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Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from <i>Chlamydomonas reinhardtii</i>

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Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from Chlamydomonas

reinhardtii

FRANZEN, Lars Gunnar, et al.

Abstract

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges. The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively.

FRANZEN, Lars Gunnar, et al. Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from Chlamydomonas reinhardtii. Plant Molecular Biology, 1989, vol. 12, no. 4, p. 463-474

DOI : 10.1007/BF00017585

Available at:

http://archive-ouverte.unige.ch/unige:148883

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

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Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from Chlamydomonas reinhardtii

Lars-Gunnar Franz6n, 1. Gerhard Frank, 2 Herbert Zuber 2 and Jean-David Rochaix 1

1 Departments of Molecular Biology and Plant Biology, University of Geneva, Geneva, Switzerland;

2 Institute of Molecular Biology and Biophysics, EidgenOssische Technische Hochschule, HiJnggerberg, Zfirich, Switzerland; * address for correspondence: L.-G. Franzdn, D~partement de Biologie Moldculaire, Universit~ de Gen~ve, 30 quai Ernest-Ansermet, CH-1211 Gen~ve 4, Switzerland

Received 29 November 1988; accepted in revised form 2 February 1989

Key words: cDNA sequence, Chlamydomonas reinhardtii, chloroplast, nuclear-encoded subunits, Photo- system I, transit peptide

Abstract

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecu- lar masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges.

The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively.

Abbreviations: OEE1, 2 and 3, oxygen evolution enhancer proteins 1, 2 and 3; Rubisco, ribulose bis- phosphate carboxylase/oxygenase; PS, photosystem; P21 and P30, C. reinhardtii thylakoid polypeptides 21 and 30

Introduction

In the photosynthetic electron transport chain, Photosystem I (PS I) catalyses the light-driven electron transfer from plastocyanin to ferredoxin.

The absorption of a light quantum results in an electron transfer from the reaction-centre chloro- phyll P700 to a series of electron acceptors, namely Ao (probably a chlorophyll molecule), A 1 (probably a quinone) and the iron-sulphur centres

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X13495 and X13496.

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X, B and A. P700 is then re-reduced by plasto- cyanin and the electron on the PSI acceptor side is transferred to ferredoxin. P700 and the first acceptors Ao, A~ and FeS x appear to be located on two chloroplast-encoded large subunits (80-85kDa). The PSI core complex also contains several smaller subunits (<25 kDa), most of which are nuclear-encoded. The number of low-molecular-mass subunits varies between PSI preparations and may depend on species and purification procedure. (For recent reviews on PS I, see [14, 20].

The role of the small subunits is unclear. A chloroplast-encoded 8-9 kDa protein has been identified as an iron-sulphur protein [12] and probably binds the iron-sulphur centres A and B.

The sequences of eDNA clones encoding four nuclear-encoded subunits have been reported:

subunit II (20-22 kDa) ofPS I from spinach [ 18]

and tomato [11], subunits IV (18kDa) and V (12 kDa) of spinach PSI [30] and a 10.8 kDa subunit from barley PSI [24]. The deduced amino acid sequences predict that the proteins are extrinsic membrane proteins. A 20 kDa subunit (probably identical to subunit II) has been identi- fied as the subunit that binds ferredoxin [33].

Removal of subunit III (20 kDa) has been shown to block the electron transfer from plastocyanin to P700 [2]. A 19 kDa subunit (probably identical to subunit III) has recently been identified as the subunit that binds plastocyanin [31 ]. The roles of the other nuclear-encoded subunits are unknown.

The unicellular green alga Chlamydomonas reinhardtii is a facultative phototroph, which can also be grown "on a reduced carbon source (acetate) in the dark. This allows the isolation of photosynthetic mutants. Many PSI defective mutants have been isolated in C. reinhardtii (e.g.

[8]). Therefore, this alga provides unique oppor- tunities for studying the cooperation of the nuclear and chloroplastic genomes in the assembly of

* The numbering system of C. reinhardtii thylakoid poly- peptides has been introduced by Chua and Bennoun [4] and later been extended to include also low-molecular-mass poly- peptides [8, 25]. The polypeptides are numbered according to their apparent molecular mass in SDS electrophoresis, beginning from the high-molecular-mass region.

