Richard Kuras

The Chloroplast Gene ycf9 Encodes a Photosystem II (PSII) Core Subunit, PsbZ, That Participates in PSII Supramolecular Architecture

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.

Molecular Genetic Identification of a Pathway for Heme Binding to Cytochrome b 6

Heme binding to cytochromeb 6 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 b 6 is tightly bound to the polypeptide. We exploited this property to define a pathway for the conversion of apo- to holocytochrome b 6, 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 b 6, which allowed us to specifically identify cytochrome b 6 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 b 6 and (ii) the additional band present in strains carrying bhsite-directed mutations corresponds to a bl-heme-dependent intermediate in the formation of holocytochrome b 6. Five nuclear mutants (ccb strains) that are defective in holocytochromeb 6 formation display a phenotype that is indistinguishable from that of strains carrying site-directed bh ligand mutants. The defect is specific for cytochromeb 6 assembly, because the ccbstrains can synthesize other b cytochromes and allc-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.

MRL1, a Conserved Pentatricopeptide Repeat Protein, Is Required for Stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis

The expression of the chloroplast genome requires specialized proteins that are coded in the nucleus and imported into the organelle. We have identified such a protein that binds the leading end of the mRNA for the most abundant chloroplast enzyme. The function of this novel stabilization factor is conserved from green algae to land plants. We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 5 ′ untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 5 ′ >3 ′ degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.

Evidence for regulatory function of nucleus-encoded factors on mRNA stabilization and translation in the chloroplast

A salient feature of organelle gene expression is the requirement for nucleus-encoded factors that act posttranscriptionally in a gene-specific manner. A central issue is to understand whether these factors are merely constitutive or have a regulatory function. In the unicellular alga Chlamydomonas reinhardtii, expression of the chloroplast petA gene-encoding cytochrome f, a major subunit of the cytochrome b6f complex, depends on two specific nucleus-encoded factors: MCA1, required for stable accumulation of the petA transcript, and TCA1, required for its translation. We cloned the TCA1 gene, encoding a pioneer protein, and transformed appropriate mutant strains with tagged versions of MCA1 and TCA1. In transformed strains expressing decreasing amounts of MCA1 or TCA1, the concentration of these factors proved limiting for petA mRNA accumulation and cytochrome f translation, respectively. This observation suggests that in exponentially growing cells, the abundance of MCA1 sets the pool of petA transcripts, some of which are TCA1-selected for an assembly-dependent translation of cytochrome f. We show that MCA1 is a short-lived protein. Its abundance varies rapidly with physiological conditions that deeply affect expression of the petA gene in vivo, for instance in aging cultures or upon changes in nitrogen availability. We observed similar but more limited changes in the abundance of TCA1. We conclude that in conditions where de novo biogenesis of cytochrome b6f complexes is not required, a rapid drop in MCA1 exhausts the pool of petA transcripts, and the progressive loss of TCA1 further prevents translation of cytochrome f.

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.

A specific c-type cytochrome maturation system is required for oxygenic photosynthesis

Oxygenic photosynthesis is an important bioenergetic process that maintains the Earths atmosphere and allows carbon fixation. A critical enzyme in this process, the cytochrome b6f complex, differs from other protein complexes of the same family by an unusual covalently attached cofactor chemically defined as a c′ heme. We have identified a set of pioneer proteins that carry the biogenesis of this c′ heme and started their characterization. They are encoded by the genomes of all organisms performing oxygenic photosynthesis, whatever their phylogenetic distances. These proteins are thus among the few that distinguish photosynthetic cells evolving oxygen from other types of living cells.

Specific mutagenesis of the rieske iron-sulfur protein in Rhodobacter sphaeroides shows that both the thermodynamic gradient and the pK of the oxidized form determine the rate of quinol oxidation by the bc(1) complex.

In the Rieske iron-sulfur protein (ISP) of the ubiquinol:cytochrome c(2) oxidoreductase (bc(1) complex) of Rhodobacter sphaeroides, residue Tyr 156 is located close to the iron-sulfur cluster. Previous studies of the equivalent residue in both Saccharomyces cerevisiae [Denke, E., Merbitz-Zahradnik, T., Hatzfeld, O. M., Snyder, C. H., Link, T. A., and Trumpower, B. L. (1998) J. Biol. Chem. 273, 9085-9093] and Paracoccus denitrificans [Schroter, T., Hatzfeld, O. M., Gemeinhardt, S., Korn, M., Friedrich, T., Ludwig, B. , and Link, T. A. (1998) Eur. J. Biochem. 255, 100-106] have indicated that mutations at this site can lead to modifications in the redox potential of the ISP. To study the effect of similar modifications on the thermodynamic behavior and kinetics of partial reactions of the bc(1) complex upon flash activation, we have constructed four mutant strains of Rb. sphaeroides where Tyr 156 was mutated to His, Leu, Phe, or Trp. The bc(1) complex was assembled and able to support photosynthetic growth in all mutants. Three substitutions (Leu, Phe, Trp) led to alteration of the midpoint potential (E(m)) of the ISP and a slowing in rate of quinol oxidation, suggesting that electron transfer from quinol to the oxidized ISP controls the overall rate and that this step includes the high activation barrier. The Trp mutation led to an increase of approximately 1 pH unit in the pK value of the oxidized ISP. The pH dependence of the rate of quinol oxidation in this mutant was also shifted up by approximately 1 pH unit, showing the importance of the protonation state of the ISP for quinol oxidation. This provides support for a model in which the dissociated form of the oxidized ISP is required for formation of the enzyme-substrate complex [Ugulava, N., and Crofts, A. R. (1998) FEBS Lett. 440, 409-413].

