Nuclear genes required for post-translational steps in the biogenesis of the chloroplast cytochrome b6f complex in maize

Nuclear genes essential for the biogenesis of the chloroplast cytochrome b ₆ f complex were identified by mutations that cause the specific loss of the complex. We describe four transposon-induced maize mutants that lack cytochrome b ₆ f proteins but contain normal levels of other photosynthetic complexes. The four mutations define two nuclear genes. To identify the step at which each mutation blocks protein accumulation, mRNAs encoding each subunit were examined by Northern hybridization analysis and the rates of subunit synthesis were examined in pulse-labeling experiments. In each mutant the mRNAs encoding the known subunits of the complex were normal in size and abundance and the major subunits were synthesized at normal rates. Thus, these mutations block the biogenesis of the cytochrome b ₆ f complex at a post-translational step. The two nuclear genes identified by these mutations may encode previously unknown subunits, be involved in prosthetic group synthesis or attachment, or facilitate assembly of the complex. These mutations were also used to provide evidence for the authenticity of a proposed fifth subunit of the complex and to demonstrate a role for the cytochrome b ₆ f complex in protecting photosystem 11 from light-induced degradation.     

Biosynthesis of P700-Chlorophyll a Protein Complex, Plastocyanin, and Cytochrome b(6)/f Complex

Changes in the amount of P700-chlorophyll a protein complex, plastocyanin, and cytochrome b₆/f complex during greening of pea (Pisum sativum L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) leaves were analyzed by an immunochemical quantification method. Neither subunit I nor II of P700-chlorophyll a protein complex could be detected in the etiolated seedlings of all three plants and the accumulation of these subunits was shown to be light dependent. On the other hand, a small amount of plastocyanin was present in the etiolated seedlings of all three plants and its level increased about 30-fold during the subsequent 72-hour greening period. Furthermore, cytochrome f, cytochrome b₆, and Rieske Fe-S center protein in cytochrome b₆/f complex were also present in the etiolated seedings of all three plants. The level of each subunit component increased differently during greening and their induction pattern differed from species to species. The accumulation of cytochrome b₆/f complex was most profoundly affected by light in pea leaves, and the levels of cytochrome f, cytochrome b₆, and Rieske Fe-S center protein increased during greening about 10-, 20-, and more than 30-fold, respectively. In comparison to the case of pea seedlings, in wheat and barley leaves the level of each subunit component increased much less markedly. The results suggest that light regulates the accumulation of not only the chlorophyll protein complex but also the components of the electron transport systems.     

Mutations in the UQCC1-Interacting Protein,UQCC2, Cause Human Complex III Deficiency Associated with Perturbed Cytochrome b Protein Expression

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.     

Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups

To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.     

Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803

The cytochrome b₆f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b₆f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.     

Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b6f Complex in Thermosynechococcus elongatus

The cyanobacterial cytochrome b₆f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b₆f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700⁺ reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b₆f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b₆f complexes with different functions that might be correlated with supercomplex formation.     

Heliobacterial Rieske/cytb complex

Data on structure and function of the Rieske/cytb complex from Heliobacteria are scarce. They indicate that the complex is related to the b ₆ f complex in agreement with the phylogenetic position of the organism. It is composed of a diheme cytochrome c, and a Rieske iron–sulfur protein, together with transmembrane cytochrome b ₆ and subunit IV. Additional small subunits may be part of the complex. The cofactor content comprises heme c _i, first discovered in the Q_i binding pocket of b ₆ f complexes. The redox midpoint potentials are more negative than in b ₆ f complex in agreement with the lower redox midpoint potentials (by about 150 mV) of its reaction partners, menaquinone, and cytochrome c ₅₅₃. The enzyme is implicated in cyclic electron transfer around the RCI. Functional studies are favored by the absence of antennae and the simple photosynthetic reaction chain but are hampered by the high oxygen sensitivity of the organism, its chlorophyll, and lipids.     

Photocontrol of the Accumulation of Plastid Polypeptides during Greening of Tomato Cotyledons : Potentiation by a Pulse of Red Light

