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Biogenesis of PSI involves a cascade of translational autoregulation in the chloroplast of Chlamydomonas

2004; Springer Nature; Volume: 23; Issue: 13 Linguagem: Inglês

10.1038/sj.emboj.7600266

1460-2075

Katia Wostrikoff, Jacqueline Girard‐Bascou, Françis-André Wollman, Yves Choquet,

ATP Synthase and ATPases Research

Article10 June 2004free access Biogenesis of PSI involves a cascade of translational autoregulation in the chloroplast of Chlamydomonas Katia Wostrikoff Katia Wostrikoff CNRS/UPR 1261, ass. Univ. Paris VI, Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Jacqueline Girard-Bascou Jacqueline Girard-Bascou Search for more papers by this author Francis-André Wollman Francis-André Wollman Search for more papers by this author Yves Choquet Corresponding Author Yves Choquet CNRS/UPR 1261, ass. Univ. Paris VI, Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Katia Wostrikoff Katia Wostrikoff CNRS/UPR 1261, ass. Univ. Paris VI, Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Jacqueline Girard-Bascou Jacqueline Girard-Bascou Search for more papers by this author Francis-André Wollman Francis-André Wollman Search for more papers by this author Yves Choquet Corresponding Author Yves Choquet CNRS/UPR 1261, ass. Univ. Paris VI, Institut de Biologie Physico-Chimique, Paris, France Search for more papers by this author Author Information Katia Wostrikoff1, Jacqueline Girard-Bascou, Francis-André Wollman and Yves Choquet 1 1CNRS/UPR 1261, ass. Univ. Paris VI, Institut de Biologie Physico-Chimique, Paris, France *Corresponding author. CNRS/UPR 1261, ass. Univ. Paris VI, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France. Tel.: +33 1 58 41 50 75; Fax: +33 1 58 41 50 22; E-mail: [email protected] The EMBO Journal (2004)23:2696-2705https://doi.org/10.1038/sj.emboj.7600266 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Photosystem I comprises 13 subunits in Chlamydomonas reinhardtii, four of which—the major reaction center I subunits PsaA and PsaB, PsaC and PsaJ—are chloroplast genome-encoded. We demonstrate that PSI biogenesis involves an assembly-governed regulation of synthesis of the major chloroplast-encoded subunits where the presence of PsaB is required to observe significant rates of PsaA synthesis and the presence of PsaA is required to observe significant rates of PsaC synthesis. Using chimeric genes expressed in the chloroplast, we show that these regulatory processes correspond to autoregulation of translation for PsaA and PsaC. The downregulation of translation occurs at some early stage since it arises from the interaction between unassembled PsaA and PsaC polypeptides and 5′ untranslated regions of psaA and psaC mRNAs, respectively. These assembly-dependent autoregulations of translation represent two new instances of a control by epistasy of synthesis process that turns out to be a general feature of protein expression in the chloroplast of C. reinhardtii. Introduction Many enzymatic functions are carried out by hetero-oligomeric proteins. The assembly of their constitutive subunits in an appropriate stoichiometry can be regarded as spontaneous, being a thermodynamically favoured process. Still, biological systems have optimised the rate of protein production in order to avoid wasteful accumulation of unassembled subunits. For instance, in prokaryotes, the operonal organisation of many genes allows a fine-tuning of the coupled transcription/translation rates of subunits from the same protein complex. In many other instances, a proteolytic disposal of unassembled subunits operates as a backup system in biogenesis pathways. The biogenesis of oligomeric protein complexes in the energy transducing membranes of organelles has unique features of particular complexity. Most of these protein complexes comprise more than 10 distinct subunits, some of which are nucleus-encoded, whereas the others are encoded by the organellar genome (Fox, 1996; Wollman et al, 1999). Because the gene copy numbers for nucleus- versus organelle-encoded subunits from a same protein complex can differ by as much as four orders of magnitude, organelle-based regulation processes, as well as crosstalks between the