TCA1, a single nuclear-encoded translational activator specific for petA mRNA in Chlamydomonas reinhardtii chloroplast.

We isolated seven allelic nuclear mutants of Chlamydomonas reinhardtii specifically blocked in the translation of cytochrome f, a major chloroplast-encoded subunit of the photosynthetic electron transport chain encoded by the petA gene. We recovered one chloroplast suppressor in which the coding region of petA was now expressed under the control of a duplicated 5' untranslated region from another open reading frame of presently unknown function. Since we also recovered 14 nuclear intragenic suppressors, we ended up with 21 alleles of a single nuclear gene we called TCA1 for translation of cytochrome b(6)f complex petA mRNA. The high number of TCA1 alleles, together with the absence of genetic evidence for other nuclear loci controlling translation of the chloroplast petA gene, strongly suggests that TCA1 is the only trans-acting factor. We studied the assembly-dependent regulation of cytochrome f translation--known as the CES process--in TCA1-mutated contexts. In the presence of a leaky tca1 allele, we observed that the regulation of cytochrome f translation was now exerted within the limits of the restricted translational activation conferred by the altered version of TCA1 as predicted if TCA1 was the ternary effector involved in the CES process.

Assembly-controlled regulation of chloroplast gene translation.

Studies of the biogenesis of the photosynthetic protein complexes in the unicellular green alga Chlamydomonas reinhardtii have pointed to the importance of the concerted expression of nuclear and chloroplast genomes. The accumulation of chloroplast- and nuclear-encoded subunits is concerted, most often as a result of the rapid proteolytic disposal of unassembled subunits, but the rate of synthesis of some chloroplast-encoded subunits from photosynthetic protein complexes, designed as CES proteins (Controlled by Epistasy of Synthesis), is regulated by the availability of their assembly partners from the same complex. Cytochrome f, a major subunit of the cytochrome b(6)f complex is a model protein for the study of the CES process. In the absence of subunit IV, another subunit of the cytochrome b(6)f complex, its synthesis is decreased by 90%. This results from a negative autoregulation of cytochrome f translation initiation, mediated by a regulatory motif carried by the C-terminal domain of the unassembled protein [Choquet, Stern, Wostrikoff, Kuras, Girard-Bascou and Wollman (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4380-4385]. Using site-directed mutagenesis, we have characterized this regulatory motif. We discuss the possible implications regarding the mechanism of the CES process for cytochrome f expression. We have studied the possible generalization of this mechanism to other CES proteins.

Identification of the initiation codon for the atpB gene in Chlamydomonas chloroplasts excludes translation of a precursor form of the beta subunit of the ATP synthase.

The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP synthase, contains three in-frame ATGs that are candidate translation initiation codons. An earlier study revealed that the N terminus of the assembled beta subunit maps at the +2 position with respect to the second in-frame methionine codon (Fiedler et al. 1995). Using chloroplast transformation, we have examined the possibility that either of the two additional in-frame ATG codons is competent for translation initiation. We provide evidence that translation of atpB is initiated exclusively at the second ATG codon. We conclude that the beta subunit is not synthesized with an N-terminal leader before its assembly into a functional ATP synthase complex.

Synthesis, assembly and degradation of thylakoid membrane proteins.

The thylakoid membrane of chloroplasts contains four major protein complexes, involved in the photosynthetic electron transfer chain and in ATP synthesis. These complexes are built from a large number of polypeptide subunits encoded either in the nuclear or in the plastid genome. In this review, we are considering the mechanism that couples assembly (association of the polypeptides with each other and with their cofactors) with the upstream and downstream steps of the biogenetic pathway, translation and proteolytic degradation. We present the contrasting images of assembly that have emerged from a variety of approaches (studies of photosynthesis mutants, developmental studies and direct biochemical analysis of the kinetics of assembly). We develop the concept of control by epistasy of synthesis, through which the translation of certain subunits is controlled by the state of assembly of the complex and address the question of its mechanisms. We describe additional factors that assist in the integration and assembly of thylakoid membrane proteins.

In vivo evidence for 5'-->3' exoribonuclease degradation of an unstable chloroplast mRNA.

The acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5' untranslated region (UTR). However, when this 5' UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1-1 background. Together, these results suggest that the 5' end of the petD 5' UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5' UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5'-->3' exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5' UTR protects the mRNA from 5'-->3' degradation in wild-type cells.

Translation of cytochrome f is autoregulated through the 5' untranslated region of petA mRNA in Chlamydomonas chloroplasts.