A pulse of red light acting through phytochrome accelerates the formation of chlorophyll upon subsequent transfer of dark-grown seedlings to continuous white light. Specific antibodies were used to follow the accumulation of representative subunits of the major photosynthetic complexes during greening of seedlings of tomato (Lycopersicon esculentum). The time course for accumulation of the various subunits was compared in seedlings that received a red light pulse 4 h prior to transfer to continuous white light and parallel controls that did not receive a red light pulse. The light-harvesting chlorophyll-binding proteins of photosystem II (LHC II), the 33-kD extrinsic polypeptide of the oxygen-evolving complex (OEC1), and subunit II of photosystem I (psaD gene product) all increased in the light, and did so much faster in seedlings that received the inductive red light pulse. The red light pulse had no significant effect on the abundance of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), nor on several plastid-encoded polypeptides: the large subunit of Rubisco, the β subunit of the CF₁ complex of plastid ATPase, and the 43- and 47-kD subunits of photosystem II (CP43, CP47). Subunits I (cytochrome b₆f) and III (Rieske Fe-S protein) of the cytochrome b₆f complex showed a small or no increase as a result of the red pulse. The potentiation of greening by a pulse of red light, therefore, is not expressed uniformly in the abundance of all the photosynthetic complexes and their subunits.     

On the mode of integration of plastid-encoded components of the cytochrome bf complex into thylakoid membranes

Four distinct integration/translocation routes into/across thylakoid membranes have recently been deduced for nuclear-encoded polypeptides of the photosynthetic membrane. Corresponding information for the plastid-encoded protein complement is lacking. We have investigated this aspect with in-organello assays employing chimeric constructs generated with codon-correct cassettes for genes of plastid-encoded thylakoid proteins, and appropriate transit peptides from six nuclear genes, representing three targeting classes, as a strategy. The three major plastid-encoded components of the cytochrome b ₆  f complex, namely pre-apocytochrome f, (including apocytochrome f, and pre-apocytochrome f lacking the C-terminal transmembrane segment), cytochrome b ₆ , and subunit IV, which differ in the number of their transmembrane segments, were studied. Import into chloroplasts could be observed in all instances but with relatively low efficiency. Thylakoid integration can occurr post-translationally, but only components with secretory/secretory pathway (SEC)-route-specific epitopes were correctly assembled with the cytochrome complex, or competed with this process. Inhibitor studies were consistent with these findings. Imported cytochrome b ₆ and subunit IV operated with uncleaved targeting signals for thylakoid integration. The corresponding determinant for cytochrome f is its signal peptide; its C-terminal hydrophobic segment did not, or did not appreciably, contribute to this process. The N-termini of cytochrome b ₆ and subunit IV appear to reside on the same (lumenal) side of the membrane, consistent with the currently favored four-helix model for the cytochrome, but in disagreement with the topography proposed for both components. The impact of the findings for protein routing, including for applied approaches such as compartment-alien transformation, is discussed. Received: 18 September 1996 / Accepted: 15 October 1996     

Nitric Oxide–Triggered Remodeling of Chloroplast Bioenergetics and Thylakoid Proteins upon Nitrogen Starvation in Chlamydomonas reinhardtii

Starving microalgae for nitrogen sources is commonly used as a biotechnological tool to boost storage of reduced carbon into starch granules or lipid droplets, but the accompanying changes in bioenergetics have been little studied so far. Here, we report that the selective depletion of Rubisco and cytochrome b₆f complex that occurs when Chlamydomonas reinhardtii is starved for nitrogen in the presence of acetate and under normoxic conditions is accompanied by a marked increase in chlororespiratory enzymes, which converts the photosynthetic thylakoid membrane into an intracellular matrix for oxidative catabolism of reductants. Cytochrome b₆f subunits and most proteins specifically involved in their biogenesis are selectively degraded, mainly by the FtsH and Clp chloroplast proteases. This regulated degradation pathway does not require light, active photosynthesis, or state transitions but is prevented when respiration is impaired or under phototrophic conditions. We provide genetic and pharmacological evidence that NO production from intracellular nitrite governs this degradation pathway: Addition of a NO scavenger and of two distinct NO producers decrease and increase, respectively, the rate of cytochrome b₆f degradation; NO-sensitive fluorescence probes, visualized by confocal microscopy, demonstrate that nitrogen-starved cells produce NO only when the cytochrome b₆f degradation pathway is activated.     

The Rieske/cytochrome b complex of Heliobacteria

Heliobacteria have a Rieske/cytochrome b complex composed of a Rieske protein, a cytochrome b_6, a subunit IV and a di-heme cytochrome c. The overall structure of the complex seems close to the b₆f complex from cyanobacteria and chloroplasts to the exception of the di-heme cytochrome. We show here by biochemical and biophysical studies that a heme c_i is covalently attached to the Rieske/cytochrome b complex from Heliobacteria. We studied the EPR signature of this heme in two different species, Heliobacterium modesticaldum and Heliobacillus mobilis. In contrast to the case of b₆f complex, a strong axial ligand to the heme is present, most probably a protonatable amino acid residue.     