nucleo-cytosolic and organelle compartments, should be at work to regulate the level of expression of the various subunits of these oligomeric proteins. Studies of respiratory mutants from yeast or of photosynthetic mutants from the unicellular green alga Chlamydomonas reinhardtii have been instrumental in this major issue in cell biology (Fox, 1996; Wollman et al, 1999). These studies showed that the accumulation of the various subunits of these oligomeric proteins is a concerted process: most mutant strains deficient for the expression of a major protein subunit present a pleiotropic loss of the whole set of subunits of the complex. Two main mechanisms are responsible for this concerted accumulation in the chloroplast of C. reinhardtii (reviewed in Wollman et al, 1999; Choquet and Vallon, 2000). Many unassembled subunits undergo a rapid proteolytic degradation but others show an assembly-dependant regulation of their rates of synthesis. We have defined this assembly-dependent regulatory process as a 'control by epistasy of synthesis' or CES process (Wollman et al, 1999; Choquet and Vallon, 2000). In the case of cytochrome f, encoded by the chloroplast petA gene, we were able to characterise the molecular mechanism underlying the CES process as an autoregulation of petA mRNA translation (Choquet et al, 1998) that involves negative feedback from the C-terminal domain of the unassembled polypeptide (Choquet et al, 2003). In the present study, we provide evidence that assembly-dependent autoregulation of translation initiation may be a central mechanism in the biogenesis of chloroplast oligomeric proteins in C. reinhardtii. Photosystem I (PSI) is a thylakoid-embedded pigment–protein complex that performs light-induced charge separation and drives electron transfer from plastocyanin to ferredoxin. In C. reinhardtii, PSI is made of 13 subunits: PsaA, B, C and J are chloroplast-encoded, whereas PsaD, E, F, G, H, K, L, N and O are nucleus-encoded (for a review, see Webber and Bingham, 1998). The core components of PSI are two large transmembrane subunits, PsaA and PsaB, which share strong sequence similarity and may have arisen from gene duplication (reviewed in Baymann et al, 2001). Both subunits comprise 11 transmembrane helices. The N-terminus of each subunit faces the chloroplast stroma, whereas their C-termini protrude on the lumenal side of the thylakoid (Sun et al, 1997). PsaA and PsaB assemble at an early step of PSI biogenesis, forming the chlorophyll a–protein complex I (CPI) that binds most of the pigments and redox cofactors of PSI. CPI is the template for assembly of the extrinsic PsaC subunit on the stromal side of the membranes. PsaC, a 9 kDa polypeptide, coordinates the Fe–S clusters FA and FB through two cysteine-rich domains. The stromal subunits PsaD and PsaE then assemble coordinately around PsaC (Yu et al, 1995). PSI mutants lacking either PsaA, PsaB or PsaC display the same severe drop in the accumulation of all PSI subunits, demonstrating that accumulation of these proteins is a concerted process. Strains deleted for psaC show wild-type rates of synthesis of PsaB and PsaA, which are then rapidly degraded (Takahashi et al, 1991). In contrast, psaA mutants show wild-type rates of PsaB synthesis (Girard-Bascou et al, 1987; Goldschmidt-Clermont et al, 1990) but reduced rates of PsaC synthesis (Takahashi et al, 1991). Finally, mutants lacking expression of PsaB show no detectable PsaA synthesis, whether the strains contain mutations in the chloroplast psaB gene itself (Girard-Bascou et al, 1987), or in the nuclear TAB1 gene, which is required for psaB mRNA translation initiation (Stampacchia et al, 1997). A chloroplast mutation in the psaB 5′ untranslated region (UTR) suppresses the effect of this nuclear defect and restores translation of both PsaB and PsaA subunits, arguing for a role of PsaB availability in PsaA translation (Stampacchia et al, 1997). In the present work, we used chimeric genes expressing reporter proteins translated under the control of psaA or psaC 5′ UTRs to provide evidence that the biogenesis of PSI involves a cascade of autoregulation of translation, most likely at the level of initiation, mediated by the unassembled CES subunits