a nuclear-encoded function essential for translation of ... · a nuclear-encoded function essential...

The Plant Cell, Vol. 9, 773-782, May 1997 O 1997 American Society of Plant Physiologists

A Nuclear-Encoded Function Essential for Translation of the Chloroplast psaB mRNA in Chlamydomonas

Otello Stampacchia,a Jacqueline Girard-Bascou,b Jean-Luc Zanasco,a William Zerges,a Pierre Bennoun,b and Jean-David Rochaixas1 a Department of Molecular and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, CH 121 1 Geneva 4, Switzerland

Curie, 75005 Paris, France lnstitut de Biologie Physico-Chimique, Centre National de Ia Recherche Scientifique, UPR 9072, 13 rue Pierre et Marie

We report the analysis of a photosystem I-deficient mutant of Chlamydomonas reinhardtii, F15, that contains a mutation at the TA67 (for translation of psa& mRNA) nuclear locus. Pulse labeling of chloroplast proteins revealed that the synthesis of the two photosystem I reaction center polypeptides PSAA and PSAB was undetectable in this mutant. The mRNA levels of these proteins were only moderately reduced, suggesting that the primary defect occurs at a step during or after translation. We constructed chimeric genes consisting of the psaA and psa6 5' untranslated region (5' UTR) fused to the aminoglycoside adenyltransferase (aadA) coding sequence, which confers spectinomycin resistance. In- sertion of these genes into the chloroplast genome through biolistic transformation and analysis of their expression in the TA67 mutant nuclear background revealed that the psa6 (but not the psaA) 5' UTR is the target of the wild-type TAB7 function. This suggests that TA67 is required for the initiation of psa6 mRNA translation. The dependence of PSAA synthesis or accumulation on PSAB synthesis is strongly suggested by the identification of a suppressor mutation within the psa6 5' UTR. The suppressor specifically restores the synthesis of both proteins in the presence of the tab7-Fl5 mutation. The location of the suppressor mutation within a putative base-paired region near the psa6 initiation codon suggests a role for TA67 in the activation of translation of the psa6 mRNA.

INTRODUCTION

Much attention has been focused recently on the regulation of gene expression in chloroplasts, the photosynthetic or- ganelles of plants and algae. Chlamydomonas reinhardtii has proven to be especially useful in this area because this green unicellular alga is amenable to biochemical, genetic, and molecular analyses. The establishment of reliable nuclear and chloroplast transformation systems has further enhanced the versatility of this organism. Molecular genetic analysis has identified severa1 post-transcriptional steps as essential for the production of chloroplast-encoded proteins (reviewed in Mayfield, 1990; Rochaix, 1992, 1996; Gillham et al., 1994; Mayfield et al., 1995). These steps include RNA splicing (Choquet et al., 1988; Goldschmidt-Clermont et al., 1990) and RNA stability (Kuchka et al., 1988; Drapier et al., 1992; Monod et al., 1992; Nickelsen et al., 1994) and translation (Rochaix et al., 1989; Zerges and Rochaix, 1994). It has been possible to identify genetically nuclear-encoded functions that are involved in each of these steps. In the ma- jority of the mutants analyzed, the defect appears to be specific for the expression of a single chloroplast gene.

To whom correspondence should be addressed. E-mail jean-david. rochaixQmolbio.unige.ch; fax 41 -22-702-6868.

Nuclear-encoded functions needed for translation have been shown to be essential for the expression of at least three genes encoding polypeptide subunits of photosystem II (PSII). These are psbC, psbl), and psbA (Kuchka et al., 1988; Rochaix et al., 1989; Girard-Bascou et al., 1992; Zerges and Rochaix, 1994). The mutants affected in psbC gene expression are deficient in translation initiation or preinitiation (Zerges and Rochaix, 1994) because the psbC mRNA leve1 is unaffected in the.mutants, and expression of a chimeric fusion of thepsbC 5' untranslated region (5' UTR) to the aminoglycoside adenyltransferase (aadA) reporter gene, conferring spectinomycin resistance, is dependent on the wild-type nuclear-encoded functions involved in psbC expression. In addition, biochemical evidence for the in- volvement of a multiprotein complex in the light-regulated translation of the psbA mRNA has been reported (Danon and Mayfield, 1991, 1994a, 1994b).

Here, we report the characterization of a nuclear mutant, F15, affected in the synthesis of the two reaction center subunits of photosystem I (PSI), the PSAB and PSAA proteins. We have named the corresponding nuclear locus TAB7 (for translation of psaJ mRNA). We demonstrate that the primary defect in this mutant occurs during preinitiation or initiation of psaB mRNA translation. This finding is supported

774 The Plant Cell

WT F15 B WT F15 suF15 C WT F15

2a.2b-

Cyt.F.D1 .D2-

MgJ^^^^^^^^^^^H

%PsaA 100 50 25 12.5 0 60 08

Figure 1. The F15 Mutant Is Affected in the Synthesis and/or Accu-mulation of the PSI Core Subunits.

