Reference
Analysis of the genes of the OEE1 and OEE3 proteins of the photosystem II complex from Chlamydomonas reinhardtii
MAYFIELD, Stephen Patrick, et al.
Abstract
The sequences of the nuclear genes of the 33 kDa (OEE1) and the 16 kDa (OEE3) polypeptides of the oxygen evolving complex of Chlamydomonas reinhardtii have been established. Comparison between the OEE1 protein sequences of C. reinhardtii and higher plants and cyanobacteria reveals 67 and 47% homology. In contrast, C. reinhardtii and higher plants have only 28% overall homology for OEE3 which is mostly limited to the central portion of the protein. The transit peptides of the C. reinhardtii proteins consist of 52 (OEE1) and, most likely, 51 (OEE1) amino acids. They have a basic amino terminal region and, at least in the case of OEE1, a hydrophobic segment at their carboxy terminal end typical of thylakoid lumen proteins. Comparison of the genomic and cDNA clones indicates that the OEE1 and OEE3 genes contain five and four introns, respectively, some of which are located within the coding sequences of the transit peptides.
MAYFIELD, Stephen Patrick, et al . Analysis of the genes of the OEE1 and OEE3 proteins of the photosystem II complex from Chlamydomonas reinhardtii . Plant Molecular Biology , 1989, vol. 12, no. 6, p. 683-693
DOI : 10.1007/BF00044159
Available at:
http://archive-ouverte.unige.ch/unige:148876
Disclaimer: layout of this document may differ from the published version.
1 / 1
Analysis of the genes of the OEE1 and OEE3 proteins of the photosystem II complex from Chlamydomonas reinhardtii
Stephen P. Mayfield, 1" 3 Mich61e Schirmer-Rahire, 1 Gerhard Frank, 2 Herbert Zuber 2 and Jean-David Rochaix ~ *
l Departments of Molecular Biology and Plant Biology, University of Geneva, Geneva, Switzerland (* author for correspondence); 2 Eidgen6ssische Technische Hochschule, H6nggerberg, CH-8093 Zarich, Switzerland; 3 Present address: Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Rd, La Jolla, CA 92037, USA
Received 8 November 1988; accepted in revised form 1 March 1989
Key words: Chlamydomonas reinhardtii, chloroplast, oxygen evolving complex, photosystem I1, transit pep- tides
Abstract
The sequences of the nuclear genes of the 33 kDa (OEE1) and the 16 kDa (OEE3) polypeptides of the oxygen evolving complex of Chlamydomonas reinhardtii have been established. Comparison between the OEE1 protein sequences of C. reinhardtii and higher plants and cyanobacteria reveals 67 and 47%
homology. In contrast, C. reinhardtii and higher plants have only 28 % overall homology for OEE3 which is mostly limited to the central portion of the protein. The transit peptides of the C. reinhardtii proteins consist of 52 (OEE1) and, most likely, 51 (OEE1) amino acids. They have a basic amino terminal region and, at least in the case of OEE 1, a hydrophobic segment at their carboxy terminal end typical ofthylakoid lumen proteins. Comparison of the genomic and cDNA clones indicates that the OEE1 and OEE3 genes contain five and four introns, respectively, some of which are located within the coding sequences of the transit peptides.
Introduction
The photolysis of water and subsequent evolution of oxygen during photosynthesis take place within the chloroplasts of higher plants and algae on membrane-bound photosystemlI (PSII) par- ticles. These particles are composed of a mem- brane embedded core, which includes the reaction center, an oxygen-evolving enhancer complex and a light-harvesting system. Isolated oxygen-
* The nucleotide sequence data reported will appear in the EMBL, the accession number X13826 (OEE1) and X13832 (OEE3).
evolving particles are complex assemblies containing chlorophylls a and b, carotenoids, quinones, lipids, iron, a catalytic manganese cluster and an array of at least nine polypeptides [1, 2]. PSII proteins can be grouped into two distinct classes. The first includes the core PSII proteins which are integral membrane proteins encoded by the chloroplast genome. The second includes the three extrinsic proteins of the oxygen- evolving complex called oxygen-evolving enhan-
GenBank and DDBJ Nucleotide Sequence Databases under
cer proteins 1, 2 and 3 (OEE1, 2 and 3) [1, 3];
these proteins are coded for by nuclear genes, synthesized as precursors on cytoplasmic ribo- somes [4] and post-translationally transported across the two chloroplast envelope membranes and the thylakoid membrane into the lumen of the thylakoid where they bind to the core PSII par- ticle. It has recently been shown that additional polypeptides are associated with PSII [5, 6, 7].
In vitro reconstitution experiments with isolated oxygen-evolving particles have shown that the PSII extrinsic proteins play important roles in photosynthetic oxygen evolution (reviewed in [ 1, 8]). These studies have suggested that two of the three extrinsic polypeptides (OEE2 or 24 kDa protein and OEE3 or 16kDa protein) are involved in the binding of calcium and chloride ions which are required cofactors in oxygen evolu- tion. Removal of the OEE2 and OEE3 proteins from the PSII particle by salt treatment results in a decrease in oxygen-evolving activity, but not in a reduction of manganese ion content. Removal of the OEEI (or 33 kDa protein) results in both the loss of oxygen-evolving activity and a re- duction of manganese ions bound to the thylakoid membrane although a high concentration of chloride can partially substitute for OEE1 [1].