A process that we refer to as control by epistasy of synthesis (CES process) occurs during chloroplast protein biogenesis in Chlamydomonas reinhardtii: the synthesis of some chloroplast-encoded subunits, the CES subunits, is strongly attenuated when some other subunits from the same complex, the dominant subunits, are missing. Herein we investigate the molecular basis of the CES process for the biogenesis of the cytochrome b6f complex and show that negative autoregulation of cytochrome f translation occurs in the absence of other complex subunits. This autoregulation is mediated by an interaction, either direct or indirect, between the 5' untranslated region of petA mRNA, which encodes cytochrome f, and the C-terminal domain of the unassembled protein. This model for the regulation of cytochrome f translation explains both the decreased rate of cytochrome f synthesis in vivo in the absence of its assembly partners and its increase in synthesis when significant accumulation of the C-terminal domain of the protein is prevented. When expressed from a chimeric mRNA containing the atpA 5' untranslated region, cytochrome f no longer showed an assembly-dependent regulation of translation. Conversely, the level of antibiotic resistance conferred by a chimeric petA-aadA-rbcL gene was shown to depend on the state of assembly of cytochrome b6f complexes and on the accumulation of the C-terminal domain of cytochrome f. We discuss the possible ubiquity of the CES process in organellar protein biogenesis.

Mutations of cytochrome b6 in Chlamydomonas reinhardtii disclose the functional significance for a proline to leucine conversion by petB editing in maize and tobacco.

We have introduced a proline codon in place of a leucine codon at position 204 of the petB gene of Chlamydomonas reinhardtii. This gene modification mimics the presence of proline codons at the same position in the petB genes of maize and tobacco, which are subsequently edited to leucine codons at the RNA level. Following transformation, we observed no editing at this position in C. reinhardtii, independent of the type of proline codon we have used: the CCA codon, edited in maize, or a CCT codon. Strains carrying the introduced mutation were non phototrophic and displayed a block in photosynthetic electron transfer, consistent with a lack of cytochrome b6f activity. Thus the presence of a proline residue at position 204 in cytochrome b6 is detrimental to photosynthesis. We show that the mutant phenotype arose from a defective assembly of cytochrome b6f complexes and not from altered electron transfer properties in the assembled protein complex. Biochemical comparison of the proline-containing transformants with a cytochrome b6 mutant deficient in heme-attachment indicates that their primary defect is at the level of assembly of apocytochrome b6 with the bh heme, thereby preventing assembly of the whole cytochrome b6f complex.

Molecular genetic identification of a pathway for heme binding to cytochrome b6.

Heme binding to cytochrome b6 is resistant, in part, to denaturing conditions that typically destroy the noncovalent interactions between the b hemes and their apoproteins, suggesting that one of two b hemes of holocytochrome b6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holocytochrome b6, and to identify mutants that are blocked at one step of this pathway. Chlamydomonas reinhardtii strains carrying substitutions in either one of the four histidines that coordinate the bh or bl hemes to the apoprotein were created. These mutations resulted in the appearance of distinct immunoreactive species of cytochrome b6, which allowed us to specifically identify cytochrome b6 with altered bh or bl ligation. In gabaculine-treated (i.e. heme-depleted) wild type and site-directed mutant strains, we established that (i) the single immunoreactive band, observed in strains carrying the bl site-directed mutations, corresponds to apocytochrome b6 and (ii) the additional band present in strains carrying bh site-directed mutations corresponds to a bl-heme-dependent intermediate in the formation of holocytochrome b6. Five nuclear mutants (ccb strains) that are defective in holocytochrome b6 formation display a phenotype that is indistinguishable from that of strains carrying site-directed bh ligand mutants. The defect is specific for cytochrome b6 assembly, because the ccb strains can synthesize other b cytochromes and all c-type cytochromes. The ccb strains, which define four nuclear loci (CCB1, CCB2, CCB3, and CCB4), provide the first evidence that a b-type cytochrome requires trans-acting factors for its heme association.

Isolation of a psaF-deficient mutant of Chlamydomonas reinhardtii: efficient interaction of plastocyanin with the photosystem I reaction center is mediated by the PsaF subunit.