(A) and (B) Analysis of chloroplast protein synthesis in the wild-type(WT) and mutant strains is shown. Cells were pulse labeled with 14C-acetate for 45 min, as described in Methods, and thylakoid mem-branes were purified and their proteins fractionated by electrophore-sis on a 7.5 to 15% SDS-polyacrylamide gel. The 2a and 2b bandscorrespond to the PSAB and PSAA polypeptides, respectively.Cyt.F, cytochrome f; D1 and D2, two PSIl reaction center polypep-tides.(C) All steps were performed as described in (A) and (B), except thatpulse labeling was for 5 min and total cell extracts were used forelectrophoresis. LS, large subunit of ribulose-1, 5-bisphosphate car-boxylase.(D) Gel blot analysis of the PSAA protein in the F15 and suF15strains is shown. Serial dilutions of wild-type protein extracts wereincluded. The ApsaA and ApsaC lanes contain extracts from strainshomoplasmic for a deletion of psaA-exon III and of the psaC gene,respectively (Fischer et al., 1996). Amounts of protein were deter-mined by densitometric scans of the blot.

by the isolation of a chloroplast suppressor mutation withinthe psaB 5' UTR that suppresses the fab-F15 mutation. Ourresults also indicate that the apparent absence of PSAAsynthesis in the mutants is a consequence of the absence ofPSAB synthesis and is mediated by an event that follows ini-tiation of psaA mRNA translation and may involve a block in

translation elongation or accelerated turnover of PSAA in theabsence of the PSAB protein.

RESULTS

Characterization of a Nuclear Mutant Deficient in theSynthesis of the PSAA and PSAB Reaction CenterPolypeptides of PSI

Several nonphotosynthetic nuclear mutants lacking PSIwere isolated previously (Girard et al., 1980). In this study,we examined the PSI-deficient mutant F15. This mutant car-ries a recessive mutation at the nuclear TAB 1 locus and isunable to grow photoautotrophically.

To test whether the synthesis of any of the chloroplast-encoded subunits of PSI is affected in this mutant, chloroplast-encoded proteins were pulse labeled for 45 min in the presenceof cycloheximide, an inhibitor of cytosolic translation, andthylakoid membrane proteins were analyzed by PAGE. It canbe seen in Figures 1A and 1B that the core subunits of PSI,the PSAA and PSAB proteins, were undetectable in F15.Similar patterns were observed for cells pulse labeled foronly 5 min (Figure 1C).

The levels of psaA and psbC mRNAs were similar to thoseof a wild-type strain in the F15 mutant (Figure 2), whereas thepsaB mRNA in F15 accumulated to ^20% of that of wild-typelevels. Other strains have been isolated in which the PSABprotein is reduced to 10 to 15% of that of the wild-type level,and yet these strains are still able to grow photoautotrophi-cally (K. Redding, N. Fischer, and J.-D. Rochaix, unpub-lished data); therefore, it appears that the fab7-F15 mutationaffects primarily post-transcriptional steps of psaA or psaBexpression.

A strain unable to synthesize the PSAB or PSAA proteinwould not be expected to accumulate PSI complexes, because

WT F15 suFIS

psaB

psaA

psbC

Figure 2. RNA Gel Blot Analysis of psaA and psaB mRNAs in theWild-Type, F15, and suF15 Strains.

Five micrograms of RNA was loaded per lane. The hybridizationprobes psaA, psaB, and psfaC are indicated. The fainter lower mo-lecular weight band observed in F15 and suF15 with the psaA probecorresponds to the psaA exon III transcript and is also seen occa-sionally in the wild type (WT).

psaS mRNA Translation 775

HSM TAPWT FuDSO WT FuDSO

~ff • \ X

TAP+Spc100WT FuDSO

F15su F15 F15su F15 F15su F15

TAP+Spc150WT FuDSO

TAP+Spc200WT FuDSO

TAP+Spc300WT FuDSO

\F15su F15 F15su F15 F15su F15

Figure 3. Analysis of the Expression of the Chimeric Genes Containing the Wild-Type and Suppressor psaB 5' UTRs in the fab7-F15 NuclearBackground.Wild-type (WT), FudSO (FuDSO), F15, and suF15 (F15su) strains that were used as controls were tested on each plate. Two representative tetradsare shown in the center of the plates (second and third rows). The upper tetrad results from cross 1 (WT 5' UTR psaB-aadA [+] x F15 [-]), andthe lower tetrad results from cross 2 (su 5' UTR psaB-aadA [+] x F15 [-]) (cf. Table 1). HSM, minimal medium; TAP, acetate-containing me-dium; Spc, spectinomycin. The concentration of the antibiotic is indicated in micrograms per milliliter (100, 150, 200, and 300).

the absence of any of the PSI core subunits leads to deg-radation of the other subunits (Girard et al., 1980; Takahashiet al., 1991). Figure 1D shows that PSAA protein accumula-tion is undetectable in F15 (<5% of that of wild-type levels)as well as in a strain with a deletion of thepsa/4 exon III (ApsaAin Figure 1D).