PSI and the detailed function of the various com- ponents in the intact complex. Six low-molecular- mass PSI subunits have been identified in C. reinhardtii [8], namely the thylakoid poly- peptides 20 and 21 (both approx. 18 kDa), 28 (13 kDa), 30 (11 kDa), and 35 and 37 (both smaller than 10kDa).* Here, we present the analysis of eDNA clones encoding polypeptides 21 (P21) and 30 (P30). Analysis of the deduced amino acid sequences of the mature proteins and the transit peptides predicts that P21 and P30 are extrinsic membrane proteins located on the lume- nal and the stromal sides of the thylakoid mem- brane, respectively. Comparison between the pro- tein sequences of P21 and P30 and corresponding proteins from higher plants [30, 24] reveals highly conserved regions which may be functionally important.

Materials and methods Isolation of proteins

PSI particles were isolated from the PS II- deficient mutant FuD7 [ 3 ]. The cells were broken in a French press and thylakoid membranes were purified by flotation [4]. PS I particles were iso- lated from Triton X-100 solubilized thylakoids by sucrose gradient centrifugation [8, 22]. The PS I particles were loaded on preparative SDS gels using the SDS-urea system described by Piccioni et al. [25]. Before use, the urea was deionized with the mixed-bed ion-exchange resin Amberlite MB-1 to avoid modification of amino groups in the proteins. The proteins were visualized by Coomassie Blue staining, electro-eluted in 50 mM Tris-HC1 (pH 8.55) and 0.1% SDS, precipitated with 20% trichloroacetic acid and dried under vacuum. The N-terminal amino acid sequences were determined using an Applied Biosystems model 470A protein sequenator according to standard procedures.

eDNA cloning

For each protein, two oligonucleotides cor- responding to different parts of the N-terminal

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Table l. Oligonucleotide probes used to screen eDNA libraries.

21A (corresponds to AsprThr6 in P21)

GT CAG (GA) CC (_Q_.A_) GC (GA) AT GTC 21B (corresponds to Serlt-Leut7 in P21)

CAG CTT (GA) GC GTA (GA) GC CTT _C_CTT 30A (corresponds to Val3-Lys s in P30)

CTT G G G (GA) GC (GA) GC CTT (GC) AC 30B (corresponds to Lysg-Pro~3 in P30)

G G G (GA) CC (GC) AC CTC CTT

The oligonucleotides were made complementary to mRNA, assuming the codon usage of several nuclear genes of C. reinhardtii [26]. Bases that do not match with the eDNA sequences are underlined. The two mismatches in oligo- nucleotide 21B are due to an error in the protein sequence, and the mismatches in oligonucleotides 21A and 30A are due to unusual codons that were neglected when the oligonucle- otides were synthesized.

sequence were synthesized (Table 1) using an Applied Biosystems model 381A DNA synthe- sizer. The oligonucleotides were used to screen two eDNA libraries, a 2gtl0 library [10] and a 2gtl 1 library that had been prepared from gamete

poly(A) ÷ RNA. The 2gtl 1 library was a kind gift from Dr W. S. Adair (Washington University, St, Louis, MO). Nitrocellulose replicas of phage plaques grown on Escherichia coil strain C600 or Y1090 were hybridized with 32p-labelled oligo- nucleotides as described in [9], except that E. coli carder DNA was replaced by yeast tRNA. For each protein, only plaques hybridizing with the two oligonucleotide probes were purified and the inserts were subcloned into the plasmid pUC18.

To isolate new eDNA clones, restriction frag- ments (Fig. 1) from the pUC18 clones were labelled using the method described by Feinberg and Vogelstein [7] ('random oligo labelling') and used to screen the eDNA libraries.