The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membrane and its precise role is not yet fully understood. Here we describe the isolation of a psaF-deficient mutant of the green alga Chlamydomonas reinhardtii generated by co-transforming the nuclear genome of the cw15-arg7A strain with two plasmids: one harboring a mutated version of the psaF gene and the other containing the argininosuccinate lyase gene conferring arginine prototrophy. This psaF mutant still assembles a functional PSI complex and is capable of photoautotrophic growth. However, electron transfer from plastocyanin to P700+, the oxidized reaction center chlorophyll dimer, is dramatically reduced in the mutant, indicating that the PsaF subunit plays an important role in docking plastocyanin to the PSI complex. These results contrast with those obtained previously with a cyanobacterial psaF-, psaJ- double mutant where no phenotype was apparent.

The trans-spliced intron 1 in the psaA gene of the Chlamydomonas chloroplast: a comparative analysis.

In the secondary structure model that has been proposed for the trans-spliced intron 1 in the Chlamydomonas reinhardtii psaA gene, a third RNA species (tscA RNA) interacts with the 5' and 3' intron parts flanking the exons to reconstitute a composite structure with several features of group-II introns. To test the validity of this model, we undertook the sequencing and modelling of equivalent introns in the psaA gene from other unicellular green algae belonging to the highly diversified genus Chlamydomonas. Our comparative analysis supports the model reported for the C. reinhardtii psaA intron 1, and also indicates that the 5' end of the tscA RNA and the region downstream from the psaA exon 1 cannot be folded into a structure typical of domain I as described for most group-II introns. It is possible that a fourth RNA species, yet to be discovered, provides the parts of domain I which are apparently missing.

A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii.

The light-independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild-type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light-independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.

Nucleotide sequences of the continuous and separated petA, petB and petD chloroplast genes in Chlamydomonas reinhardtii.

We have mapped and sequenced the petA (cytf), petB (cytb6) and petD (subunit IV) genes on the chloroplast genome of Chlamydomonas reinhardtii. At variance with the pet genes in higher plant chloroplasts, the petB and petD genes are continuous, not adjacent and not located next to the psbB gene. The corresponding polypeptide sequences are highly conserved when compared with their counterparts from other sources but have a few features specific of algal cytb6/f complexes. In particular the transit sequence of cytf displays unique characteristics when compared with those previously described for cytf in higher plants.

A small chloroplast RNA may be required for trans-splicing in Chlamydomonas reinhardtii.

In C. reinhardtii, the mature psaA mRNA is assembled by a process involving trans-splicing of three separate transcripts encoded at three widely scattered loci of the chloroplast genome. At least one additional chloroplast locus (tscA) is required for trans-splicing of exons 1 and 2. We have mapped this gene by transformation of a deletion mutant with a particle gun. The 0.7 kb region of the chloroplast genome that is sufficient to rescue tscA function has been subjected to insertion mutagenesis, showing that it does not contain significant open reading frames. We suggest from these experiments that the product of the tscA gene may be a small chloroplast RNA that acts in trans in the first trans-splicing reaction of psaA. A model for the mode of action of this RNA is presented, in which the characteristic structure of group II introns is assembled from three separate transcripts.

Trans-splicing mutants of Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii the three exons of the psaA gene are widely scattered on the chloroplast genome: exons 1 and 2 are in opposite orientations and distant from each other and from exon 3. The mature mRNA, encoding a core polypeptide of photosystem I, is thus probably assembled from separate precursors by splicing in trans. We have isolated and characterized a set of mutants that are deficient in the maturation of psaA mRNA. The mutants belong to 14 nuclear complementation groups and one chloroplast locus that are required for the assembly of psaA mRNA. The chloroplast locus, tscA, is remote from any of the exons and must encode a factor required in trans. The mutants all show one of only three phenotypes that correspond to defects in one or other or both of the joining reactions. These phenotypes, and those of double mutants, are consistent with the existence of two alternative splicing pathways.

Mutant phenotypes support a trans-splicing mechanism for the expression of the tripartite psaA gene in the C. reinhardtii chloroplast.

The chloroplast psaA gene of the green unicellular alga Chlamydomonas reinhardtii consists of three exons that are transcribed from different strands. Analysis of numerous nuclear and chloroplast mutants that are deficient in photosystem I activity reveals that roughly one-quarter of them are specifically affected in psaA mRNA maturation. These mutants can be grouped into three phenotypic classes, based on their inability to perform either one or both splicing reactions. The data indicate that the three exons are transcribed independently as precursors which are normally assembled in trans and that the splicing reactions can occur in either order. While some chloroplast mutations could act in cis, the nuclear mutations that fall into several complementation groups probably affect factors specifically required for assembling psaA mRNA.

Structural and transcription analysis of two homologous genes for the P700 chlorophyll a-apoproteins in Chlamydomonas reinhardii: evidence for in vivo trans-splicing.