TAB1 Is Required for the Initiation of psaBmRNA Translation

The protein pulse-labeling pattern in the F15 mutant, inwhich the PSAA and PSAB proteins were undetectable, re-sembles that observed earlier for a chloroplast mutant with aframeshift mutation within psaB (Girard-Bascou et al., 1992).In this mutant, a shorter PSAB protein was detected by us-ing pulse-labeling analysis, and no PSAA protein was de-tectable. In contrast, mutants deficient in psaA geneexpression are able to synthesize normal amounts of PSABprotein, although the protein turns over rapidly (Girard-Bascou et al., 1992). Based on these results, we predictedthat the primary defect in F15 is in the expression of psaB.

To test this hypothesis, we analyzed the requirement forpsaB gene expression by means of chimeric reporter genes.The psaB 5' UTR together with the upstream promoter re-

gion were translationally fused to the aadA reporter gene,which confers spectinomycin resistance (Goldschmidt-Clermont, 1991). The chimeric gene fusion was integratedinto the chloroplast genome at the atpB locus of the FudSOstrain by using biolistic transformation, as described previ-ously (Nickelsen et al., 1994; Zerges and Rochaix, 1994).Transformants were first selected for the ability to grow onminimal medium and then scored for their ability to grow onspectinomycin-containing medium. The psaB 5' UTR-aad/Aconstruct conferred resistance to high concentrations of theantibiotic (we tested up to 1 mg/mL). After four consecutivesubcloning steps on spectinomycin-containing medium, thehomoplasmicity of the insertions was verified by DMA gelblot analysis (data not shown).

The transformants (mating type [+]) containing the psaB 5'UTR-aacW chimeric gene were crossed to the F15 mutant(mating type [-]). In this cross, all of the progeny inherit thechloroplast genome containing the chimeric gene, whereasthe nuclear mutation fat>7-F15 segregates 2:2. The progenywere scored for photoautotrophic growth and antibiotic resis-tance. In all progeny of these crosses, the ability to grow onminimal medium cosegregated with the resistance to the anti-biotic (Figure 3 and Table 1). Conversely, all of the photosyn-thetic-deficient progeny were sensitive to the antibiotic.Accumulation of the chimeric psaB-aadA mRNA was found

776 The Plant Cell

Table 1. Crosses between Strains Containing the Chimeric Genes with the Wild-Type and SuppressorpsaB 5‘ UTR and the F15 Mutant Strain

Cross No. Cross Phenotypea, No. of Tetrads

1 WT 5’ UTR psaB-aadA (+) X F15 (-) 2 (WT, Spc?: 2 (PSI, SPCS) 160f 16 2 su 5’ UTRpsaB-aadA (+) x F15 (-) 2 (WT, spcr):2 (PSI, Spc? 13 Of 13 3 5‘ UTRpsaA-aadA (+) X F15 (-) 2 (WT, Spc?:2 (PSI:Spc? 12 of 12 4 su (+) X F15 (-p 4 (wT):o (PSI) 6 of 6

aGrowth was tested on Tris-acetate-phosphate plates supplemented with increasing concentrations of spectinomycin (up to 1 mg/mL). WT, wild type; Spc‘, spectinomycin resistant; Spcs, spectinomycin susceptible. bThe numbers refer to the progeny within the tetrads with the phenotype indicated. Csu designates the chloroplast suppressor strain containing the chloroplast mutation from suF15 and the TA67 wild-type allele.

to be similar in all of the progeny from this cross (Figure 4A), and the AAD protein was detectable in the wild-type progeny but not in the F15 mutant progeny (Figure 48). These results indicate that the psaB 5’ UTR contains the target(s) for the TAB7 function. Together with the RNA gel blot analysis shown in Figure 2, these data show that the tab7-FI5 mutation affects the preinitiation or the initiation of psaB mRNA translation.

The protein pulse-labeling results (Figure 1A) show that the synthesis of both PSAB and PSAA was undetectable in the F15 mutant. The same result was obtained with a shorter 5-min pulse (Figure lC), suggesting that F15 could also be affected in PSAA protein synthesis at either a translational or an early post-translational step. To test whether this effect on PSAA synthesis was mediated through the psaA 5’ UTR, we constructed a psaA 5’ UTR-aadA chimeric gene, which was introduced into the chloroplast genome as described above. The transformants were then crossed to the F15 strain. All of the progeny from the tetrads examined were spectinomycin resistant (Table 1, cross 3), and none showed any reduction in the expression of the reporter gene in the mutant background up to the maximal concentrations of the antibiotic tested (1 mg/mL). These results support the idea that the nuclear-encoded function TAB7 is not required for translation from the psaA 5‘ UTR (see Discussion). However, they do not rule out the possibility of a functional interaction between TABl and psaA exon sequences downstream from the initiation codon.