PsaA or PsaC. Results Assessment of regulation of translation initiation in the CES behaviour of PsaA, using the aadA reporter gene One of the two major reaction centre (RCI) subunits of PSI, PsaA, is a CES subunit since it displays a reduced rate of synthesis in the absence of the other RCI subunit, PsaB. This may result from regulation of translation initiation, controlled by the 5′ UTR of the psaA gene. To test that hypothesis, we constructed a chimeric gene bearing the psaA 5′ UTR fused immediately upstream of the bacterial aadA gene coding sequence, which confers resistance to the antibiotics spectinomycin and streptomycin (Goldschmidt-Clermont, 1991). By biolistic transformation, this chimeric gene was inserted downstream of the chloroplast petA gene in the wild type and in mutant strains unable to accumulate PsaB (Figure 1A). We chose the tab1-F15 nuclear mutant strain, which is deficient for translation of the psaB messenger (Stampacchia et al, 1997), and the chloroplast mutant strain C3 (Girard-Bascou et al, 1987), which expresses a truncated version of PsaB that is rapidly degraded (see Supplementary Figure I). Transformants were recovered from each recipient strain on spectinomycin (100 μg ml−1)–Tris-acetate-phosphate (TAP) plates. The level of antibiotic resistance conferred by the chimeric gene was determined by plating the transformants on TAP medium supplemented with increasing concentrations of antibiotics, as illustrated in Figure 1B (see also Supplementary Figure II and Supplementary Table I). While the chimeric gene allowed the growth of strains derived from the wild type on antibiotic concentrations higher than 1000 μg ml−1 of spectinomycin plus 100 μg ml−1 of streptomycin, transformants devoid of PsaB (tab1-F15 and C3 in Figure 1B) no longer grew when the concentration of antibiotics reached 500+50 μg ml−1 of spectinomycin+streptomycin. Since the chimeric 5′psaA-aadA mRNAs accumulated to the same level in all transformed strains (Figure 1C), these results point to a specific downregulation of translation of the psaA 5′ UTR-driven aadA gene when expressed in the absence of PsaB. Figure 1.The psaA 5′ UTR confers a PsaB-dependent expression to the reporter gene aadA. (A) Schematic map of the petA-petD chloroplast region where the 5′psaA-aadA chimeric gene has been inserted, in direct orientation with respect to the petA gene, at the neutral EcoRV site (V). ↱ indicates transcription start sites. (B) Growth of independent transformants, derived from the recipient strains listed in the left, in the presence of increasing concentrations of antibiotics. The PsaATr and maa-F31 strains are devoid of PsaA expression (see later). (C) Accumulation of the chimeric aadA (and petA, as a loading control) mRNAs in the wild-type strain and in some of the transformants presented in panel B. Download figure Download PowerPoint We could exclude that the reduced resistance to antibiotics observed in strains lacking PsaB was a mere consequence of PSI deficiency by transforming mutant strains lacking expression of PsaA with the same chimeric gene: the transformed strains that were recovered, although deficient for PSI accumulation, presented the same growth properties on TAP–antibiotics plates as those derived from the wild-type recipient strain (PsaATr and maa-F31 in Figure 1B and C). The psaA 5′ UTR is sufficient to confer a PsaB-dependent rate of synthesis to the cytochrome f reporter protein In the above experiments however, we monitored only indirectly expression of the chimeric 5′psaA-aadA gene through resistance of the transformed strains to antibiotics. As an alternative, we constructed another chimeric gene, 5′psaA-petA, allowing cytochrome f to be translated under the control of the psaA 5′ UTR (Figure 2A). We introduced the chimera in the chloroplast genome of the ΔpetA strain (Table I). Recovery of phototrophic transformants aAf indicated that the psaA 5′ UTR allowed high enough expression of cytochrome f to sustain photoautotrophic growth. Figure 2.The CES behaviour for the PsaA subunit corresponds to a translational regulation mediated by the psaA 5′ UTR. (A) Map of the chloroplast petA gene in wild-type and aAf strains. Relevant restriction sites are