Restoration of oxygen-evolving activity accom- panies the rebinding of these extrinsic peptides to the PSII reaction center [ 1 ].
Using a molecular genetic approach we have shown that in Chlamydomonas reinhardtii the OEE1 and OEE2 proteins are required for high levels of photosynthetic oxygen evolution in vivo [3, 10]. The absence of the OEE2 protein does not affect the accumulation of the other PSII core or peripheral proteins [3]. The absence of the OEE 1 protein greatly decreases the stability of the PSII core particle and only about 25 ~o of the core PSII proteins accumulate in mutants deficient for the expression of the OEE1 polypeptide [ 10]. The other peripheral proteins, OEE2 and OEE3, ac- cumulate in OEE 1-deficient strains, although they are probably not anchored to the photosynthetic membrane.
As a first step in identifying domains within the OEE proteins which are involved in photosyn-
thetic oxygen evolution and in stabilizing the PSII core complex we undertook the characterization of the OEE proteins and their genes in C. rein- hardtii. Here we present the sequence of the OEE1 and OEE3 cDNAs, the deduced amino acid se- quence of these proteins, and the characterization of the single psbl (OEE1) and psb3 (OEE3)genes from C. reinhardtii. The mature OEE1 and OEE3 proteins from C. reinhardtii are compared with the homologous proteins from spinach [10, 12, 13]
and cyanobacteria. We also present the transit peptide sequence of the OEE1 and OEE3 pro- teins and a comparison of these sequences with two other transit peptides from the OEE2 protein [ 3 ] and the small subunit ofribulose bisphosphate carboxylase (Rubisco) [14, 15] of C. reinhardtii.
Materials and methods
Isolation of cDNAs and genomic clones encoding OEE1 and OEE3
A C. reinhardtii DNA library constructed in 2-gtll [3] was independently screened with antisera raised against mature C. reinhardtii OEE1 and OEE3 proteins as described by Young and Davis [16]. Several plaques containing phage expressing one or the other of the proteins were identified and purified to homogeneity. The largest of the three cDNAs identified for the OEE1 protein was nick-translated and used to identify additional cDNAs from a C. reinhardtii 2-gtl0 library [15]. One of these cDNAs was of sufficient length (1.6 kb) to be a full-length clone.
This cDNA was subcloned into the Eco RI site of plasmid pUC19 to form plasmid pPII-12P4.
Three phage were identified which expressed the OEE3 protein. The largest cDNA obtained (pPII-24.3) was 1.3 kb in size and was subcloned into the Eco RI site of plasmid pUC8. It was used to isolate a full-length cDNA clone (pPII-24.5) from the C. reinhardtii 2-gtl0 library.
To isolate genomic clones encoding either the OEE1 or OEE3 genes, plasmids pPII-12P4 and pPII-24.3 were nick-translated and used as probes to identify genomic clones [17] from a
C. reinhardtii genomic library cloned into 2- EMBL3 [18]. Several overlapping clones were identified for each probe.
Sequencing of the OEE1 and OEE3 cDNAs, and determination of the primary amino acid sequence of the OEE1 and OEE3 proteins
The cDNA inserts of plasmids pPII-12P4, pPII-24.3 and pPII-24.5 were isolated, digested with restriction endonucleases (as diagrammed in Fig. 1), labeled at either the 5' or 3' end, and sequenced by the method of Maxam and Gilbert [ 19]. Both strands of the cDNAs were sequenced and all restriction site junctions were sequenced across by using other restriction enzymes. The sequences for the 5' ends of the psbl and psb3 genes were determined by sequencing genomic clones containing that portion of the gene. A com- posite sequence containing the 5' genomic and the entire cDNA sequence are shown for OEE1 and OEE3 in Figs 2 and 3 respectively.
Identification of mRNA-coding regions of the psb l and psb3 genes
digested with restriction enzymes which re- cognized common sites in both the genomic clones and cDNAs and then labeled at the 5' and 3' ends. The labeled DNA was heat denatured and allowed to renature in the presence of an excess of C. reinhardtii RNA. The resulting RNA/DNA hybrids were digested with S 1 nucle- ase by the method of Berk and Sharp [20]. The 5' ends of the transcripts were determined by using a sequence ladder as reference.
Isolation of DNA, gel electrophoresis and blotting For Southern analysis, DNA was isolated from wild type strain 137c by pronase digestion and phenol extraction followed by banding on CsCI gradients as described [21]. The DNA was digested with restriction endonucleases, se- parated on agarose gels and blotted to nylon membrane filters as described [22]. Prehybridi- zation and hybridization was as described by Johnson etaL [23]. All filters were washed in 1 x SSPE, 0.1% SDS at 60 °C. Autoradiogra- phy was carried out at - 70 °C with the use of an intensifying screen (Dupont, Cronex).