A Point Mutation within the psaB 5‘ UTR Suppresses the Nuclear tabl-Fl5 Mutation

To gain further insights into the targets of the TAB7 function, we isolated a phenotypic revertant of the F15 strain, suF15, which is photoautotrophic. This reversion phenotype was inherited uniparentally from the mating type (+) parent in crosses, indicating that it is dueto a suppressor mutation located within the chloroplast genome (for details, see Meth- ods). From the results obtained with the chimeric genes described above, the region likely to be altered in the suppressor strain is the psaB 5’ UTR. Therefore, we sequenced

this chloroplast DNA region from the wild-type, F15, and suF15 strains (Figure 5A). Primer extension analysis was also performed with wild-type mRNA to identify the 5’ end of the psaB mRNA located 373 nucleotides upstream of the translation initiation site (Figure 5A). Although the 5’ UTR sequences of the wild-type and F15 strains were identical, the sequence of suF15 revealed a single base change, a G-to-A transition. This mutation is located adjacent to a putative Shine-Dalgarno sequence and is 17 bases upstream of the AUG translation initiation site (Figure 5A).

Although the location of the mutation in the psaB 5‘ UTR in the suF15 strain implied that it is responsible for the suppressor phenotype, we could not exclude the possibility that the suppressor mutation was actually in another region of the chloroplast genome and that the base change detected in the 5’ UTR of psa6 produces no phenotype. To confirm unambiguously that the single base change in the psa6 5’ UTR of the suF15 strain is responsible for the suppression phenotype, we introduced this mutation into the psaB gene of the F15 strain by using chloroplast transformation. This was achieved by inserting an aadA cassette into the EcoRl site upstream of the wild-type psaB gene and by replacing the Eco47111-BspHI fragment with its counterpart from the suF15 suppressor strain (Figure 58; see Methods). The resulting plasmid, pOS12, was used to transform F15 by se- lecting for the spectinomycin resistance conferred by aadA. After subcloning the transformants severa1 times on antibiotic-containing medium until they were homoplasmic, the restoration of PSI activity was tested by measuring the fluorescence transients. Among the 15 transformants tested, 12 displayed a wild-type fluorescence pattern. The three re- maining transformants were still PSI deficient, probably because the recombination event during integration of the transforming DNA had occurred between the site of insertion of the aadA cassette and the suppressor mutation.

To test further whether the effect of the suppressor mutation was restricted to the psaB 5’ UTR, we fused the suppressor psa6 5’ UTR to aadA and introduced the chimeric gene into Fud50 cells bearing a deletion of the atpB 3‘ coding sequence. In the cross of the transformants with F15, the PSI-deficient and wild-type progeny segregated 2:2. This was expected because the endogenous psaB gene in

psaS mRNA Translation 777

the transformants contains the wild-type 5' UTR (Figure 3and Table 1). The suppressor mutation markedly increasedthe resistance of the F15 mutant progeny to spectinomycin(Figure 3). The observation that the psaB 5' UTR of thesuF15 strain confers increased antibiotic resistance in prog-eny that inherited the fa£>7-F15 mutation further confirmsthat the single base change described above is responsiblefor the suppression phenotype.

F15 F15 WT WT

aadA

psaB

psbD

WTWTF15F15 FuDSO

B

AAD-

Figure 4. Levels of the Chimeric psaB 5' UTR-aad/4 mRNA and ItsProduct in the Presence of the Wild-Type TAB1 and Mutant tabl-F15Alleles.

(A) Gel blot analysis of RNA isolated from four members of a repre-sentative tetrad of cross 1 (Table 1) is shown. The wild-type (WT)and fa£>7-F15-bearing mutant progeny are indicated. RNA from anontransformed wild-type strain is also included. The hybridizationprobes used are indicated below the gels.(B) Protein gel blot analysis from total cell extracts from the fourmembers of a representative tetrad of cross 1 (Table 1) is shown.The wild-type and F15 mutant progeny are indicated. An antibodyraised against the recombinant AAD protein was used (Zerges andRochaix, 1994). The FuDSO lane contains protein from the untrans-formed strain used as recipient for the chimeric constructs. Thebands above and below the AAD band (indicated with an arrow) areproteins cross-reacting with the polyclonal serum raised against therecombinant AAD protein and are also present in the Fud50 control.