indicated: B, BglII; N*, an NcoI site introduced by site-directed mutagenesis around the petA initiation codon for cloning purposes; H, HincII. (B) Accumulation of petA, psaA and atpB (as a loading control) transcripts in a representative tetrad progeny (out of seven) of the cross aAf × tab1-F15 and from parental and wild-type strains, revealed by hybridisation to petA, psaA (exon 3) and atpB-specific probes. (C) Accumulation of cytochrome f, PsaA and OEE2 (as a loading control), detected with specific antibodies on whole-cell proteins extracted from those strains. Lack of PsaA signs the tab1 progeny (marked by *). (D) Synthesis of cytochrome f, determined by 5 min pulse labelling with [14C]acetate in the presence of 8 μg ml−1 cycloheximide preventing cytosolic synthesis, in the same strains. Positions of cytochrome f and CP43 (providing an incorporation and loading control) are marked. Download figure Download PowerPoint Table 1. Transformation experiments Transformed strains Recipient strainsa Plasmid used Selection WT::aAK WT (SpS) pfaAK Spec. resistance F15::aAK tab1-F15 (SpS), (1) pfaAK Spec. resistance C3::aAK C3 (SpS), (2) pfaAK Spec. resistance aAf ΔpetA (SpR), (3) pKaAf Phototrophy aACf ΔpetA (SpR), (3) pKaACf Phototrophy {C3, aAf} C3 (SpS), (2) pKaAf Spec. resistance PsaATrb1 WT (SpS) pKrPsaATr Spec. resistance {PsaATr, aAf} psaATr (SpS)b2 pKaAf Spec. resistance aCf WT (SpS) pKaCf Spec. resistance {ΔpsaC, aCf} ΔpsaC (SpS) (4) pKaCf Spec. resistance a All recipient strains were mt+, and either resistant (Sp) or sensitive (Sp) to spectinomycin. (1) Stampacchia et al (1997), (2) Girard-Bascou et al (1987), (3) Kuras and Wollman (1994), (4) Takahashi et al (1991). b1 The PsaATr strain was initially selected for spectinomycin resistance due to the presence of the recycling aadA cassette. After excision of the cassette according to Fischer et al (1996), the strain became Sp and was used as a recipient strain for a second round of transformation with plasmid pKaAf, based on selection for spectinomycin resistance. Strain aAf was then crossed to the tab1-F15 nuclear mutant strain that lacks PsaB. Each member of the resulting tetrads carried the chloroplast chimeric gene, uniparentally transmitted from the mt+ parent aAf. Half of them (indicated by * in Figure 2B–D) had fluorescence transients typical of impaired PSI activity (Chua et al, 1975) (data not shown) and harboured the nuclear tab1 mutation, which shows Mendelian segregation; the other half inherited a wild-type nuclear genome. Therefore, analysis of cytochrome f expression among tetrad progeny by RNA hybridisation, protein pulse labelling and immunodetection with specific antibodies allowed us to compare the expression of the chimeric gene in the presence or absence of the PsaB subunit. We observed a 2:2 segregation in cytochrome f expression, in agreement with the above experiments using aadA as a reporter: the tab1 progeny exhibited a drastic decrease in the accumulation of the reporter protein, reaching only 10–15% of that observed in the parental strain aAf or in the progeny with a wild-type nuclear genome (a preliminary report of this experiment has been presented at the 673rd meeting of the Biochemical Society; Choquet et al, 2001) (Figure 2C). This resulted from a decrease in translation of the 5′psaA-petA reporter gene (Figure 2D). However, the chimeric messenger, which migrates faster than wild-type petA mRNA, was less accumulated in the tab1 offspring from aAf × tab1-F15 crosses (Figure 2B). In contrast, the psaA mRNA was similarly accumulated in all progeny, even those lacking detectable PsaA product (tab1 progeny). Since RNA stability determinants can be found in coding sequences (Singh et al, 2001), we reproduced the same set of experiments with a third chimeric gene, 5′psaAC-petA, which contained an extension of 60 nucleotides (nt), corresponding to the first 20 residues of the PsaA protein fused in-frame with the petA coding sequence, in addition to the promoter and 5′ UTR of psaA. After transformation of the ΔpetA strain, phototrophic transformants aACf were recovered and crossed to the tab1-F15 mutant strain. The expression of cytochrome f was similar in strains aACf and aAf and showed the same