DNA sequencing

Restriction fragments were subcloned into the vectors M13 mpl8 and mpl9 as indicated in Fig. 1. DNA sequencing was carried out by the dideoxy chain-termination method of Sanger etal. [27], using ~-35S-dATP (Amersham) and Sequenase enzyme (US Biochemicals). For all

A ~. N Ps

5' I - ~ ~ ~ ' , ~ .\\\~

L I

v

I

13'

IO0 bp

I I

)

I

q I

B 5' ', M I ~xxx~\x-~xx\xxx\\xx~ s Pv

I .

I00 bp I I

4 I

Fig. 1. Partial restriction maps and sequencing strategy for the eDNA clones of P21 (A) and P30 (B). Restriction sites are marked: N, Nco I; Ps, Pst I; M, Msp I; S, Sal I; Pv, Pvu I. The position of the intervening sequence found in one of the clones of P30 is indicated (i). The coding regions are boxed, and the points corresponding to the terminal processing sites of the transit sequences are indicated with arrows. The restriction fragments of non-full-length clones used as probes in all Southern and Northern hybridizations are shown as black lines under the restriction maps. The horizontal arrows indicate the direction and

extent of individual DNA sequencing reactions.

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templates, reactions with normal GTP and with the analogue ITP were run in parallel. In this way, all ambiguities caused by GC-rich sequences were resolved.

DNA and RNA isolation, electrophoresis and blotting Growth of the cells, isolation of DNA and RNA, agarose gel electrophoresis, blotting to nitro- cellulose filters and hybridizations were as described in [9]. The restriction fragments that were used as probes (Fig, 1) were labelled as described in [7].

Results

Cloning and sequence of cDNAs encoding poly- peptides 21 and 30

Except for the 9kDa iron-sulphur protein [ 12] the low-molecular-mass subunits ofPS I appear to be nuclear-encoded. To clone the genes of the C. reinhardtii P S I subunits we decided to use oligonucleotide probes, as such probes can be made very specific due to the very restricted codon usage found in several nuclear genes of C. reinhardtii [26]. We therefore isolated the P S I subunits and determined their N-terminal amino acid sequences. For each protein, two inde- pendent oligonucleotide mixtures were synthe- sized. These mixtures were composed of 4-8 oligonucleotides and corresponded to regions encoding different parts of the N-terminal protein sequences (Table 1). The oligonucleotides were labelled and used to screen a 2gtl0 eDNA library.

Two clones hybridizing with the two probes for P21 and eight clones hybridizing with the two probes for P30 were isolated. The longest inserts were subcloned into the plasmid pUC18. There were also clones which hybridized with the probes 21B and 30A but which did not hybridize with the probes 21A or 30B. These clones were not analysed. As the clones obtained from the 2gtl0 library were found not to be full-length, we used restriction fragments of the inserts (Fig. 1) to

screen an independently prepared 2gtl 1 library.

For both proteins, two eDNA clones encoding the complete protein were isolated from this library and subcloned into pUC18. The eDNA sequences and the deduced amino acid sequences are presented in Figures 2 and 3.

The eDNA clones of P30 encode a polypeptide of 97 amino acids. Comparison with the N-termi- nal amino acid sequence of the mature protein shows that the first 24 amino acids represent the transit sequence. Thus the mature protein con- sists of 73 amino acids (8.1kDa), although its apparent molecular mass on SDS gels is 11 kDa.

In the cDNA sequence of P21 there are two possible in-frame start codons at positions 1 and 13. If we assume that translation starts at the first AUG, the translation product consists of 227 amino acids. The N-terminus of the mature pro- tein corresponds to residue 63 in this sequence and thus the molecular mass of the mature protein can be calculated to be 17.9kDa (165 amino acids), in good agreement with the apparent molecular mass on SDS gels: 18kDa.

Both genes share the unusual eodon bias found in other C. reinhardtii nuclear genes [26]. The most striking feature is that adenine is not found in the third position of any codon. The 3' untrans- lated regions are long, as are those of the genes coding for the small subunit of Rubisco [ 10] and the OEE2 protein [21]. A likely component of the polyadenylation signal, TGTAA, [28] is found 13 bases upstream of the poly(A) tail in both sequences.