Ai46

91

136

181

226

271

316

361

406

451

496

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586

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676

721

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atatgcagt tgggtaagg cacttgtcc tcttatact

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cgtacgatg cagtaaata tatggccaa tggctcctc

gttctcaac ttaaaatgc aatctctta acgagaatcr*

gaaatatat ttgaattaa aatttccca caggattat

ataatatca actaaaaaa tctttttoo attttaaaa

ttacgcttt gtatgcaaa gtttgcttt gcacctgaa

taaattttt atttaatgg tagtttaat agtagtaat

ttaaacaaa aaaaatcct aattgttta tccctttaa

taaagtttt tttacttag tgaagtaaa aataccgct

tattttttc ttttgattt aacaattag cattttaac

ttctctcag tgttatact gcttaaaag tttttaggt$

atatttaat aatattaca tatagggag taagacaatoliool

B

ACTAAACTG TTTCCAAAA TTTAGCCAA CGTCTAGCTT K L F P K F S Q G L AACTACACGT AGAATTTGG TATGGGCTT GCTATGGCTT T R R I W Y G L A M AGAAAGCCAT GATGGTATG ACAGAAGAA AATCTTTACE S H D G M T E E N L YTTTCGTTCG CACTTCGGT CAATTATCT ATCATTTTCF R S H F G Q L S I I FTCTGGAAAC TTATTCCAC GTAGCATGG CAAGGTAACS G N L F H V A H Q G NTGGGTAACT GACCCAGTT CATATTCGT CCAATTGCTW V T D P V H I R P I ATGCGATCClW D BamHI

BspHI

Spel BamHI

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cttcgaagc 45

tcagaaata 90

gtatttagg 135

t t t tagcaa 180

taaagctca 225

ggcgtagtc 270

tttactttt 315

tagttttat 360

ttacttcaa 405

aagagcgct 450

cccttctgg 495

cttttactt 540

cattagata 585

t t tATGGCA 630__M A

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Pvul——I

aadA

R15 R18

Figure 5. Sequence of the psaB 5' UTR from the suF15 Strain Re-veals a Single Base Change Close to the Translation Initiation Site.

(A) Sequence of the psaB promoter region, 5' UTR (lowercase let-ters), and N-terminal portion of the coding sequence (uppercase let-ters). The mature end of the psaB 5' UTR site is indicated with anarrow, and the position of the mutation found in the suF15 strain ismarked (asterisk). The oligonucleotide used for primer extension(oligo 1) and the putative Shine-Dalgarno sequence are indicated.The boxed residues are the recognition sequences for the EcoRI andBamHI restriction enzymes.(B) Insertion vector pOS12 used for reintroducing the suppressormutation into the F15 mutant. Arrows show the 5' end of the maturepsaB mRNA and the direction of transcription of the psaB gene andof the aadA selectable marker. The aadA coding sequence (hatchedbox) is preceded by the psbD promoter region (stippled box) andpsbD5' UTR (black box). R15and R18 refer to the chloroplast EcoRIfragments (Rochaix, 1978). The Eco47lll-BspHI fragment used to in-troduce the suF15 suppressor mutation is indicated (open triangles).

778 The Plant Cell

DlSCUSSlON

Target Site of the TASI Function

-80

U U

A U

A C

c u ' CU

In this study, we have characterized the phenotype of a nuclear mutant, F15, that is specifically affected in PSI accumulation. Protein pulse-labeling experiments revealed that this mutant is deficient in the synthesis of the two PSI reaction center polypeptides, PSAA and PSAB. Although we also used short 5-min pulses, the interpretation of the protein labeling patterns was hampered by the difficulty in distinguish- ing between a defect in translation initiation, elongation, or enhanced degradation of either PSAA or PSAB after translation. Furthermore, the PSAA protein migrated as a diffuse band in the electrophoretic gel system used; therefore, it is difficult to assess whether the PSAA labeling in the pulse analyses was strongly reduced or completely absent. To cir- cumvent some of these problems, we used chimeric genes in which the psaB and psaA 5' UTRs were fused to the coding sequence of a heterologous protein, AAD, which confers resistance to spectinomycin. We introduced these reporter constructs into the chloroplast genome of Chlamydomonas and crossed the transformants to the F15 mutant. Analysis of the progeny with the psaB 5' UTR fusion constructs reveated cosegregation between antibiotic resistance and photoautotrophic growth or antibiotic sensitivity and lack of photoautotrophic growth (Table 1). These results clearly indicate that the target site of the TAB7 nuclear-encoded function is within thepsaB 5' UTR and that it is essential for initiation of translation of the psaB mRNA. In contrast, the expression of the psaA 5' UTR-aadA chimeric genes was not detectably affected by the presence of the mutant allele fabf-Fl5 (Table i), indicating that the TAB7 function is not required for translation from the psaA 5' UTR.