To determine the mRNA-coding regions of the psbl and psb3 genes, genomic clones were
Bs Bn Bs
D I Hf I O IO Bn Bn Hf B n O Hf Hf D
I I I I I I II II II I I I I
Ii1 II,
I
Hf D Bs
B S \/ D So Hf D I D So D
I I II I II III I i I
4 ~ 9 ~ 41
b 9
Fig. 1. Sequencing strategy for OEEI (A) and OEE3 (B) cDNAs. The cDNA inserts were digested with the enzymes as indicated, labeled at either the 5' or 3' end and sequenced by the method of Maxam and Gilbert. Restriction endonuclease sites are indicated
by D, Dde I; Bs, Bst NI; Hf, HinfI; Bn, Ban I; S, SalI, Sa, Sau 3A.
Protein sequencing
Mature OEE1 polypeptide was isolated from the C. reinhardtii mutant F54-14 which lacks both the PSI and the chloroplast ATPase complexes [24] as described [3]. The amino acid sequence was determined by using on Applied Biosystem model 470 A protein sequenator according to standard procedures.
Results and discussion
Cloning and sequence of the cDNAs encoding the OEE1 and OEE3 polypeptides of photosystem H Both the OEE1 and OEE3 polypeptides asso- ciated with oxygen-evolving photosystem II par- ticles (called the 33 kDa and 16 kDa proteins in spinach) had been shown to be nuclear encoded in spinach [4] as was indicated in C. reinhardtii [25]. We therefore constructed a library in the 2gtl 1 expression vector containing C. reinhardtii cDNA inserts [3]. The library was screened with rabbit polyclonal antisera specific for either the OEE1 or OEE3 polypeptides. Several plaques containing phage expressing one or the other of these polypeptides were identified and purified to homogeneity. In order to obtain full-lenght cDNA clones the inserts obtained were used as probes to identify additional clones from a C. reinhardtii 2gtl0 cDNA library [ 15]. Two full-length clones corresponding to OEE1 and OEE3 were identi- fied and their inserts subcloned into pUC 19 giving rise to plasmids pPII-12P4 and pPII-24.5, respec- tively. The nucleotide sequence of 12P4 and the deduced amino acid sequence of the OEE1 pro- tein are presented in Figure 2, and the nucleotide sequence of cDNA pPII-24.5 and the deduced amino acid sequence of the OEE3 protein are presented in Figure 3.
As a way of positively identifying the cDNAs as those encoding the authentic OEE1 and OEE3 polypeptides, and as a means of identifying the amino terminus of the mature protein, we isolated mature OEE1 and OEE3 protein for sequence analysis [3]. The amino terminal end of the
mature OEE 1 protein corresponds to amino acid 52 of the precursor OEE1 protein and is marked by an * in Fig. 2. We were unable to obtain an amino acid sequence for the mature OEE3 pro- tein, but have estimated the end of the transit peptide (amino terminus of the mature protein) from a comparison with the ends of transit se- quences of nuclear-encoded chloroplast proteins that are localized on the lumenal side of the thylakoids: OEE1 and OEE2 proteins of C. rein- hardtii (see Fig. 7) and plastocyanin of higher plants [26, 27]. The carboxy terminal part of the transit peptides of these proteins is hydrophobic and ends with AXA.
Examination of the cDNAs reveals that both share the unusual codon bias found in other C. reinhardtii nuclear genes [3, 15, 28]. Note that A is not used in the third position of any amino acid codon. This codon bias, with a few de- viations, has been observed in all of the C. rein- hardtii nuclear-encoded genes sequenced to date that give rise to abundant transcripts.
Both OEE1 and OEE3 are encoded by single-copy genes
To determine the number of genes encoding OEE1 and OEE3 in C. reinhardtii, genomic DNA was digested with restriction endonucleases, fractionated on agarose gels, blotted to nitro- cellulose and hybridized with either the labeled 750 bp Hind III fragment (at the 5' end of psbl), Fig. 5) or pPII-24.3 plasmid. As shown in Figure 4, both of these probes hybridize to only one or two fragments for each of the restriction enzymes used, suggesting that both OEE1 and OEE3 are encoded by single genes. To further characterize the psbl (encoding the OEE1 pro- tein) and psb3 (encoding the OEE3 protein) genes we isolated genomic clones for both of these genes. An EMBL3 C. reinhardtii genomic library [18] was screened using nick-translated pPII- 12P4 or pPII-24.3 as probes. Several overlapping clones were isolated for each of the cDNAs and a map of the chromosomal section containing the psbl and psb3 genes was compiled using com-
