Characterization of oxidative phosphorylation enzymes in Euglena gracilis and its white mutant strain WgmZOflL (original) (raw)
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Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2002
The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, a-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives. D
Journal of Biological Chemistry, 2000
The algae of the family Chlamydomonadaceae lack the gene cox3 that encodes subunit III of cytochrome c oxidase in their mitochondrial genomes. This observation has raised the question of whether this subunit is present in cytochrome c oxidase or whether the corresponding gene is located in the nucleus. Cytochrome c oxidase was isolated from the colorless chlamydomonad Polytomella spp., and the existence of subunit III was established by immunoblotting analysis with an antibody directed against Saccharomyces cerevisiae subunit III. Based partly upon the N-terminal sequence of this subunit, oligodeoxynucleotides were designed and used for polymerase chain reaction amplification, and the resulting product was used to screen a cDNA library of Chlamydomonas reinhardtii. The complete sequences of the cox3 cDNAs from Polytomella spp. and C. reinhardtii are reported. Evidence is provided that the genes for cox3 are encoded by nuclear DNA, and the predicted polypeptides exhibit diminished physical constraints for import as compared with mitochondrial-DNA encoded homologs. This indicates that transfer of this gene to the nucleus occurred before Polytomella diverged from the photosynthetic Chlamydomonas lineage and that this transfer may have occurred in all chlamydomonad algae.
Plant and Cell Physiology, 2000
The expression of the Chlamydomonas reinhardtii cytochrome c gene was studied at the steady-state mRNA level. The inclusion of acetate under illumination produced a marked increase in cytochrome c transcripts. This effect was not affected by two inhibitors of mitochondrial energy metabolism. Three different obligate photoautotrophic mutants with defective mitochondria showed normal levels of induction, suggesting that utilization of acetate for respiration is not required for this process. Light, in the presence or absence of acetate, also promoted an increase in cytochrome c transcript levels. This effect could be abolished by treatment of the cells with an inhibitor of the photosynthetic electron transport chain, suggesting that light acts through photosynthesis to promote the induction. In addition, a genomic clone encompassing the Chlamydomonas cytochrome c gene has been isolated and analyzed. The gene contains three introns, two of which are located at positions similar to those in the rice and Arabidopsis cytochrome c genes, indicating the existence of an evolutionary link. It is concluded that the cytochrome c gene from C. reinhardtii is subject to metabolic regulation through a mechanism that responds to the intracellular level of either acetate or a compound derived from its metabolization through a pathway different from mitochondrial respiration.
Current Genetics, 2002
In Chlamydomonas reinhardtii several nucleus-encoded proteins that participate in the mitochondrial oxidative phosphorylation are targeted to the organelle by unusually long mitochondrial targeting sequences. Here, we explored the components of the mitochondrial import machinery of the green alga. We mined the algal genome, searching for yeast and plant homologs, and reconstructed the mitochondrial import machinery. All the main translocation components were identified in Chlamydomonas as well as in Arabidopsis thaliana and in the recently sequenced moss Physcomitrella patens. Some of these components appear to be duplicated, as is the case of Tim22. In contrast, several yeast components that have relatively large hydrophilic regions exposed to the cytosol or to the intermembrane space seem to be absent in land plants and green algae. If present at all, these components of plants and algae may differ significantly from their yeast counterparts. We propose that long mitochondrial targeting sequences in some Chlamydomonas mitochondrial protein precursors are involved in preventing the aggregation of the hydrophobic proteins they carry.