Synthesis and assembly of the cytochrome b-f complex in higher plants

The cytochrome b-f complex is composed of four polypeptide subunits, three of which, cytochrome f, cytochrome b-563 and subunit IV, are encoded in chloroplast DNA and synthesised within the chloroplast, and the fourth, the Rieske FeS protein, is encoded in nuclear DNA and synthesised in the cytoplasm. The assembly of the cytochrome b-f complex therefore requires the interaction of subunits encoded by different genomes. A key role for the nuclear-encoded Rieske FeS protein in the assembly of the complex is suggested by a study of cytochrome b-f complex mutants. The assembly of individual subunits of the complex may be regulated by the availability of prosthetic groups. The genes for the chloroplast-encoded subunits and cDNA clones for the Rieske FeS protein have been isolated and characterised. Cytochrome f and the Rieske FeS protein are synthesised initially with N-terminal presequences required for their correct assembly within the chloroplast. The deduced amino acid sequences of the four subunits have been used to suggest models for the arrangement of the polypeptides in the thylakoid membrane.     

The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii

Two genes encoding cytochrome c oxidase subunits, Cox2a and Cox2b, are present in the nuclear genomes of apicomplexan parasites and show sequence similarity to corresponding genes in chlorophycean algae. We explored the presence of COX2A and COX2B subunits in the cytochrome c oxidase of Toxoplasma gondii. Antibodies were raised against a synthetic peptide containing a 14-residue fragment of the COX2A polypeptide and against a hexa-histidine-tagged recombinant COX2B protein. Two distinct immunochemical stainings localized the COX2A and COX2B proteins in the parasite's mitochondria. A mitochondria-enriched fraction exhibited cyanide-sensitive oxygen uptake in the presence of succinate. T. gondii mitochondria were solubilized and subjected to Blue Native Electrophoresis followed by second dimension electrophoresis. Selected protein spots from the 2D gels were subjected to mass spectrometry analysis and polypeptides of mitochondrial complexes III, IV and V were identified. Subunits COX2A and COX2B were detected immunochemically and found to co-migrate with complex IV; therefore, they are subunits of the parasite's cytochrome c oxidase. The apparent molecular mass of the T. gondii mature COX2A subunit differs from that of the chlorophycean alga Polytomella sp. The data suggest that during its biogenesis, the mitochondrial targeting sequence of the apicomplexan COX2A precursor protein may be processed differently than the one from its algal counterpart.     

DAC Is Involved in the Accumulation of the Cytochrome b6/f Complex in Arabidopsis