21 41 61 81 I01 121 141 161 181 201 221 241 261 281
.-150 .-100
CCGCGCGCGCAAGCACCGATCGGGCTGTAEGGCTAGCGAGATCAATTTCCGTACCGCGTCGGGGCAGGCATGAA 51 .-50
GCGCGTTGTTTCTCTTACGGAGTTGTCCCCCGTCAACCGCTAGTCGACCATTTCTCCTTTGCACACCACAGCAGCAAAG
.1 .30 .60
ATG GCC CTC CGC GCT GCC CAG TCC GCT AAG GCC GGT GTC CGT GCC GCC ~G CCC ~C CGT ] M A L R A A Q S A K A G V R A A R P N R
.90 .120
GCC ACC GCC GTG GTG TGC AAG GCG CAG AAG GTC GGC CAG GCC GCC GCT GCC GCT GCT CTG A T A V V C K A Q K V G Q A A A A A A L
i2 .150 (O) .180
GCC ACC GCC ATG GTT GCC G~ TCG GCC AAC GCC CTG ACC TTC GAC GAG ATT CAG GGC CTG
A T A M V A G S A N A L T F D E I Q G L
.210 .240
ACC TAC CTG CAG GTC ~G GGC TCC GGC ATT GCC AAC ACC TGC CCC GTG CTG GAG AGC GGC T Y L Q V K G S G I A N T C P V L E S G
• 270 .300
ACC ACC AAC CTG AAG GAG CTG AAG GCC GGC TCC TAC ~G CTG GAG AAC TTC TGC ATT GAG
T T N L K E L K A G S Y K L E N F C I E
• 330 .360
CCC ACC TCT TTC ACC GTG AAG GAG GAG AGC CAG TTC AAG GGT GGC GAG ACT GAG TTT GTC P T S F T V K E E S Q F K G G E T E F V
.390 i3 .420
AAG ACC AAG CTC ATG ACC CGC CTG ACC TAC ACC CTG GAC GCCTATG TCC GGC TCG TTC ~G K T K L M T R L T Y T L D A M S G S F K
.450 .480
GTC GGC TCT GAC GGC TCC GCT GAG CTG AAG GAG GAT GAC GGC ATT GAC TAC GCC GCC ACC V G S D G S A E L K E D D G I D Y A A T
.510 .540
ACC GTC CAG CTG CCC GGT GGC GAG CGC GTG GCT TTC CTG TTC ACC ATC AAG CAG TTC GAT
T V Q L P G G E R V A F L F T I K Q F D
.570 •600
GGC AAG GGC ACC CTG GAC GGC ATC ~G GGT GAC TTC CTG GTG CCC TCG TAC CGC GGC TCG
G K G I L D G I K G D F L V P S Y R G S
i4 .630 .660
TCC TTC CTG GAC CCC ~G~GC CGC GGI GGC TCG ACC GGC TAC GAC AAC GCC GIG GCT CIG S F L D P K R G G S T G Y D N A V A L
.690 .720
CCC GCC CGC GCT GAT GCC GAG GAG CTG CTG AAG GAG AAC GTG AAG ATC ACC AAG GCC CTG
P A R A D A E E L L K E N V K I T K A L
i5 .780
AAG GGC TCT GCC GTG TTC TCC GTT GCC AAGVGTG GAC CCC GTG ACC GGC GAG ATC GCT GGT
K G S A V F S V A K V D P V T G E I A G
.810 .840
GTG TTC GAG TCC ATC CAG CCC TCG GAC ACT GAC CTG GGC GCC AAG CCC CCC AAG GAC ATC V F E S I Q P S D T D L G A K P P K D I
.870 .900
AAG GTC ACC GGC CTG TGG TAC GCC CAG CTG AAG TAA GCGCTTTCGCCCCCCGAAGATAAACACTTCT K V T G L W Y A Q L K - -
.950
GGAGGAGGAGGACGCTACTGCTTAGCATGGTCGACCGCTGCGAGGAGGAGACGTGAGCCAGGATGCTGGAGG~GCTCTA
• .1000 . . . 1050
GCGGGTGTACATCGIAGGTGCTGCTTCCGGGATTGGAAGGGTAGGCAAAGCAGTGCTGCGGGCGGGCAGACGG~GTGTC
.1100
AAAACGCTCGCCTGTGTGCGGTGCAGTGCTGGAGCGITHTGTTGAGGACGCTCAAACTCGGGTGGATTCAGAG~TCGG
.1150 .1200
GGCGCGGAGGTTGCGTGGTTGAICGAGCGGGGGCCAAACGIIGCCGCIGCCGCACGCCTGCCGIATGCGCGCCCG~C~
• . .1250 . . 1300
CAGCCGTGGTGCTGTCGGAGCGCGAGTGCCGGTTTTGGAGGTCTAGGAGTGCTAGG~TGGTGCGAG~GCCGTGTCA~CC
.1350
GGCTTTTCGAGAAGAAAGATACAGTGTGCCCAAAAGIGTGTGATTATIGTGTTTCATAGTGGCTCTC~AAACAAGGC~G
.1400 .1450
CTIICCCAGTGTAGCCTGACACTTCGGAGCCCTATTTCCCCGACTCGCTICTCGTCGTGCATGCGCTITGCIGGCAGTA
.1500
GTGCAGCACCGCTGCTCICAGTCAATAACAGGCTGGCAAAGGCCCTGTGCACAGATTAG~CATGAGGCG~GTTTGCGGC
.1550
GGCGTGTAACACCCAAGATGAGAAAAAAAAAAAAAAAAAAA
Fig. 2. Complete nucleotide and amino acid sequence of the OEE1 cDNA and protein. The nucleotide sequence was derived from cDNA pPII-12P4 and is shown from the arrow at - 50 bp to the poly(A) tail, while the 5' end of the psbl gene was derived from sequencing genomic clones and is shown to - 150 bp from the initiation codon. The location of the transcription start is indicated by the S1 mark located 55 bp from the initiation codon. The start of the mature OEE1 protein is designated by an
* at amino acid 52. The locations of intervening sequences within the single psbl gene are shown by arrowheads numbered il through i5. The likely poly(A) addition sequence TGTAA is underlined by a wavy line.