decrease in expression in the tab1 progeny of the crosses. With this construct, the drop in the rate of expression of the reporter protein in the absence of PsaB was not accompanied by any change in the mRNA level (see Supplementary Figure III). Thus we conclude that both the aadA gene product and cytochrome f reporters, expressed under control of the 5′psaA UTR, showed a drop in translation in the absence of PsaB. In the subsequent experiments, we chose to use the chimeric gene 5′psaA-petA, because it contains only sequences from the psaA 5′ UTR. In order to rule out the possibility that the decrease in expression of the chimeric gene in the absence of PsaB resulted from a pleiotropic effect of the tab1 mutation on translation of both psaA and psaB messengers, we used the chloroplast mutant strain C3, which expresses a truncated PsaB, but has a wild-type nuclear genome. Upon transformation with the chimeric gene 5′psaA-petA, the resulting strains, hereafter named {C3, aAf}, were used in pulse-labelling experiments. We observed a low rate of cytochrome f translation in all {C3, aAf} transformants tested when compared to the aAf strain (Figure 3C), similar to that observed in the aAf, tab1 progeny indicated by * in Figure 2D. Figure 3.The expression of the 5′psaA-petA reporter gene is no longer repressed in the absence of PsaB when the endogenous PsaA subunit is lacking. (A) Accumulation of petA, psaA and petD (as a loading control) transcripts in progeny from the cross {C3, aAf} × maa-F31 and in wild-type and parental strains, detected with probes specific for petA, psaA (exon 1) and petD. Absence of mature psaA mRNA signs the maa progeny, designated by *. (B) Accumulation and (C) translation of cytochrome f in the same strains. OEE2 accumulation provides a loading control. In (C), the translation of the 5′psaA-petA chimeric gene in the presence of PsaB (lane aAf) is shown for comparison. (Inset) Longer exposure of the gel for the second progeny of the tetrad, which incorporated poorly radiolabelled 14C. Positions of neosynthesised CP43, truncated PsaB (Tr) and cytochrome f are indicated. In the two progeny with a wild-type genome, 14C incorporation is lower in cytochrome f than in CP43, while it is higher in the maa progeny. Download figure Download PowerPoint Together, these experiments proved that the absence of PsaB causes decreased expression of not only psaA but also of chimeric genes translated under the control of the psaA 5′ UTR. Therefore, the CES regulation in psaA expression occurs before translation elongation or before cotranslational degradation of the nascent PsaA polypeptide, which would both depend on the coding sequence. Rather, some early step in the initiation process is regulated, either an activation step prior to initiation, translation initiation itself or a transition step between initiation and elongation of translation. Downregulation of PsaA expression in the absence of PsaB is due to autoregulation of translation In the absence of the PsaB protein, downregulation of psaA translation could occur either because PsaB would act as an activator for PsaA translation ('transactivation' hypothesis; Figure 4, right panel) or because unassembled PsaA, which may accumulate to some extent, would expose a translational repressor domain (autoregulation hypothesis; Figure 4, left panel). Because the psaA 5′ UTR was sufficient to confer the PsaA CES behaviour to chimeric genes, these two hypotheses could be discriminated by looking at the expression of the 5′psaA-driven cytochrome f reporter in the absence of both the PsaA and PsaB subunits (Figure 4, bottom). In the case of a transactivation hypothesis, the absence of the positive regulator brought along with the PsaB subunit should result in poor expression of the chimeric gene, either in the presence or in the absence of PsaA. By contrast, in the autoregulation hypothesis, a strain lacking the repressor domain carried by the unassembled CES protein, PsaA, should show a high expression of the chimera, even in the absence of the assembly partner PsaB. Figure 4.The two models for the repression of psaA mRNA translation in the absence of the dominant protein PsaB can be discriminated by looking at the