Gene organization and expression

To determine the number of genes encoding P21 and P30, genomic DNA was digested with restric- tion enzymes, fractionated on agarose gels, blotted onto nitrocellulose and hybridized with labelled eDNA fragments (Fig. 1). As shown in Figure 4 these probes hybridized with only one fragment for each of the restriction enzymes, suggesting that both proteins are encoded by single-copy genes. The possibility that these genes are present in more than one copy within large

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CGC AAC CCG

1 R N P

ATG GCC CTC ACT ATG .1 M A L T M

CGT CGT GCT CTG CGC GTC GCC TGC 21 R R A L R V A C

TCC GCT CTG

41 S A L

61

81

GGC CTG ACC G L T

G T

ACT GCC CTG GCT GCG T A L A A ATG GCC G~C ATC GCG

M A D I A D I A AJ~G AAG GAG CTG AAG

K K E L K K K X L K GCC GTG GCC CTG AAG

A V A L K

ACC CTG GAG

T E

T X

GCC ACC ATG

101 A T M

GCC GGC CTG CTG TGC GGC AAC GAC 121 A G L L C G N D

CAGTCCGAACTTGCAGAA GCC GTC AAG GCT TCC TCG CGC GTG GCG .30

A V K A S S R V A CAG GCC CAG .90 AAG AAC GAG ACC GCC TCC

Q A Q K N E T A S .150

GCC GCT GCC GTG TCG CTG TCT GCC CCC A A A V S L S A P

.210

CCC TGC TCC GAG AGC AAG GCT TAC GCC P C S E S K A Y A P T X E ~ K A Y A

-.270

AAG CGC CTG AJ~G CAG TAC GAG GCG GAC K R L K Q Y E A D K R L K

.330

GAG CGC ACC P.~G GCC CGC TTC GCC AAC E R T K A R F A N

.390 GGT CTG CCC

G L P .450 GTC TTC ATE

V F I .510 CAG TAC CTG

Q Y L .570

CAC CTG ATT GCT GAC CCC H L I A D P CTG AAG TAT GGC CAC GCT GGC GAG CCC ACC TTT GGC TTC CTC 141 L K Y G H A G E P T F G F L

