Yves Choquet

An atypical haem in the cytochrome b6f complex

Photosystems I and II (PSI and II) are reaction centres that capture light energy in order to drive oxygenic photosynthesis; however, they can only do so by interacting with the multisubunit cytochrome b6f complex. This complex receives electrons from PSII and passes them to PSI, pumping protons across the membrane and powering the Q-cycle. Unlike the mitochondrial and bacterial homologue cytochrome bc1, cytochrome b6f can switch to a cyclic mode of electron transfer around PSI using an unknown pathway. Here we present the X-ray structure at 3.1 Å of cytochrome b6f from the alga Chlamydomonas reinhardtii. The structure bears similarities to cytochrome bc1 but also exhibits some unique features, such as binding chlorophyll, β-carotene and an unexpected haem sharing a quinone site. This haem is atypical as it is covalently bound by one thioether linkage and has no axial amino acid ligand. This haem may be the missing link in oxygenic photosynthesis.

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.

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.

Trans-splicing mutants of Chlamydomonas reinhardtii

SummaryIn 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.

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 ∼50 kb from exon 2 (198 bp), which is located ∼ 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.

Translational regulations as specific traits of chloroplast gene expression.

Studies of protein synthesis in the chloroplast compartment have revealed a unique combination of translational autoregulations and trans‐regulations due to the delivery of a variety of nuclear factors that act post‐transcriptionally. We show how these two characteristics concur to set the major step in the regulation of chloroplast gene expression at the translational level, leading to a surprisingly low sensitivity of chloroplast protein synthesis in response to extensive changes in plastome copy number and transcript concentration.

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.

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.

Chloroplast Biogenesis of Photosystem II Cores Involves a Series of Assembly-Controlled Steps That Regulate Translation

The biogenesis of photosystem II, one of the major photosynthetic protein complexes, involves a cascade of assembly-governed regulation of translation of its major chloroplast-encoded subunits. In Chlamydomonas reinhardtii, the presence of the reaction center subunit D2 is required for the expression of the other reaction center subunit D1, while the presence of D1 is required for the expression of the core antenna subunit apoCP47. Using chimeric genes expressed in the chloroplast, we demonstrate that the decreased synthesis of D1 or apoCP47 in the absence of protein assembly is due to a genuine downregulation of translation. This regulation is mediated by the 5′ untranslated region of the corresponding mRNA and originates from negative feedback exerted by the unassembled D1 or apoCP47 polypeptide. However, autoregulation of translation of subunit D1 is not implicated in the recovery from photoinhibition, which involves an increased translation of psbA mRNA in response to the degradation of photodamaged D1. De novo synthesis and repair of photosystem II complexes are independently controlled.

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

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.