A Chloroplast Suppressor Mutation within the psaB 5' UTR

We have isolated a phenotypic revertant of F15 that grows photoautotrophically and in which PSAA and PSAB synthesis is restored. This strain contains a mutation within the psaB 5' UTR that changes a G to an A at position -17 relative to the AUG start codon (see Figure 5A). To ascertain that this single base change is indeed responsible for the suppression of the fabl-Fl5 mutation, we performed two in- dependent experiments. First, this mutation was reintro- duced into the psaB 5' UTR of the chloroplast genome of the F15 mutant, and suppression of the photosynthetic defect was observed. Second, the psaB 5' UTR from the suppressed strain, when fused to aadA, conferred antibiotic resistance in the presence of the fab7-FlS allele.

The wild-type psaB 5' UTR can be folded into a secondary structure [AG (free energy change) = -23 kcal/mol] in which a putative Shine-Dalgarno sequence near the AUG

5 ' 4 4 -

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A - U U - A - -20

C u U - A C - G '& -F A suF1S C - G C - G U - A C - G

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Figure 6. Putative Secondary Structure in thepsaB 5' UTR Near the Translation lnitiation Site.

The secondary structure obtained with the MulFold program from the Genetics Computer Group (Madison, WI) package is shown. The AG of the structure calculated by the program is approximately-23 kcalhol. The suppressor mutation in suF15 is indicated (asterisk). Dots mark G U base pairs. The initiation codon is boxed. Numbers refer to boxes relative to the initiation codon.

translation start is occluded (Figure 6). Although this structure is hypothetical, it is interesting that the suppressor mutation could have a destabilizing effect by creating a mismatch in the lower stem. This raises the possibility that the TAB7 function is required for opening the stem to allow for translation initiation. The presence of structured regions immedi- ately upstream of translation initiation regions is a common feature in prokaryotes (reviewed in Gold, 1988; McCarthy and Gualerzi, 1990). These organisms are considered to be the ancestors of chloroplasts. In particular, these features of the psaB 5' UTR resemble those of the mom gene of bacteriophage Mu in which the presence of a secondary structure blocks translation. This inhibition can be alleviated by a translational activator, the com gene product (Wulczyn et al., 1989).

Coordinated Synthesis andlor Accumulation of the PSAA and PSAB Subunits

The pulse-labeling experiments (Figure 1) clearly show that the fack of PSAB synthesis drastically affects PSAA synthesis. The effect on PSAA synthesis appears to result exclu- sively from the absence of PSAB synthesis and not from the presence of the fabl-Fl5 mutation, as shown by the fact that the suFl5 mutation in the psaB 5' UTR is capable of re- storing the synthesis of both proteins (-60% of that of wild-

psaB mRNA Translation 779

type levels; Figure 1 D). Taken together, these data point to a coordinated synthesis and/or accumulation of the PSAA and PSAB subunits. The results obtained with the chimeric genes rule out a link between the synthesis of these two polypeptides mediated by the psaA 5’ UTR during initiation of translation. One possibility is that PSAA and PSAB synthesis is coordinated during translation elongation. The ex- istence of a mechanism coordinating synthesis and/or accumulation of different subunits of the same photosynthetic complex has been suggested for PSll polypeptides (de Vitry et al., 1989) and for the ATPase complex (Drapier et al., 1992) and has recently been demonstrated for the cytochrome b,fcomplex. In this case, the rate of synthesis of cytochrome f is strongly reduced in the absence of the PETB or PETD subunits of the same complex (Kuras and Wollman, 1994; Kuras et al., 1995a, 199513).

Alternatively, our failure to detect PSAA synthesis in the absence of PSAB may have been due to rapid turnover of the nascent or complete PSAA polypeptide. This result, together with the fact that the PSAB polypeptide can be detected by pulse labeling of mutant cells deficient in PSAA (Girard-Bascou et al., 1987), indicates a clear asymmetry in the roles of PSAA and PSAB in the assembly of the PSI reaction center. The data are compatible with a role of the PSAB protein as the primary anchor in PSI assembly. In its absence, all other newly synthesized PSI core subunits might be rapidly degraded. Such a pivotal role for PSAB in PSI accumulation is supported by the observation that the PSAA polypeptide is undetectable in F15 (Figure lD), whereas a strain lacking PSAC (APSAC in Figure 1 D), which carries the terminal elec- tron acceptors of PSI, still has detectable amounts of PSAA polypeptide. A similar tight coordination also occurs in the assembly of other photosynthetic complexes of Chlamydomo- nas (de Vitry et al., 1989; Drapier et al., 1992) and in membrane-bound multiprotein complexes of yeast mito- chondria (Crivellone et al., 1988; Schmitt and Trumpower, 1991). Therefore, it could be a universal mechanism.

How Many Nuclear-Encoded Functions Are Required for Translation of Chloroplast mRNAs?