The biogenesis and assembly of photosynthetic multisubunit protein complexes is assisted by a series of nucleus-encoded auxiliary protein factors. In this study, we characterize the dac mutant of Arabidopsis (Arabidopsis thaliana), which shows a severe defect in the accumulation of the cytochrome b₆/f complex, and provide evidence suggesting that the efficiency of cytochrome b₆/f complex assembly is affected in the mutant. DAC is a thylakoid membrane protein with two predicted transmembrane domains that is conserved from cyanobacteria to vascular plants. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation analyses revealed a specific interaction between DAC and PetD, a subunit of the cytochrome b₆/f complex. However, DAC was found not to be an intrinsic component of the cytochrome b₆/f complex. In vivo chloroplast protein labeling experiments showed that the labeling rates of the PetD and cytochrome f proteins were greatly reduced, whereas that of the cytochrome b₆ protein remained normal in the dac mutant. DAC appears to be a novel factor involved in the assembly/stabilization of the cytochrome b₆/f complex, possibly through interaction with the PetD protein.The cytochrome b₆/f (Cyt b6/f) complex is a multisubunit complex that resides in the thylakoid membrane and functions in linear and cyclic electron transport. In the linear process, the complex receives electrons from PSII and transfers them to PSI, a process that is accompanied by the generation of a proton gradient, which is essential for ATP synthesis (Mitchell, 1961; Saraste, 1999). The native form of this complex is present as a dimer with a mass of 310 kD that can be converted into a 140-kD monomer with increasing detergent concentrations (Huang et al., 1994; Breyton et al., 1997; Mosser et al., 1997; Baniulis et al., 2009). In higher plants, the Cyt b6/f monomer contains at least eight subunits: Cyt f, Cyt b₆, PetC, PetD, PetM, PetL, PetG, and PetN (Wollman, 2004). PetC and PetM are encoded by nuclear genes, whereas the others are encoded by plastid genes. It has been shown that PetG and PetN are necessary for complex stability in tobacco (Nicotiana tabacum; Schwenkert et al., 2007). By contrast, PetL is not required for the accumulation of other subunits of the Cyt b6/f complex, even though it is involved in the stability and formation of the functional dimer (Bendall et al., 1986; Schwenkert et al., 2007). Inactivation of PetC in Arabidopsis (Arabidopsis thaliana) resulted in significantly reduced amounts of Cyt b6/f subunits and completely blocked linear electron transport, indicating that PetC participates in the formation of the functionally assembled Cyt b6/f complex (Maiwald et al., 2003). In Synechocystis sp. PCC 6803, the PetM subunit has no essential role in Cyt b6/f complex electron transfer or accumulation; however, the absence of this subunit apparently affects the levels of other protein complexes involved in energy transduction (Schneider et al., 2001). In addition to the other proteins, FNR was identified as a subunit of the Cyt b6/f complex isolated from spinach (Spinacia oleracea) thylakoid membranes (Zhang et al., 2001).Previous research has revealed how the Cyt b6/f complex assembles into a functional dimer (Bendall et al., 1986; Lemaire et al., 1986; Kuras and Wollman, 1994). In the Cyt b6/f complex, Cyt b₆ and PetD form a mildly protease-resistant subcomplex that serves as a template for the assembly of Cyt f and PetG, producing a protease-resistant cytochrome moiety (Wollman, 2004). The PetC and PetL proteins then participate in the assembly of the functional dimer (Schwenkert et al., 2007). PetD becomes more unstable in the absence of Cyt b₆, and the synthesis of Cyt f is greatly reduced when either Cyt b₆ or PetD is inactivated, indicating that both Cyt b₆ and PetD are prerequisite for the synthesis of Cyt f (Kuras and Wollman, 1994). The reduced synthesis of Cyt f can be explained by the so-called CES (for controlled by epistasy of synthesis) mechanism. It is suggested that, in this mechanism, the synthesis rate of some chloroplast-encoded subunits of photosynthetic protein complexes is regulated by the availability of their assembly partners from the same complexes (Choquet et al., 2001). The mechanism of CES for Cyt f has been studied in detail in Chlamydomonas reinhardtii (Choquet et al., 1998; Choquet and Vallon, 2000). In it, the unassembled Cyt f inhibits its own translation through a negative feedback mechanism, and MCA1 and TCA1 have been demonstrated to be involved in the regulation of Cyt f synthesis (Boulouis et al., 2011).Many studies have focused on understanding the conversion of apocytochrome to holocytochrome via the covalent binding of heme in Cyt f and Cyt b₆ during the assembly of Cyt b6/f through the CCS and CCB pathways (Nakamoto et al., 2000; Wollman, 2004; de Vitry, 2011). The CCS pathway was originally discovered in the green alga C. reinhardtii through genetic studies of ccs mutants (for cytochrome c synthesis) that display a specific defect in membrane-bound Cyt f and soluble Cyt c₆, two thylakoid lumen-resident c-type cytochromes functioning in photosynthesis (Xie and Merchant, 1998). In the CCS pathway, six loci that include plastid ccsA and nuclear CCS1 to CCS5 have been found in C. reinhardtii (Xie and Merchant, 1998). In these mutants, the apocytochrome is normally synthesized, targeted, and processed, but heme attachment is perturbed. The CCB pathway is involved in the covalent attachment of heme c(i) to Cyt b₆ on the stromal side of the thylakoid membranes (Kuras et al., 2007). The ccb mutants show defects in the accumulation of subunits of the Cyt b6/f complex and covalent binding of heme to Cyt b₆ (Lyska et al., 2007; Lezhneva et al., 2008). However, heme binding is not a prerequisite for the assembly of Cyt b₆ into the Cyt b6/f complex, although the fully formed Cyt b6/f showed an increased sensitivity to protease (Saint-Marcoux et al., 2009).The assembly of the Cyt b6/f complex is a multistep process, and current studies have shown that the covalent binding of heme to Cyt f and Cyt b₆ is highly regulated. Thus, it is reasonable to speculate that, similar to the other photosynthetic protein complexes (Mulo et al., 2008; Nixon et al., 2010; Rochaix, 2011), the assembly of the Cyt b6/f complex is also assisted by many nucleus-encoded factors. In this study, we characterized an Arabidopsis protein, DAC (for defective accumulation of Cyt b6/f complex), that seems to be involved in the assembly of the Cyt b6/f complex. In addition, we provide evidence that DAC interacts directly with PetD before it assembles within the Cyt b6/f complex.     

A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc₁ complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc₁ subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc₁ complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c₁ carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c₅₅₂, mediating electron transfer to the ba₃ oxidase. Identification of this cytochrome bc₁ complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.