21 v GTT
41 V GTC
61 R CGC
81 O CAG
lOl V GIG i4 121 W 141 1 ACC
161 F TIC
181 A GCC
.-300 .-Z50
GCMGCGACTGACTCCTTGCGCG,aSu4GCIUL4TGCGTGAGCGCGCG,~J~GGTCGCTTGGGCCCTCGCGAC .-200
CCCGCCCCGCGCACCICCAGCCCATACCAAGACATATGCAACCAAATGCACAAGCCGACGCACCTCCACAGCGCCCAGA
.-150 .-I00
GAGCCCGCAAGCAIAAGGTACGACGGTACGGCGGCAGAGATCGAGCCGGCATGGGCAGCGCGACCATTTTGCCICCGGC
51 .-50
AAGCAGGITTICCGTCACAGCTCTCGCTCGCAAGCAICGTCTGTCACTICTCCGCAACCACTTCAACCTAATAGCAAAG
.1 .30
ATG GCC CTC GCC lCI AAG GTT GCI ACC CGC CCC GCC GIG GCC TCC CGC CG~
] M A L A S K V A T R P A V A S R R 9 n i2
TCC GGC GAG ICG CGC CGCUGCGTGTG CTG'GGC GGT CIG GIG GTG CGC GCC
V R A GCC GCC GIG
A A V TCG GII CGC
S V R AAC GTC CGC
N V R AAG GAG Tel
K E S ACT GAG GeT
T E A CTG GCC TCG
L A S ATe CAG GCC
I q A AAG AAG CTG
K K L
S G E S R R A .150 GCC CCC ~G GCT GCC CTT GCT
A P K A A L A .210 GAC CGC GGC lie GAC CIG AIC
D R G F D L I .270 GAG GGC TTC ACE CAG GeT CGC
E G F T Q A R GAG GCC CGC ATe GAC GeT GAC .~0
E A R I D A D .390 CGC GAG CAG CIG CGC CGC CAG R E Q L R R Q
.450 ACC ~G GAG ~G GAG GCC ~G
I K E K E A K .510 GTT GAG GAC CIG GAC TTC GCC
V E O L O F A .570 GAG AIC Ace AAG GCC ~G CTG
E I T K A K L
V L G G L l (e) CTG ACC•CCC GTC GAC CTG
L T P V O L i3 TAC GAG'GCC CG~ GAC CIG
Y E A R O L GeT TCC CTG GAC GAG Ace
A S l D E T CIG GAC GTG TIC AIC CAG L O V F I Q
GTC GGC ACC CTG CGC Tl'C V G T L R F AAG GCC GCC CIG GGC CTG
K A A L G t CTG CGC GAG AAG GAC CAG
t R E K D Q GAC TCC GIC CIG GCC GCC
D S V L A A
• 60
GGT GCC GIG G A V
.120 GCG AGC GeT
A S A
. 1 8 0
TIC GAT GAC F D D
.240 GAC CTG CCC
O L P
• 300 AAG AAG CGC
K K R
.360
AAG TCC TAC K S Y
.420 GAC CTG AAC
D t N .480 CGC AAG GAG
R K E .540 GCC TCC GCC
A S A
• 500
GTC CTG TAA V L ---
.650
GCGCCCTCTTCTTTCCACCTAACAGCTGAATTTTCGGCCATCTCTGGAGCTGCTGCAGCAGCAGCAIGAGGATGGGGAG
.700 .750
~ATGTAAG~GGCTTTGGAGTGAGGAG~AGCGGTTGCG~CGGTTGCAATGCAAGCGGTGGTGTGA~GCGT~GGAACCTAG .800
AA~A~GTGTGACTG~GTGT~AA~T~GGGICCGTGTGCGAAGGAcGCGC~GTACGGGTGCA/~CCT~CGGTGG~CTAAGA
.850 ,900
G~GGGCGCCTGGAGGAGTGGTACTGAGGGCAGTGCACGGGAGTGCGCGGGTGTGCCGG~GCAGTTI~TGTTGTCTGCAC
.950
IGGGCCGCCCTGCCGCTCCGCITGGATGATGIGGCTTGIGIGTTTGGCCGCACGCIGCTTCCA~CGCGTTGCG~GCCTG
.I000 .1050
IGIGCGGII~GAIGGAIIAGGCGGTI~CCCCCAAGAGAAGACGAICAACGTAGAAAACAGGACAGGAGATAATCAAIC
.1100 .1150
TCCGCACAIIIGGCAACCCCAGITIGCCACGIGIATICICAAGCTTAGGCAIGCAGCICIICIGGAGTGGCICGCAACA .1200
GCACGGCGIGTAAIACACACGCAGTGATACATAATICIGCICIGIACATGTICCTI%GICCTIGGIGTGIAACCACGGC .1250
AAG T CAAIU~AAAAAAAAAAAAAAAAAA
Fig. 3. Complete nucleotide and amino acid sequence of the OEE3 eDNA and protein. The nucleotide sequence was derived from cDNAs pPII-24.3 and pPII-24.5 and is shown from the arrow at - 35 bp to the poly(A) tail, while the 5' end of the psb3 gene was derived from sequencing genomic clones and is shown to - 300 bp from the initiation codon. The location of the transcription start is indicated by the S 1 mark located 65 bp from the initiation codon. The likely start of the mature OEE3 protein is designated by an * at amino acid 52. The locations of intervening sequences within the single psb3 gene are shown
by arrowheads numbered il through i4. The likely poly(A) addition sequence TGTAA is underlined by a wavy line.