expression of a 5′psaA-driven reporter gene in the absence of both PsaA and PsaB. Download figure Download PowerPoint We thus crossed the {C3, aAf} strain, which expresses the 5′psaA-petA reporter gene, but does not accumulate PsaB, because it expresses only a truncated and unstable polypeptide, with the maa-F31 strain, defective for PsaA expression. This mutant is impaired in trans-splicing of precursor transcripts carrying exon 1 and exon 2 of psaA. Therefore, it lacks full-length psaA mRNA but accumulates the exon 1 precursor RNA (Goldschmidt-Clermont et al, 1990). As a preliminary control, we first checked that the maa mutation did not prevent expression of the chimeric gene by crossing the aAf strain with the maa-F31 strain. The rates of synthesis and accumulation of the reporter cytochrome f were identical in all tetrad progeny (see Supplementary Figure IV). Thus cytochrome f expressed from the chimeric 5′psaA-petA gene remains insensitive to the maa nuclear mutation. Analysis of cytochrome f expression in one out of five tetrads from the cross {C3, aAf} × maa-F31 is shown in Figure 3A–C. Each tetrad progeny harboured both the chimeric gene, as demonstrated by the higher mobility of petA mRNAs (Figure 3A), and the psaB mutation, as revealed by the synthesis of a truncated PsaB polypeptide (Figure 3C). Only half of the progeny, identified by the absence of mature psaA mRNA (Figure 3A), inherited the maa nuclear mutation and are indicated by * in Figure 3. The two progeny with a wild-type nuclear genome were similar to the parental strain {C3, aAf}: they showed low synthesis and accumulation of the psaA-driven cytochrome f (Figure 3B and C) due to the absence of PsaB. In contrast, the maa progeny, which express neither full-length PsaB nor PsaA, recovered a high expression of the reporter gene, similar to that observed in the aAf strain. Therefore, translation of the 5′psaA-driven cytochrome f is no longer decreased in the absence of the PsaB subunit, when the CES protein PsaA cannot accumulate in the thylakoid membranes. These experiments strongly support an autoregulation of psaA translation rather than a transactivation hypothesis. A truncated PsaA polypeptide escapes the psaA CES control We noted that accumulation of chimeric 5′psaA-petA transcripts was higher in the maa nuclear background (Figure 3A). This has been observed previously with other 5′psaA-containing transcripts in various psaA trans-splicing mutants, for example with the psaA exon 1 precursor itself (Choquet et al, 1988). The high expression of the 5′psaA-driven cytochrome f in the maa progeny from the cross {C3, aAf}, observed in the above experiment, may be due to the overaccumulation of the chimeric messenger. In addition, some nucleus-encoded factors, required for the translation of psaA mRNA, may not be able to bind to the precursor transcript of psaA exon 1 and thus become fully available for translation of the sole chimeric 5′psaA-petA mRNA. For a critical assessment of these alternative hypotheses, we caused a premature termination of translation 155 residues after the initiation methionine by introducing a frameshift in the third exon of psaA, thereby preventing accumulation of full-length PsaA (Figure 5A). In this case, the putative nuclear regulatory factors participate normally in the translation of a truncated psaA product, which should be rapidly degraded because of its inability to assemble into a PSI complex. Figure 5.Expression of the 5′psaA-petA reporter gene in strains expressing a truncated PsaA. (A) Strategy used to introduce a mutation (*) in the third exon of psaA. Relevant restriction sites are shown, as well as transmembrane helices coding regions (grey boxes). Coding sequences are indicated by horizontal hatches. Due to the mutation *, most of the psaA mRNA (after trans-splicing) is not translated in the mutant strain (white rectangle). (B) Pulse labelling of the PsaATr strain (time 0) followed by a chase for the indicated times in the presence of an excess of nonradioactive acetate and 200 μg ml−1 chloramphenicol. The position of truncated PsaA is marked with a dot. As already described (Delepelaire, 1983), the AtpF protein is short-lived. (C) Accumulation of petA, psaA