GGC TAC ATC GGC TAC GTC GGT CGC 161 G Y I G Y V G R

ACT GAC AAG GAG ATC ATT ATT GAC GTG CCC CTG 181 T D K E I I I D V P L

GGC TGG CCC CTG GCC 201 G W P L A

AAC ATC ACC GTG TCT 221 N I T V S

.630 GCT GTG CAG GAG CTG CAG

A V Q E L Q

ATT GCC GTC AAG GGC GAG I A V K G E GCC ACC ~G CTG GCG TGG

A T K L A W CGC GGC ACC CTG CTG GAG R G T L L E

CCT TCG TCG .60 P S S

.120 AAG GTC GGC

K V G .180 TCC GCG GCG

S A A .240 AAG CTG GAG

K L E K L X

.300 AGC GCC CCC

S A P .360 TAC GCT AAG

Y A K .420 GGC CTG GCC

G L A .480 TAC GTT GCT

Y V A .540 GCT AAG CCC

A K P .600 CAG GGC GCT

Q G A .660 AAG GAG GAG

K E E .70O

CCC CGC TAA ATGCACCAGCAGCCTGCGGGCCGCAACGCTTGATCAGCCTTCTGTAT P R --o

.750 .800

CTCGTTCGGAGGAACGGAGAGCTGCTCTGGCGCTGCGTGTTCTCACCACATGCAGCTGACGCGGATGGGACCCTGCTTC .850

AGTCGGCGTGACGGTCTGAGCCTGGCTTTGCTGGTGTCGCTTGTACGCCGGCGGCCGGAGGAGCGGTTGCGTGGACTCA

• 900 .950

GGAAGCAGCAGGAGAGCGCTTGGTAGCGTTGGATGGGACGGCCCTGGCCCCGCTCTGGGTCGTCCCCGTCACGTTTTTG .1000

AGCGAGCGGGG/~AGGAGTTGTTTGATGCCGTGTCTATCCCGGTCTTGACTGAcTAcGGGCGAAAGGCTGGcATCTGGGT

.I050 .1100

TGTCAGGCGGTGGCT~TGGCCGCGTCAGGCGGGGTTGCAGGCGCTGGCCTGTGTAGCTAGAGCCTAGGGTAA~GTC

.1150 .1200

CGCGGGACCATGTGTGGAAGGGGCCTATGGCCTAACCACGCACCGTGGCATCGAGAAGACTATGACTGTAAACCAATTT TCGTiA) n

Fig. 2. cDNA sequence, predicted amino acid sequence and N-terminal sequence of the mature protein for P21. The N terminus of the mature protein is 63 amino acids (*) from the N terminus of the deduced preprotein, and differs at two points (underlined), probably due to errors in the protein sequencing. Amino acids that could not be identified in the protein sequence are shown as X. A likely component of the polyadenylation signal, TGTAA, is underlined. The sequence shown in the figure represents the clones isolated from the 2gt 11 library. The 2gt I 0 clone differs at two points. The G at position 500 is changed to an A, changing

the amino acid from Gly to Asp, and there is one extra T at base 1210 just before the poly(A) tail.

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GTGACACTTCGCCTTTCATCTCCCCACCAAAACCGGAAA

.I .30 i

ATG CAG GCC (~l'G TCG TCT C(~C GTG AAC ATC GCG ~C AAG (~CC CAG CGC G(~T CAG CGC CTG 60 I M Q A L S S R V N I A A K P O R A Q R L

* .gO .120

GTG GI'C CGC GCC GAG GAG Gi'T AAG GeT GCC CCC AAG AAG GAG GTC GGC CCC AAG CGC GGC 21 V V R A E E V K A A P K K E V G P K R G

X X V K A A P K K E V G P K R G

.150 .180

TCG CTG GTG ~kAG ATC CTG CGC CCC GAG TCC TAC TGG TTC IkAC CAG GTC GGC AAG GTC GTC 41 S L V K I S L L R P E S Y W F N 0 V G K V V

.210 .240

TCC GTC GAC tAG AGC GGC GTC CGC TAC CCC GTC GTT GTC CGT TTC GAG AAC CAG AAC TAC 61 S V D Q S G V R Y P V V V R F E N Q N Y

.270 .300

GCT GGT GTC ACG ACG AAC AAC TAC GCT CTG GAT GAG GTT GTT GCC GCC AAG TAA ATGGCTG 81 A G V T T N N Y A L D E V V A A K ---

.350

G~TT~G~GcAG~AT~A~GGAATG~GAGAGGAG~GG~AAGTGT~G~TA~GGGA~TAG/kGAAGAGAAGATTAG~GGG~T

.400 .450

GGCCTTAGGGCAAGTCGCGATTTTGTGAGCTAGTTTTGGGCGCGCTTGCGCGACCCCAGCCAAGCATAGACTTGGCGAT

.500

CGGACACATACAGGTGTCGGACACTGACCATGAGCTAAGGAGCGGTGACGAGGGAGCAGGCGGGTGTAATACTAAATGC TG(A)n

Fig. 3. cDNA sequence, predicted amino acid sequence and N-terminal sequence of mature protein for P30. The N terminus of the mature protein is 25 amino acids (*) from the N terminus of the deduced preprotein. Amino acids that could not be identified in the protein sequence are shown as X. The location of the intervening sequence in one eDNA clone is shown by an arrowhead

marked L A likely component of the polyadenylation signal, TGTAA, is underlined.

repeat units (> 15 kb) cannot be ruled out by these hybridization data. However, copy number reconstruction experiments also indicate that these genes are present in single copies (data not shown).