Archives of Biochemistry and Biophysics, 1998
The dithionite-reduced spectra of the purified bc 1 complexes from the colorless alga Polytomella spp. and the closely related green alga Chlamydomonas reinhardtii were compared. The spectrum of the bc 1 complex from C. reinhardtii showed a profile similar to those of the bc 1 complexes from other species. In contrast, the bc 1 complex from Polytomella spp. exhibits a double-peak spectrum in the ␣-band region, where the absorption bands of cytochrome c 1 and cytochrome b are completely resolved. To further understand the molecular basis of these spectroscopic differences, the mitochondrial gene encoding cytochrome b of Polytomella spp. was cloned, sequenced, and compared with that of C. reinhardtii. The Polytomella spp. cytochrome b gene is 1113 bp long and does not contain introns. The deduced protein sequence exhibits 56% identity and 68% similarity with the cytochrome b of C. reinhardtii, and in a phylogenetic analysis it clearly affiliated with the b-type cytochromes of C. reinhardtii and C. smithii. A comparison of the primary sequences of the Polytomella spp. cytochrome b with other b-type cytochromes, and its analysis based on the structure featuring eight transmembrane stretches, allowed the identification of a tyrosine in position 114, which substitutes for a tryptophan present in all mitochondrial b-type cytochromes sequenced to date. In addition, the primary sequence of the cytochrome b from Polytomella spp. has a serine at position 36, instead of a nonpolar residue (alanine or leucine) found in all other species. In the proposed model for cytochrome b, both residues Tyr 114 and Ser 36 are in close proximity to the highpotential b H heme. The above data suggest that the polar residues Y 114 and S 36 , each one by itself or in combination, may interact with heme b H of Polytomella spp. and, thus, may be responsible for the unique spectroscopic characteristics of cytochrome b.
Polytomella spp. growth on ethananol
European Journal of Biochemistry, 2000
A defined medium with ethanol as sole carbon source was devised for growth of the colorless, unicellular alga Polytomella spp. Cell density on this carbon source was related to extracellular pH. An acidic pH was required for ethanol utilization; best yields were obtained at pH 3.7. Spectroscopic analysis of the cells showed that the concentration of cytochrome c per cell was 40% higher than at pH 6.0; the concentrations of cytochrome a 606 (cytochrome c oxidase) and b 566 (cytochrome bc 1 complex) were the same. A soluble cytochrome c 550 was purified from cells grown at pH 3.7 and characterized by peptide sequencing as the 12-kDa cytochrome c 550 of the mitochondrial respiratory chain. Immunoblots of total cell proteins showed higher accumulation of cytochrome c 550 at pH 3.7 than at pH 6.0. RNA blot analysis gave clear evidence of the abundance of c 550 transcript in cells grown at pH 3.7. The amount of mitochondrial proteins obtained from cells grown at pH 3.7 was twofold higher than that of cells grown at pH 6.0. Mitochondria isolated from both cell types readily oxidized succinate, malate or ethanol. The rates of oxygen uptake were 20±25% higher in mitochondria from cells grown at pH 3.7. Cyanide and antimycin A inhibited respiration with succinate up to 95% in both types of mitochondria. The participation of cytochrome c 550 in mitochondrial electron transport from succinate to oxygen was shown by spectral measurements.
Journal of Biological Chemistry, 2001
The mitochondrial genomes of Chlamydomonad algae lack the cox2 gene that encodes the essential subunit COX II of cytochrome c oxidase. COX II is normally a single polypeptide encoded by a single mitochondrial gene. In this work we cloned two nuclear genes encoding COX II from both Chlamydomonas reinhardtii and Polytomella sp. The cox2a gene encodes a protein, COX IIA, corresponding to the N-terminal portion of subunit II of cytochrome c oxidase, and the cox2b gene encodes COX IIB, corresponding to the C-terminal region. The cox2a and cox2b genes are located in the nucleus and are independently transcribed into mRNAs that are translated into separate polypeptides. These two proteins assemble with other cytochrome c oxidase subunits in the inner mitochondrial membrane to form the mature multi-subunit complex. We propose that during the evolution of the Chlorophyte algae, the cox2 gene was divided into two mitochondrial genes that were subsequently transferred to the nucleus. This event was evolutionarily distinct from the transfer of an intact cox2 gene to the nucleus in some members the Leguminosae plant family.