mon restriction enzymes (Fig. 5). Comparison of the restriction map with the pattern of the DNA fragments on the Southern blots confirmed that both psbl and psb3 are single-copy genes, as is the psb2 (OEE2 protein) gene in C. reinhardtii [ 3 ].
These results agree with the fmding that a muta- tion in the OEE1 gene segregates as a single nu- clear mutation [ 10].
To define the coding regions and to determine the 5' and 3' boundaries of the two genes,
genomic fragments were digested with restriction enzymes that recognized sites in both the cDNA and the genomic clones, labeled at either the 5' or 3' end, hybridized with an excess of C. reinhardtii mRNA, and digested with S1 nuclease. The transcription initiation site, as defined by these S 1 protection experiments, lies about 55 base pairs upstream of the ATG start codon of the psb 1 gene (shown in Fig. 2), while the transcription start site of the psb3 gene lies 65 base pairs upstream of the
OEE1 and OEE3 are adjacent to the sequence TGTAA which has been proposed as a poly(A) addition sequence in C. reinhardtii [28]. Compari- son of the cDNA and corresponding genomic sequences reveals that the psbl and psb3 genes of C. reinhardtii contain 5 and 4 exons, respectively.
Since there are no published reports on the characterization of genomic clones of the OEE1 and OEE3 genes of higher plants, it remains to be seen whether the sites of introns within these genes are the same in C. reinhardtii and higher plants.
Fig. 4. Southern blot analysis of wild type C. reinhardtii genomic DNA. The DNA was digested with the restriction enzymes as indicated at the top of the figure. Fragments were separated by agarose gel electrophoresis, blotted to nylon membrane and probed with labeled nick-translated cDNA inserts (A) from plasmid pPII-24.3 (whose 5' end is near the Sal I site shown in Fig. 5) and from the 5' end of the psbI
gene (750 bp genomic Hind III fragment in Fig. 5).
initiation codon. The psbl gene has two 3' boundaries which were visible as discrete S 1 nu- clease protected bands 485 and 685 base pairs downstream from the stop codon (data not shown). Two distinct mRNAs are also observed when denatured RNA is blotted to nitrocellulose and probed with nick-translated pPII-12P4 cDNA [ 10]. A single 3' boundary was observed for the psb3 gene approximately 640 base pairs downstream from the stop codon. Short 5' leader sequences and long 3' untranslated sequences have also been observed in other C. reinhardtii nuclear genes coding for OEE2 [3] and the small subunit of carboxylase [15]. However, this fea- ture has not been observed for the nuclear ~ and fl tubulin genes [28]. The 3' boundaries of both
OEE1 proteins from C. reinhardtii and spinach share large regions of homology, while OEE3 pro- teins share limited homology
Recently the amino acid sequence of the OEE1 protein from spinach has been deduced by protein sequencing of isolated mature OEE1 protein [ 11 ] and by the sequence of a spinach cDNA clone of OEE3 [ 12]. The 241 amino acid OEE1 protein of C. reinhardtii is 6 residues shorter that its spinach homologue. These missing amino acids are mostly located at the N-terminus of the spinach se- quence. Comparison of the primary amino acid sequence of the OEE1 protein of C. reinhardtii with those of spinach [ 11, 12], Synechocystis 6803 [29] and Anacystis nidulans [30] reveals an overall homology of 67, 48 and 46~o, respectively. The two cyanobacterial sequences are 43 and 48~o homologous to their higher-plant counterpart [29, 30]. As shown in Figure 6A several regions can be recognized where the homology is much greater, in particular a stretch of 33 identical residues (139 to 171 in Fig. 6A) between C. rein- hardtii and spinach which is 70~o conserved in cyanobacteria. Two cysteine residues at positions 23 and 47 are also conserved in these four orga- nisms. It has been noticed previously that some sequence homology exists between a region of the OEE 1 protein of spinach and a segment of bacte- rial superoxide dismutases surrounding the Mn ligand [ 11 ]. The corresponding region in C. rein- hardtii which includes residues 13 to 28 is striking- ly less well preserved raising questions as to the
OEE I region
R A P H 5 H P SA X/A P
i I i I I I I i i I i
_ _ _. ~ - _-.-.. - - --c - .