One of the two clones corresponding to P30 contained an extra insertion of 70 bases (Fig. 1B, Fig. 3). The insertion starts with GTGCGT and ends with CTGCAG (the 3' end is a Pst I site), and agrees with the terminal consensus sequences of eukaryotic introns. Thus this clone appears to correspond to a precursor mRNA still containing an intron. To determine wether this intron is indeed found in the C. reinhardtii genome, genomic DNA was digested first with Msp I and Pvu I, which cut on both sides of the intron, and this double digest was redigested with Pst I, which cuts in the 3' end of the intron (Fig. 1B). The eDNA probe (Fig. 1B) hybridized with fragments of 700 bp and 590 bp, respectively (Fig. 4C). The size difference between these fragments, 110 bp, perfectly agrees with the distance between the

Msp I and Pst I sites in the intron-containing eDNA clone (109 bp), suggesting that the puta- tive intron is present in the genomic DNA. By comparing the size of the Pst I-Pvu I fragment, 590 bp (Fig. 4C), and the size of the Pst I-Sal I fragment, 250 bp (not shown), with the eDNA sequence (Fig. 1B, Fig. 3) one can deduce that there are at least two more introns in the P30 gene.

There are about 100 bp of intervening sequence between the Pst I site in the known intron and the Sal I site, and about 60 bp of intervening sequence between the Sal I and Pvu I sites.

C. reinhardtii RNA was fractionated on agarose gels, blotted onto nitrocellulose and hybridized with the same fragments that were used to probe the DNA blots. In each case, the probe hybridized to a single mRNA species of 1400 (P21) and 740 (P30) nucleotides, respectively (Fig. 5). From the sizes of the mRNAs we deduce that the 5' leader sequences are rather short; the sum of 5' leader sequence and poly(A) tail can be calculated to be approximately 200 nucleotides for both mRNAs.

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Fig. 4. Southern blot analysis of wild-type C. reinhardtii genomic DNA. The DNA was digested with the restriction enzymes indicated at the top of the figures (M = Msp I, Pv = Pvu I, Ps = Pst I). Fragments were separated by agarose gel electrophoresis (A and B, 0.8% gel; C, 1.3 % gel), blotted to nitrocellulose and probed with labelled fragments (Fig. 1) of the cDNA clones

encoding P21 (A) and P30 (B and C).

Discussion

PS I defective mutants of C. reinhardtii have been found which lack subunit I and six low-molecular- mass polypeptides [8]. Genetic analysis of PSI mutants has shown that several nuclear genes are involved in the assembly of PS I; 25 nuclear muta- tions were localized in 13 different complemen- tation groups [8]. It is likely that some of these complementation groups correspond to structural genes of PS I subunits. Therefore, the study of

Fig. 5. Northern blot analysis of wild type C. reinhardtii RNA. Total RNA was separated on denaturing form- aldehyde gels, blotted to nitrocellulose and probed with restriction fragments (Fig. 1) of the cDNA clones of P21 and

P30.

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nuclear PS I mutants could provide information about the roles of the nuclear-encoded subunits in the assembly and function of PS I. As a first step towards analysing PSI mutants at the molecular level we have isolated and characterized cDNA clones of two PSI subunits, P21 and P30. These cDNAs will be used as probes for the screening of PSI mutants (work in progress).

Nucleotide sequence

The genes for P21 and P30 share features ex- hibited by several other nuclear genes of C. reinhardtii [ 1, 10, 21, 28, 32]. The codon usage is very restricted, and for several amino acids only one of the possible codons is used. The 3' untranslated regions of the sequences are almost as long as the coding regions. Although the function of these long 3' extensions is unclear, they may play a role in mRNA stability.

For P21, two differences were found between the 2gtll clones (Fig. 2) and the 2gtl0 clone. In the 2gtl0 clone, the G at position 500 is changed to an A, changing the amino acid from glycine to aspartate, and there is one extra T at position 1210 just before the poly(A) tail. Although a change from glycine to aspartate seems to be a drastic change, the amino acid is located next to another charged residue and therefore this region may be exposed to the aqueous phase. It is thus possible that these differences are due to poly- morphism, since the two libraries were made from different laboratory strains of C. reinhardtii

There are two possible start codons in the gene for P21 at positions 1 and 13. However, trans- lation is initiated at the first AUG in 95 ~o of all eukaryotic mRNAs [5, 15]. The consensus se- quences found in vertebrate and higher plant mRNAs GCCACCAUGG and AACAAUGGC, respectively [ 16, 19], with a purine nucleotide at the - 3 position as the most important feature [ 16]. C. reinhardtii initiation regions appear to be similar to the higher plant consensus sequence;

the AUG is generally preceded by an A-rich tetra- nucleotide sequence (Table 2). Therefore, the first initiation codon (AGAAAUGGC) seems more

Table 2. Initiation codons in C. reinhardtii nuclear genes.