The analysis of mutants affected in the translation of thepsbC mRNA has revealed at least three distinct nuclear loci involved in this process (Rochaix et al., 1989; Wu and Kuchka, 1995; Zerges et al., 1997). There are similarities between the psbC and psaB systems. Both 5‘ UTRs are unusually long (547 and 373 bases, respectively) compared with other chloroplast leaders. In both cases, a suppressor mutation has been identified in the 5’ UTR that is specific for one of the required nuclear functions. Other nuclear-encoded functions have been identified for the translation of the psbA (Girard- Bascou et al., 1992) and psbD (Kuchka et al., 1988; Wu and Kuchka, 1995) mRNAs. These nuclear-encoded functions appear to act in a highly specific manner, as shown by the

fact that the translation of only one or sometimes two polypeptides is reduced, whereas the other chloroplast proteins are synthesized at wild-type levels. This could also be seen by crossing the transformants containing the chimeric psaB-aadA genes to the F34 and F64 mutants deficient in nuclear functions required forpsbC synthesis: no effect on the expression of the chimeric reporter could be detected (O. Stampacchia, W. Zerges, and J.-D. Rochaix, unpublished results). One possibility is that these numerous nuclear-encoded functions may be required to meet the specific temporal and spatial requirements for the proper synthesis and integration of each subunit into its particular photosynthetic complex.

It is not clear whether the TA67 function has additional roles other than that required for the translation of the psaB mRNA and to what extent its activity is modulated by exter- na1 stimuli, such as light. In this respect, it is interesting that in Chlamydomonas, biochemical analysis of the proteins in- teracting with the psbA 5‘ UTR has identified factors whose binding activity is light dependent (Danon and Mayfield, 1991, 1994a), and it has been shown in tobacco that the light-dependent stimulation of translation of the psbA mRNA is mediated in large part through its 5 ’ UTR (Staub and Maliga, 1993). It will be of considerable interest to investi- gate whether the nuclear TAB function is also involved in light-dependent stimulation of translation.

METHODS

Strains, Culture Conditions, and Genetic Analysis

Chlamydomonas reinhardtii strains were grown in Tris-acetate- phosphate medium. The induction of gametes, crosses, maturation of zygotes, and dissection of tetrads were performed according to Harris (1 989). Viability tests on antibiotic-containing and control plates were performed by aliquoting 1 O pL of each culture onto Petri plates containing the various media solidified with 2% agar. Individual clones were characterized by their fluorescence transients, as described by Eiennoun and Delepelaire (1 982). The Fud50 and F15 mutant strains have been described by Woessner et al. (1984) and Girard et al. (1980), respectively. The suF15 strain was isolated from F15 after mutagenesis with 5-fluorodeoxyuridine (Bennoun and Delepelaire, 1982) and selection for photoautotrophic growth. The suF15 mating- type (+) strain was crossed with wild-type mating-type (-) strain. All four progeny from each of 14 tetrads examined grew photoautotrophically. These progeny were backcrossed to the wild type to obtain suF15 (-) and su (+) strains. The latter does not produce a discernible phenotype in strains that are wild type for TA67. The cross between suF15 (-) and the wild type (+) yielded 11 tetrads in which two progeny grew photoautotrophically and two did not. The results of the cross suF15 (+) x F15 (-) are given in Table 1 (cross 4).

Pulse Labeling and Gel Electrophoresis

Pulse labeling of whole cells was performed in minimal medium with 14C-acetate for 45 or 5 min in the presence of cycloheximide (8 p.g/

780 The Plant Cell

mL), as described previously (Girard-Bascou et al., 1987). At the end of the labeling period, 200 mL of culture was used for thylakoid membrane purification. For Figure IC, the cells were pelleted and resuspended directly in gel loading buffer. The extract was fractionated by electrophoresis on a 7.5 to 12% SDS-polyacrylamide gel, as described previously (Girard-Bascou et al., 1987); it was then dried and subjected to autoradiography.

Nucleic Acids Manipulations

Standard techniques were used to manipulate and analyze nucleic acids (Sambrook et al., 1989). For RNA gel blot experiments, 5-pg samples of total RNA were electrophoresed through a 1 % agarose gel containing formaldehyde and Mops buffer, transferred to a Hybond- C+ membrane (Amersham), and probed with double-stranded DNA probes, which had been labeled with w3'P-dATP, using random primers. The 5' end of the psaB mRNA was determined by primer extension, using oligonucleotide 1 (Figure 5A) as the primer.TheaadA probe was a 0.81 -kb Ncol-Pstl cloned DNA fragment corresponding to the aadA structural gene (Goldschmidt-Clermont, 1991). The psbC probe was a 0.95-kb Hindlll fragment derived from the chloroplast DNA EcoRl fragment R9. The psaB probe was the 1.1-kb BamHI-BaOl fragment from the psaB gene (Rochaix, 1978). The psaA probe was the complete exon 111 sequence ofpsa4 (a 2.3-kb Ncol-Eco47111 fragment). The relative mRNA abundances were quantified with a Phos- phorlmager (Molecular Dynamics, Sunnyvale, CA) and standardized to the leve1 of thepsbC signal.