... ---;.-:::..-~ --.-.-...
P A K
I[ I l iooo
El E2 E3 E4
OEE3
B HPA PH S
I If I 11"1
D 9 i JJ i
El E2 E3 E4
E5 E6 i ioo i
region
R PA H P A R rooo
E5
Fig. 5. Restriction map of the chromosomal region of wild-type C. reinhardtii containing the OEEI (psbl) and OEE3 (psb3) genes.
The restriction enzymes shown are (R) Eco RI, (A) Ava I, (P) Pst I, (H) Hind III, (S) Sal I, (X) Xho I, (K) Kpn I, (D) Dde I, and (B) Barn HI. The coding regions are shown in dark bars, the 5' and 3' non-coding regions of the mRNA are shown in lined and dotted bars (corresponding to the two 3' ends ofpsbI mRNA; see text), and the intervening sequences are shown in white bars.
Scales are indicated at the right of each diagram in base pairs.
A C.r. LTFDEIQGLTYLQUKGSGIANTCPULESGITNLKELKAGSYKLENFCIEPTSFTVKEESQ ,I0 ,20 .30 .40 .50 ,60 II IIII III III I I'I II I I I I I II III I II I Sp. EGGKRLTYDEIQSKTYLEVKGTGTANQCPTVEGGVDSF-AFKPGKYTAKKFCLEPTKFAVKAEGI
C,P,
Sp,
,70 .80 .90 .100 .110 ,120
FKGGETEFUKTKLM~RL~YT~DAMSGSFKUGS6GSAELKEDDGT6YAAT~OQZPGGEROA I I IIIIIIIIIIII I I I III I llIllll IIIIIIIIII SKNSGPDFQNTKLMTRLTYTLDEIEGPFEVSSDGTVKFEEKDGIDYAAVTUQLPGGERVP
C,/~ ,
Sp,
.130 • ,140. eJe .150 •eeeo e* .160 el• el• ,170 ,180
~LRflKQFDGKGTLDGIKGDFLVPSYRGSSFLDPKGRGGSTGYDNAVALPA--RADAEEL IIIIIII I lllllllllllllllllllIllllllIllllll I I Ill FLFTIKQLVASGKPESFSGDFLUPSYRGSSFLDPKGRGGSTGYDNAVALPAGGRGDEEEL
C,~,
Sp.
.190 ,200 .210 ,220 .230 ,240
LKENVKITKALKGSAUFSOAKUDPUfGEIAGU~ESIQ~§6~6LGAKPPKDIKVTGLW~AQLK III I II II I III III I IIIIIIIIII III I I IIIII QKENNKNVASSKGTITLSVTSSKPETGEVIGVFQSLQPSDTDLGAKVPKDVKIEGVWYAQLEQQ
,10 .20 ,30 .40 .50 ,60
C.r. LTP--UDLFDDRSURDRGFDL I YEARDLDLPQffUREGFTQARASLDETKKRUKESEAR I D
B I I I I i l l II i i I I I I
Sp, ERRP I UUGPPPPLSGGLPGTEHSDQARDGTLP-YTKDRFVLQPLPPTEAAQRAKUS-ASE I
.70 ,80 ,90 ,100 ,110 ,120
C,r, ADLDVF I QKSYWTEAREQLRRQVGTLRFDLNTLASTKEKEAKKAALGLRKEF I QAUEDLD
II I II II II I I I I II I II
Sp, LNVKQF I DRKRWPSLQNDLRLRASYLRYDLKTV I SRKPKDEKKSLQELTSKLFSS I DNLD ,130 .140 ,150
C,r, FALREKDQASAAKKLE I TKAKLDSVLRAVL
I i I I I II I I
Sp, HAAK I KSPTEAEKYYGQTVSH I NEVL-AKLG
Fig. 6. A (top). Comparison of the primary amino acid sequences of the OEE1 proteins from C. reinhardtii (C.r) and spinach (Sp.) [ 11, 12, 13]. Residues that are also conserved in the O EE 1 proteins from Synechocystis 6803 [30] and A nacystis nidulans [31] are marked with dots. B (bottom). Comparison of the primary amino acid sequences of the OEE3 proteins from C. reinhardtii (C.r)
and spinach (Sp.). Protein comparison was made by the method of Needleman and WOnsch [37].
significance of the homology found in higher plants. Similar conclusions have been reached from the sequence comparison of the OEE1 pro- teins of Synechocystis 6803 [29] and Anacystis nidulans [30] with the higher-plant homologue.
Comparison of the amino acid sequence of the OEE3 proteins of spinach [ 13 ] and C. reinhardtii show them to contain limited, though significant, sequence homology (Fig. 6B). Both the spinach and C. reinhardtii OEE3 proteins contain the same number of residues (149) with a high pro- portion of charged amino acids (36/149 for C. reinhardtii). Overall, the OEE3 proteins share only 28% homology, and in only one section of the proteins (from residues 86-104) does the homology exceed 50%.