Small subunit of Rubisco [10]"

OEEI protein b OEE2 protein [21]

OEE3 protein b

P30 PSI subunit (this work) Alpha tubulin [28] a Beta tubulin [32]"

cytochrome c [1]

TAAAATGGC AAAGATGGC AAAAATGGC AAAGATGGC GAAAATGCA AACCATGCG AAACATGCG TAAAATGTC

" These genes are present in two copies. In each case, the region shown in the table is identical in the two copies.

b S. P. Mayfield and J.-D. Rochaix, unpublished results.

likely than the second one (CACUAUGCG), although the second one cannot be excluded.

Protein structure predictions

Both proteins are rich in charged amino acids and have net positive charges (their theoretical iso- electric points (pI) are 9.1 (P21) and 9.9 (P30)).

It is therefore likely that they are extrinsic sub- units. Analysis of hydropathy plots [ 17] supports this conclusion (Fig. 6). P30 has no hydrophobic segment sufficiently long to span the membrane.

In P21 there is one hydrophobic segment that could possibly be membrane-spanning (Fig. 6A), but most likely the protein is extrinsic.

The transit sequences conform to the general characteristics of sequences directing proteins into chloroplasts (e.g. [ 13]). Although the transit peptide of P30 is unusually short, 24 amino acids, it does share similarities with transit peptides of stromal proteins such as the small subunit of Rubisco [10]; for example, it contains several positively charged residues. Therefore, P30 is probably localized on the stromal side of the thylakoid membrane. The longer transit peptide of P21, 62 amino acids, resembles the transit pep- tides of proteins found in the thylakoid lumen such as the OEE2 protein [21 ]. Lumen-targeting transit peptides consist of two domains. The N-terminal part is similar to the complete transit peptide of stromal proteins. The C-terminal part is hydrophobic and is probably involved in routing the protein through the thylakoid mem-

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3e 29 so o -!.o -;'o -36

30 20 10 0 -10 -20 -30

A

... 4'o' ... o~ I ... i2'o' ... /6'e' ... ~o'o' ...

B

... .~, ... .~, ... ~,~, ... :~, ... ~,~, ... ~,~, ... ¥o, ... ~,~, ... ~, ...

Fig. 6. Hydropathy plots of the precursor potypeptides of P21 (A) and P30 (B) according to Kyte and Doolittle [17]. An interval of nine amino acids was used. Ordinate, hydropathic index; abscissa, amino acid residue number. The cleavage sites between transit peptide and mature protein are indicated by an arrow; the hydrophobic domain in P21 is indicated by a bar in A.

brane [29]. Thus P21 may be an extrinsic subunit localized on the lumenal side of the thylakoid membrane.

Recently, a cDNA sequence of an 18kDa subunit of spinach PS I (subunit IV) has been published [30]. In Figure 7A, the deduced amino acid sequences of C. reinhardtii P21 and the 18 kDa spinach protein are compared. The overall identity between the proteins is 56~, but there are some regions of greater similarity. The N termini of the proteins are clearly homologous while the C termini are less conserved. The hydrophobic domain in P21 (Fig. 6A) is conserved in the spinach protein (Fig. 7A). The spinach protein is eleven amino acids shorter, due to a gap of four amino acids in a less conserved region in the middle of the protein and due to the absence of seven C-terminal amino acids. P21 was also com- pared with the published N-terminal sequences of

PS I subunits from pea [6]. P21 is homologous to the 17kDa subunit of pea PS I; 14 of the first 20 amino acids are identical.

Based on its transit peptide and overall amino acid sequence, P21 is probably an extrinsic sub- unit located on the lumenal side of the thylakoid membrane. It may be involved in the electron transfer from plastocyanin to P700, providing positive charges that could facilitate binding of plastocyanin to the negatively charged PSI complex. Such a role has been suggested for subunit III of PSI [2]. P21 is however homolo- gous to subunit IV of spinach PSI [30]. Perhaps both subunits play a role in this electron transfer.