The two homologous genes for the P700 chlorophyll a-apoproteins (ps1A1 and ps1A2) are encoded by the plastom in the green alga Chlamydomonas reinhardii. The structure and organization of the two genes were determined by comparison with the homologous genes from maize using data from heterologous hybridizations as well as from DNA and RNA sequencing. While the ps1A2 (736 codons) gene shows a continuous gene organization, the ps1A1 (754 codons) gene possesses some unusual features. The discontinuous gene is split into three separate exons which are scattered around the circular chloroplast genome. Exon 1 (86 bp) is separated by approximately 50 kb from exon 2 (198 bp), which is located approximately 90 kb apart from exon 3 (1984 bp). All exons are flanked by intronic sequences of group II. Transcription analysis reveals that the ps1A2 gene hybridizes with a 2.8-kb transcript, while all exon regions of the ps1A1 gene are homologous to a mature mRNA of 2.7 kb. From our data we conclude that the three distantly separated exonic sequences of the ps1A1 gene constitute a functional gene which probably operates by a trans-splicing mechanism.

Characterization of a chloroplast mutation in the psaA2 gene of Chlamydomonas reinhardtii.

The synthesis of polypeptides related to the CPI chlorophyll-protein complex of photosystem I has been studied by pulse-labeling experiments in twenty chloroplast mutants of Chlamydomonas reinhardtii. Three mutations of the same locus (Girard-Bascou 1987) result in the absence of these CPI-related polypeptides. Among these mutations one, (FUD26) leads to the synthesis of a new polypeptide presumed to be a truncated CPI apoprotein. The molecular characterization of this mutation in the psaA2 gene has been achieved by DNA sequencing the 3' end of this gene. The FUD26 mutation is a 4 base pair deletion resulting in frameshift and premature termination of the protein.

Geometry for the Primary Electron Donor and the Bacteriopheophytin Acceptor in Rhodopseudomonas viridis Photosynthetic Reaction Centers.

The tetrapyrrole electron donors and acceptors (bacteriochlorophyll, BCh; bacteriopheophytin, BPh) within the bacterial photosynthetic reaction center (RC) are arranged with a specific geometry that permits rapid (picosecond time scale) electron tunneling to occur between them. Here we have measured the angle between the molecular planes of the bacteriochlorophyll dimer (primary donor), B(2), and the acceptor bacteriopheophytin, H, by analyzing the dichroism of the absorption change associated with H reduction, formed by photoselection with RCs of Rhodopseudomonas viridis. This angle between molecular planes is found to be 60 degrees +/- 2. This means that the ultrafast electron tunneling must occur between donors and acceptors that are fixed by the protein to have a noncoplanar alignment. Nearly perpendicular alignments have been determined for other electron tunneling complexes involving RCs. These geometries can be contrasted with models proposed for heme-heme electron transfer complexes, which have emphasized that mutually parallel orientations should permit the most kinetically facile transfers.

Effects of chronic graded ethanol consumption on the metabolism, ultrastructure, and mechanical function of the rat heart.

Three sequential sets of ethanolic rats (E) and their matched controls (C) were fed regular chow containing standard vitamins with the ethanol group in each series also receiving a progressively greater alcohol intake for 3 to 6 months: E1 5%, E2 10%, and E3 25% ethanol. Electron microscopy showed swelling of mitochondria, transverse tubules and sarcoplasmic reticulum, dehiscence of intercalated discs and disintegration of myofibrils scattered throughout the ventricular myocardium in E1 and E2 as early as 7 wk after beginning 5% ethanol; in addition, there were clumping of mitochondria and supercontraction of myofibrils in E3. Concomitant with substructural abnormalities in E3, there were slight but significant depressions of cardiac myofibrillar ATPase activity and mitochondrial function. Cardiac catecholamines, hydroxyproline, and total bound glycerol were unchanged. Alteration of isometric contraction of isolated, supported left ventricular papillary muscles occurred initially in E2 and was clearly evident in E3 by significant reduction of duration of systolic active state (time from onset to peak tension), while total tension generated and peak rate of tension rise were not yet disturbed. Extra vitamin supplementation in additional rats drinking 25% ethanol minimally lessened decline in myofibrillar ATPase activity, but otherwise provided no protection. Thus, chronic daily ingestion of graded quantities of ethanol representing 10 to 30% of total calories in well-nourished animals exerted toxic effects on microstructure, metabolism and mechanics of the ventricle. These alterations are postulated to be pertinent to early pathogenesis of clinical alcoholic cardiomyopathy.