and atpB (as a loading control) transcripts in a representative tetrad from the cross {PsaATr, aAf} × tab1-F15 and in wild-type and {PsaATr, aAf} strains, detected using probes specific for petA, psaA (exon 1) and atpB. (D) Accumulation of cytochrome f (and OEE2 as a loading control) in the strains. (E) Chloroplast translates in the same strains. Lack of PsaB synthesis signs the tab1 tetrad progeny, designated by *. Download figure Download PowerPoint A two-step procedure was used to introduce both the psaA frameshift and the 5′psaA-petA reporter into the same strain. We first associated the psaA mutation with a 'recycling' spectinomycin resistance cassette that allowed us to select transformants on spectinomycin-supplemented TAP medium before losing specifically the cassette—but not the mutant psaA allele—once the selective pressure is released (Fischer et al, 1996). The resulting strains, PsaATr, were screened for PSI deficiency by fluorescence kinetics and showed normal accumulation of the mutated psaA messenger (data not shown). A truncated PsaA polypeptide of about 16 kDa was detected by a pulse-labelling experiment, between two CF0 subunits of the ATP synthase (Lemaire and Wollman, 1989). Its rate of synthesis—as quantified by a PhosphorImager scan of the 14C labelling of the gel and corrected for the number of carbon atoms—is similar to that of the PsaB and PsaA polypeptides in a wild-type strain. In a pulse-chase experiment (Figure 5B), the truncated form of PsaA proved to be very unstable, with a half-life of less than 10 min. Therefore, it does not accumulate to any significant extent in thylakoid membranes. Next, we allowed spontaneous excision of the recycling cassette from the chloroplast genome of PsaATr transformants, and used them as recipient strains for a second round of transformation with the 5′psaA-petA reporter gene, associated with a new spectinomycin resistance cassette. Transformants {PsaATr, aAf}, selected on antibiotic-supplemented TAP medium, were crossed with the nuclear mutant strain tab1-F15 to compare expression of the reporter gene in the absence of PsaA alone or in the absence of both PsaA and PsaB. All tetrad progeny from that cross inherited the psaA 5′ UTR-driven cytochrome f (Figure 5C) and the truncated psaA allele carried by the chloroplast genome, because of the uniparental inheritance from the mt+ parent (Figure 5E). Half of the progeny inherited the tab1 nuclear mutation and were identified in pulse-labelling experiments by their lack of PsaB synthesis (Figure 5E), while the other half had a wild-type nuclear genome and translated PsaB normally. The rate of synthesis of the truncated PsaA polypeptide was identical in the four progeny (PsaATr, indicated by a dot in Figure 5E). Therefore, the translation of this truncated and unstable PsaA was no longer dependent on the presence of PsaB. Similarly, the 5′psaA-driven petA reporter gene was expressed at the same high level in all progeny of the cross {PsaATr, aAf} × tab1-F15 (Figure 5D and E), in contrast to what was observed among the progeny of the cross aAf × tab1-F15 (where translation was repressed in the tab1 members of the tetrad; Figure 2D). Therefore, when PsaA cannot accumulate in thylakoid membranes, translation of the 5′psaA-petA transcript remains high, even in the absence of the assembly partner, PsaB. That the presence of PsaB does not stimulate any 5′psaA-driven translation fully excludes the transactivation hypothesis. Our data point to an autoregulation of translation where unassembled PsaA exerts a negative feedback on the translation of psaA mRNA. Synthesis of the PsaC CES subunit is controlled at the level of translation initiation As indicated in Introduction, PSI contains another CES protein, PsaC, whose synthesis is decreased in the absence of PsaA. To determine whether translation initiation of PsaC was indeed regulated by the availability of PsaA, we constructed a 5′psaC-petA gene (including the first 30 nt of PsaC coding sequence), associated with a spectinomycin resistance cassette (Figure 6A). Transformants containing this reporter, named aCf, were selected for growth on spectinomycin–TAP plates and were found to grow phototrophically despite accu