It is also possible that the sequenced spinach subunitlV [30] corresponds to subunit III in [2], since the positions of subunits III and IV inter- change on SDS gels depending on the buffer sys- tem, phosphate or Tris/glycine [23].

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A

C.r. DIAGLTPCSE S~YAKLEKK

illillii iil il II

sp. DIAGLTPCKE $KQFAKREKQ C.r. ARFANYAKAG LLCGNDGLPH

II II I l llli lllil

sp. KRFDNYGKYG LLCGSDGLPH C.r. ~YVGRQYLI AVKGEAKPTD

ii Iii ill l i Ill

sp. IGWVGRSYLI AIRDEKKPTQ C.r. GTLLEKEENI TVSPR sp. GELVDNHF II

ELKTLEKRLK QYEADSAPAV ALIC~TMERTK

II i II l liili l ilill il

ALKKLQASLK LYADDSAPAL AIKATMEKTK LIADPGLALK YGHAGEVFIP TFGFLYVAGY

II Ill i III li

LIVSGDQR . . . . HWGEFITP GILFLYIAGW KEIIIDVPLA TKLAWQGAGW PLAAVQELQR

lllliilili I I II II il

KEIIIDVPLA SSLLFRGFSW PVAAYRELLN

C.r. - . . . EEVK ~J~PKKEVGPK RGSLVKILRP

I I Ill il IIIII

ba. AEEPT~APA EPAPAADEKP EAAVATKEPA KAKPPPRGPK RGTKVKILRR C.r. ESYWFNQVGK VVSVDQ-SGV RYPVVVRFEN QNYAGVTTNN YALDEV--VAAK

illit i II lli lltlilll iliil III lllil III

ba. ESYWYNGTGS VVTVDQDPNT RYPVVVRFAK VNYAGVSTNN YALDEIKEV~A

Fig. 7. A, Comparison between the amino acid sequences of C. reinhardtii P21 and subunit IV of spinach PS I [30]. The bydrophobic domain in P21 is indicated by the solid line; B, Comparison between the amino acid sequences of C; reinhardtii

P30 and the 10.8kDa subunit of barley PSI [24].

In Figure 7B, P30 is compared with a 10.8kDa subunit of barley P S I [24]. The N termini are very different; the barley protein is 26 amino acids longer in the N-terminal part. However, if one compares the 62 most C-terminal residues of P30 with the corresponding part of the barley protein, the identity is, 69~. The conservation of the 30 most C-terminal amino acids is particularly strik- ing, indicating a functional constraint on the evo- lution of this region. It is interesting to note that both proteins show abnormal migration in SDS gels. P30 has a molecular mass of 8.1 kDa but an apparent molecular mass of 11 kDa; the barley protein has a molecular mass of 10.8kDa and an apparent molecular mass of 16kDa. No con- vincing similarity could be found between P30 and the N-terminal sequences of P S I subunits from pea [6]. However, the N termini are not conserved between the C. reinhardtii and barley woteins. P30 probably corresponds to the 13 kDa

subunit of pea PS I, since this protein shows some sequence similarities with the barley 10.8kDa subunit.

P30 appears to be an extrinsic subunit located on the stromal side of the thylakoid membrane and may be involved in reactions on the acceptor side of PS I. Even though a 20 kDa protein (probably subunit II) has been identified as the subunit that binds ferredoxin [ 33 ], P30 could play a regulatory role in the electron transfer between PSI and ferredoxin.

Acknowledgements

We thank O. Jenni and F. Ebener for drawings and photography, W.S. Adair for the 2gtll library, D. Rifat for synthesizing oligonucleotides and M. Goldschmidt-Clermont, A. Day and S. Purton for discussions and comments on the

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manuscript. This work was supported by grant 3.328-086 from the Swiss National Foundation.

LGF was supported by a Long-Term Fellowship from the European Molecular Biology Organi- zation.

References

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Elsevier Science Publishers, Amsterdam (1987).

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