Biogenesis of cytochrome b6 in photosynthetic membranes

In chloroplasts, binding of a c′-heme to cytochrome b6 on the stromal side of the thylakoid membranes requires a specific mechanism distinct from the one at work for c-heme binding to cytochromes f and c6 on the lumenal side of membranes. Here, we show that the major protein components of this pathway, the CCBs, are bona fide transmembrane proteins. We demonstrate their association in a series of hetero-oligomeric complexes, some of which interact transiently with cytochrome b6 in the process of heme delivery to the apoprotein. In addition, we provide preliminary evidence for functional assembly of cytochrome b6f complexes even in the absence of c′-heme binding to cytochrome b6. Finally, we present a sequential model for apo- to holo-cytochrome b6 maturation integrated within the assembly pathway of b6f complexes in the thylakoid membranes

Les protéines CCB définissent une nouvelle voie de biogenèse des cytochromes de type c

Les cythochromes C sont des hémoprotéines impliquées dans de nombreux processus biologiques et notamment dans le transfert d électrons au sein des membranes transductrices d énergie. La biogénèse des cytochromes C requiert la fixation convalente du cofacteur hémique qui, in vivo, est assistée par des ensembles de protéines formant au moins trois systèmes distincts suivant les organismes ou les compartiments subcellulaires étudiés. Le complexe cytochrome B6f transfère des électrons du transporteur hydrosoluble entre les deux photosystèmes, tout en construisant un gradient transmembranaire de protons par leur translocation depuis le côté négatif vers le côté positif de la membrane photosynthétique. L une de ses sous-unités , le cytochrome b6 contient trois cofacteurs hémiques dont un hème de type C , l hème Ci.. Chez Chlamydomonas, la biogenèse du cytochrome B6 requiert un ensemble de protéines nommées CCB, spécifiquement impliquées dans la liaison covalente de l hème Ci. Nous avons identifié les gènes et caractérisés les quatre protéines CCB connues à ce jour. Pour comprendre l implication du système IV dans la biogenèse du cytochrome B6, nous avons caractérisé les interactions entre les cinq protéines mis en jeu. L apocytochrome B6 est tout d abord partiellement replié suite à l insertion supposée spontanée de ces deux hèmes B. La protéine est ensuite prise en charge par CCBI, puis probablement transféré à CCB3 qui recrute un hétérodimère stable formé des deux protéines homologues CCB2 et CCB3. Ces trois derniers composants du système IV constituent probablement le complexe de liaison de l hème Ci sur le cytochrome.Cytochrome C are heme proteins involved in numerous biological processes, notably in electron transfer that occurs in energy transducing membranes. Cytochrome C biogenesis requires covalent attachment of the heme that, in vivo, is assisted by several groups of proteins classified in three different systems depending of the subcellular compartment under study. Cytochrome B6f complex transfers electrons from a lipid soluble transporter to a water soluble protein transporter between the two photosystems while building up a transmembrane proton gradient by translocating protons from the negative side to the positive of the photosynthetic membrane.Cytochrome B6 one the eight subunits of the complex contains tree hemes, two b-types and on c -type, the heme Ci. In Chlamydomonas, cytochrome B6 biogenesis requires a set of specialized proteins, named CCB to ensure the covalent binding of heme Ci. We identified the genes and characterized the four CCB proteins currently know. In order to understand the role of system IV in cytochrome B6 biogenesis, we have characterized the interactions between the four CCB and their substrat. Cythochrome B6 is first partially folded by the spontaneous insertion of the two b-type hemes. The proteins is then chaperoned by CCB1, probably transferred to CCb3, which recruits the stable heterodimer formed by CCB2 and CCB4. These latter three proteins represent probably the heme Ci ligation complex of the system IV.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Range of transcript levels detected for each of the eleven candidate genes across all 22 conditions.

<p>Average N₀ values per gene and condition (n = 3) were normalized on the smallest value then log transformed. Data were then represented for each gene as a box-plot.</p

Average variation in transcript levels for each candidate gene.

<p>All 22 conditions tested (<b>A</b>, highlighted in blue), development group (<b>B</b>), abiotic stress group (<b>C</b>), and hormone group (<b>D</b>). Variation is represented as the average of z-scores derived from geNorm and NormFinder results. The lower the z-score, the more invariant the transcript levels between samples.</p

Tissue-types, growth conditions, and exogenous treatments tested (biological contexts).

<p>*acetone 1/10000^e</p><p>Tissue-types, growth conditions, and exogenous treatments tested (biological contexts).</p

Identification of Reference Genes for Real-Time Quantitative PCR Experiments in the Liverwort <i>Marchantia polymorpha</i>

<div><p>Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, <i>Marchantia polymorpha</i> has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. <i>MpAPT</i> and <i>MpACT</i> showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that <i>MpAPT</i> and <i>MpACT</i> are suitable reference genes in <i>M</i>. <i>polymorpha</i> and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data.</p></div

qPCR candidate reference genes in <i>M. polymorpha.</i>

<p>qPCR candidate reference genes in <i>M. polymorpha.</i></p

Estimation of the optimal number of reference genes required for accurate qPCR data normalization.

<p>Estimation was done by calculating the pairwise variation (V_n/n+1) of the normalization factors NF_n and NF_n+1 as described in Vandesomple et al., [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118678#pone.0118678.ref016" target="_blank">16</a>].</p