The Eco47111-BspHI fragment from thepsaB 5' untranslated region (5' UTR) containing the suppressor mutation suF15 was subcloned into a plasmid containing a Pvul-EcoRI fragment from RI8 (Rochaix, 1978), which is the region upstream of thepsaB gene, and an EcoRI- Spel fragment containing the promoter 5' UTR and part of the coding region of the psaf3 gene from the R I 5 fragment (Rochaix, 1978). A cassette containing the aadA reporter gene under the control of the psbD promoter was inserted into the EcoRl site (Figure 58). Fluores- cence transients were measured to assess whether the transformants had acquired the suppressor phenotype.

Construction of the Chimeric Genes

For construction of the psaB-aadA chimeric genes, two oligonucleotide primers were used to amplify by polymerase chain reaction a DNA fragment corresponding to the psaB promoter and 5' UTR from the cloned R I 5 chloroplast EcoRl DNA fragment (Rochaix, 1978). To clone the 5' UTR of psaB from the suppressor strain, we isolated the same fragment from the suF15 strain by shotgun cloning of EcoRI- digested chloroplast DNA (Sambrook et al., 1989). The primers used were oligonucleotide 2 (5'-TAAGGCACTTGTCCTCT) and oligonucleotide 3 (5'-CATAAAATTGTCTTACTCC), containing Clal and Ncol sites at the 5' and 3' ends, respectively. For constructing the psaA- aadA chimeric gene, we used a Hindlll fragment with the promoter region, 5' UTR, and coding region of exon I of psaA (described in Kück et al., 1987) that was subcloned into the Hindlll site of pBlue- script SK+ (Stratagene). The primers used for amplification of the psaA exon I 5' UTR were the pBluescript KS+ primer and the oligonucleotide 5'-AGTACTAATTGCCATGGATTTCTCC-3', which introduced a Ncol site at the translation initiation codon.

The polymerase chain reaction-amplified DNA fragments were phosphorylated with T4 polynucleotide kinase, cloned into the de-

phosphorylated Hincll site of pBluescribe (Stratagene), and sequenced from a double-stranded template with Sequenase (U.S. Biochemical Corp.). The fragments containing the UTRs were ex- cised by digestion with Clal and Ncol and translationally fused to the aadA coding sequence in the cg20-atpB-INT chloroplast transformation vector (Zerges and Rochaix, 1994). These constructs were trans- formed into the chloroplast genome of Fud50 with a microprojectile gun, as described previously (Zumbrunn et al., 1989; Zerges and Rochaix, 1994). Homoplasmic transformants were confirmed by ge- nomic DNA gel blot analyses after severa1 subcloning steps.

Protein Gel Blot Analyses

For the protein gel blot analysis, we prepared extracts from cultures (2 x 106 cells per mL) by resuspending the cells in 50 mM Tris-HCI, pH 6.8,2% SDS, incubating at 37°C for 30 min, and then centrifuging for 10 min in a microcentrifuge. Supernatants were used as protein extracts. Protein extracts were quantified according to Smith et al. (1985). An antibody raised against the N-terminal end of PSAA was produced (K. Redding, O. Stampacchia, and J.-D. Rochaix, unpublished data). Protein samples (30 1.9) were resuspended in 50 mM Tris-HCI, pH 6.8, 2% SDS, 100 mM DTT, and 0.25% bromophenol blue, electrophoresed through 15% acrylamide gels, and transferred to a Protran nitrocellulose membrane (Schleicher & Schuell). Protein transfer was verified by staining the filter with Ponceau S. Filters were blocked with 5% nonfat dry milk, 0.02% Tween-20, and 1 x PBS and incubated with primary antisera raised against the recombinant AAD polypeptide (Zerges and Rochaix, 1994). Filters were washed three times in PBS, reacted with peroxidase-linked anti-rabbit lg (Am- ersham) for 1 hr, and washed three times with PBS. Signals were revealed with a chemiluminescence detection system (Supersignal; Pierce, Rockford, IL).

ACKNOWLEDGMENTS

We thank all of the members of the Rochaix laboratory for stimulating discussions, Nicolas Fischer for help with the data in Figure 1 D, and Nicolas Roggli for preparing the figures. This work was supported by Grant No. 31-34014.92 from the Swiss National Fund to J.-D.R. and Grant No. UPR 9072 from the Centre Nationale de Ia Recherche Sci- entifique to J.G.-6. and P.B.

Received December 23, 1996; accepted March 3, 1997.

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O Stampacchia, J Girard-Bascou, J L Zanasco, W Zerges, P Bennoun and J D Rochaixchlamydomonas.

A nuclear-encoded function essential for translation of the chloroplast psaB mRNA in

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