These comparisons of algal and higher plant OEE proteins reveal that although these proteins belong to the same complex they have been con- served to a variable extent: 67% homology for OEE1, 61% for OEE2 [3, 13] and 28% for OEE3. In contrast the primary amino acid se- quences of the PSII core polypeptides of C. rein- hardtii and higher plants are significantly more conserved with a homology ranging between 86 and 93% [31, 32]. The OEE1 protein is closely associated with the core proteins. Loss of the OEE 1 protein destabilizes the PSII core complex resulting in a more rapid turnover of the reaction center proteins [10]. This is not the case for the OEE2 protein. Loss of OEE2 does not affect the accumulation of the core PSII proteins [3].
Amongst the OEE proteins OEE3 is the least conserved. Its loose association with the PSII complex may have allowed for a greater sequence
divergence. It thus appears that the amount of homology between the OEE proteins of C. rein- hardtii and higher plants could reflect the constraints imposed by the specific interactions of these proteins with the conserved PSII core.
Transit peptides of the proteins from the oxygen- evolving complex of C. reinhardtii
All nuclear-encoded chloroplast-localized pro- teins characterized to date have been shown to contain N-terminal transit peptides. These prese- quences appear to be required for the proper sorting of proteins to the chloroplast and for guiding the proteins to their correct location within the chloroplast. The routing of thylakoid lumen proteins is of special interest since they have to traverse the two chloroplast envelope membranes and the thylakoid membrane.
The 51 amino acid transit peptides of OEE1 of C. reinhardtii (Fig. 7) is significantly shorter than its spinach homologue which contains 84 [12].
Although there is some uncertainty as to the precise location of the transit sequence cleavage site of OEE3, its transit peptide also appears to be considerably shorther than its higher-plant ho- mologue [13]. Comparison of the transit se- quences of the three lumen-localized OEE pro- teins and of the stromal small subunit of Rubisco of C. reinhardtii reveals some conserved features.
The VARPA motif or some derivative of it appears in approximately the same position within each the transit peptides of C. reinhardtii except for OEE1 where it is only partially present. This
• 8 8 • e 8 45 ql,
ss IMAIAV I AK$SV SAAVARPARSSVRPMAAL KPAV KAAPVAAPAQA__N_NQMMV 51
IMAI • • • ?e • • • ~ {
OEE1 L RAAQSANAGV RAARPNRAT AV VC KAQKVGQAAAAAAL ATAMVAGSANALTF s7
OEE2 LM TA~LCNKAFAAAP~SIAVVVRAS~S FAGAAALVSSSPANAAYG
OEE3 L ASNV AT I~PAVASI~ AVVVRA ~_ ASAVAAVAPKAALALr PV
Fig. 7. Comparison of the transit peptides from the small subunit ofcarboxylase (SS), and the OEE1, OEE2 and OEE3 peptides of C. reinhardtii. Positively charged amino acids are indicated as are the sites of cleavage of the transit peptides (arrows with amino acid number), the location of intervening sequences within the gene (white arrowheads), and the position of conserved
sequences (underlined or boxed).
short sequence is not found in the transit peptides from other organisms. Other conserved features include the C-end of the transit peptides which is related to the cleavage site of signal sequences [32]. It is remarkable that four of the last five residues of the transit peptide of OEE1 from C. reinhardtii are identical to the C-terminal end GSAFA of the signal sequence of OEE1 from the cyanobacterium Synechocystis 6803 [30]. Figure 7 reveals that the transit peptides of OEE2 and OEE3 are highly related. However the conserved region is not found within any of the other transit peptides and its significance is not clear. A striking feature of the transit peptides of OEE1 and OEE2 from C. reinhardtii proteins is the presence of a basic amino terminal region and a hydrophobic region. This structure also appears to be present in the transit peptide of OEE3 although its precise end could not be determined. A similar two- domain organization was first reported for the transit peptide of plastocyanin, another thylakoid lumen protein [26]. The first domain which includes the amino terminal region is rich in basic and hydroxylated amino acids and appears to play a role in protein import into the chloroplast.
The second domain contains mostly hydrophobic residues and has been postulated to play a key role in the routing of protein towards the thylakoid lumen. There is growing evidence that the precur- sors of these lumen proteins are processed in two steps [33, 34]. Domains I and II of the transit peptide are removed sequentially by two pep- tidases localized in the stroma and on the thylakoids, respectively [ 35].
Surprisingly, the coding sequences of the transit peptides of the three OEE proteins of C. rein- hardtii are interrupted by one or two intervening sequences. It is of interest that some of the introns in psb2 and psb3 are located near the border between domains I and II which appear to corre- spond to functionally distinct domains of the transit peptide. While such a correlation between exons and protein functional domain is well docu- mented [36], it remains to be seen whether introns will be found a similar positions in other orga- nisms.
Acknowledgements
We thank O. Jenni for drawings and photogra- phy, N.H. Chua and L. Mclntosh for antibodies, and L.G. Franzen, M. Goldschmidt-Clermont and M. Kuchka for helpful comments. This work was supported by grant 3.328-086 from the Swiss National Foundation.
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