Dual functions of the nucleus‐encoded factor TDA1 in trapping and translation activation of atpA transcripts in Chlamydomonas reinhardtii chloroplasts

After endosymbiosis, organelles lost most of their initial genome. Moreover, expression of the few remaining genes became tightly controlled by the nucleus through trans-acting protein factors that are required for post-transcriptional expression (maturation/stability or translation) of a single (or a few) specific organelle target mRNA(s). Here, we characterize the nucleus-encoded TDA1 factor, which is specifically required for translation of the chloroplast atpA transcript that encodes subunit α of ATP synthase in Chlamydomonas reinhardtii. The sequence of TDA1 contains eight copies of a degenerate 38-residue motif, that we named octotrico peptide repeat (OPR), which has been previously described in a few other trans-acting factors targeted to the C. reinhardtii chloroplast. Interestingly, a proportion of the untranslated atpA transcripts are sequestered into high-density, non-polysomic, ribonucleoprotein complexes. Our results suggest that TDA1 has a dual function: (i) trapping a subset of untranslated atpA transcripts into non-polysomic complexes, and (ii) translational activation of these transcripts. We discuss these results in light of our previous observation that only a proportion of atpA transcripts are translated at any given time in the chloroplast of C. reinhardtii.

A novel pathway of cytochrome c biogenesis is involved in the assembly of the cytochrome b6f complex in arabidopsis chloroplasts.

We recently characterized a novel heme biogenesis pathway required for heme ci′ covalent binding to cytochrome b6 in Chlamydomonas named system IV or CCB (cofactor assembly, complex C (b6f), subunit B (PetB)). To find out whether this CCB pathway also operates in higher plants and extend the knowledge of the c-type cytochrome biogenesis, we studied Arabidopsis insertion mutants in the orthologs of the CCB genes. The ccb1, ccb2, and ccb4 mutants show a phenotype characterized by a deficiency in the accumulation of the subunits of the cytochrome b6f complex and lack covalent heme binding to cytochrome b6. These mutants were functionally complemented with the corresponding wild type cDNAs. Using fluorescent protein reporters, we demonstrated that the CCB1, CCB2, CCB3, and CCB4 proteins are targeted to the chloroplast compartment of Arabidopsis. We have extended our study to the YGGT family, to which CCB3 belongs, by studying insertion mutants of two additional members of this family for which no mutants were previously characterized, and we showed that they are not functionally involved in the CCB system. Thus, we demonstrate the ubiquity of the CCB proteins in chloroplast heme ci′ binding.

A Nucleus-Encoded Chloroplast Protein Regulated by Iron Availability Governs Expression of the Photosystem I Subunit PsaA in Chlamydomonas reinhardtii

A nucleus-encoded chloroplast protein, which controls the stability and translation of the messenger RNA encoding the PsaA subunit of Photosystem I, responds post-transcriptionally to iron availability. The biogenesis of the photosynthetic electron transfer chain in the thylakoid membranes requires the concerted expression of genes in the chloroplast and the nucleus. Chloroplast gene expression is subjected to anterograde control by a battery of nucleus-encoded proteins that are imported in the chloroplast, where they mostly intervene at posttranscriptional steps. Using a new genetic screen, we identify a nuclear mutant that is required for expression of the PsaA subunit of photosystem I (PSI) in the chloroplast of Chlamydomonas reinhardtii. This mutant is affected in the stability and translation of psaA messenger RNA. The corresponding gene, TRANSLATION OF psaA1 (TAA1), encodes a large protein with two domains that are thought to mediate RNA binding: an array of octatricopeptide repeats (OPR) and an RNA-binding domain abundant in apicomplexans (RAP) domain. We show that as expected for its function, TAA1 is localized in the chloroplast. It was previously shown that when mixotrophic cultures of C. reinhardtii (which use both photosynthesis and mitochondrial respiration for growth) are shifted to conditions of iron limitation, there is a strong decrease in the accumulation of PSI and that this is rapidly reversed when iron is resupplied. Under these conditions, TAA1 protein is also down-regulated through a posttranscriptional mechanism and rapidly reaccumulates when iron is restored. These observations reveal a concerted regulation of PSI and of TAA1 in response to iron availability.