Structure, organization and expression of the genes encoding mitochondrial cytochrome c(1) and the Rieske iron-sulfur protein in Chlamydomonas reinhardtii.

The sequence and organization of the Chlamydomonas reinhardtii genes encoding cytochrome c(1) ( Cyc1) and the Rieske-type iron-sulfur protein ( Isp), two key nucleus-encoded subunits of the mitochondrial cytochrome bc(1) complex, are presented. Southern hybridization analysis indicates that both Cyc1 and Isp are present as single-copy genes in C. reinhardtii. The Cyc1 gene spans 6404 bp and contains six introns, ranging from 178 to 1134 bp in size. The Isp gene spans 1238 bp and contains four smaller introns, ranging in length from 83 to 167 bp. In both genes, the intron/exon junctions follow the GT/AG rule. Internal conserved sequences were identified in only some of the introns in the Cyc1 gene. The levels of expression of Isp and Cyc1 genes are comparable in wild-type C. reinhardtii cells and in a mutant strain carrying a deletion in the mitochondrial gene for cytochrome b (dum-1). Nevertheless, no accumulation of the nucleus-encoded cytochrome c(1) or of core proteins I and II was observed in the membranes of the respiratory mutant. These data show that, in the green alga C. reinhardtii, the subunits of the cytochrome bc(1) complex fail to assemble properly in the absence of cytochrome b.

Cycloheximide resistance conferred by novel mutations in ribosomal protein L41 of Chlamydomonas reinhardtii.

Although most eukaryotic cells are sensitive to the 80S ribosome inhibitor cycloheximide (CYH), naturally occurring CYH resistance is widespread amongst yeast species. The primary determinant of resistance appears to be a single residue within ribosomal protein L41; resistance is acquired by the substitution of a conserved proline (P56) by a glutamate residue. We have isolated the L41 gene (RPL41) from the green alga Chlamydomonas reinhardtii, and investigated the molecular basis of CYH resistance in various mutant strains. In both the wild-type strain and the mutant act-1, a proline is found at the key position in L41. However, analysis of six independently isolated act-2 mutants reveals that all have point mutations that replace the proline with either leucine or serine. Of the two changes, the leucine mutation confers significantly higher levels of CYH resistance. This work identifies the ACT-2 locus as RPL41 and provides a possible dominant marker for nuclear transformation of C. reinhardtii.

Polytomella spp. growth on ethanol. Extracellular pH affects the accumulation of mitochondrial cytochrome c550.

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 a606 (cytochrome c oxidase) and b566 (cytochrome bc1 complex) were the same. A soluble cytochrome c550 was purified from cells grown at pH 3.7 and characterized by peptide sequencing as the 12-kDa cytochrome c550 of the mitochondrial respiratory chain. Immunoblots of total cell proteins showed higher accumulation of cytochrome c550 at pH 3.7 than at pH 6.0. RNA blot analysis gave clear evidence of the abundance of c550 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 c550 in mitochondrial electron transport from succinate to oxygen was shown by spectral measurements.

Two unusual amino acid substitutions in cytochrome b of the colorless alga Polytomella spp.: correlation with the atypical spectral properties of the bH heme.

The dithionite-reduced spectra of the purified bc1 complexes from the colorless alga Polytomella spp. and the closely related green alga Chlamydomonas reinhardtii were compared. The spectrum of the bc1 complex from C. reinhardtii showed a profile similar to those of the bc1 complexes from other species. In contrast, the bc1 complex from Polytomella spp. exhibits a double-peak spectrum in the alpha-band region, where the absorption bands of cytochrome c1 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 Tyr114 and Ser36 are in close proximity to the high-potential bH heme. The above data suggest that the polar residues Y114 and S36, each one by itself or in combination, may interact with heme bH of Polytomella spp. and, thus, may be responsible for the unique spectroscopic characteristics of cytochrome b.

Characterization of the alpha and beta-subunits of the F0F1-ATPase from the alga Polytomella spp., a colorless relative of Chlamydomonas reinhardtii.

The isolation and partial characterization of the oligomycin-sensitive F0F1-ATP synthase/ATPase from the colorless alga Polytomella spp. is described. Purification was performed by solubilization with dodecyl-beta-D-maltoside followed by Sepharose Hexyl ammonium chromatography, a matrix that interacts with the F1 sector of mitochondrial ATPases. The alpha-subunit, which migrates on SDS-polyacrylamide gels with an apparent molecular mass of 55 kDa, was identified by the N-terminal sequencing of 47 residues. This subunit exhibited a short extension at its N-terminus highly similar to the one described for the unicellular alga Chlamydomonas reinhardtii (Nurani, G. and Franzén L.-G. (1996) Plant Mol. Biol. 31, 1105-1116). In whole mitochondria, the alpha-subunit was susceptible to limited proteolytic digestion induced by heat. An endogenous protease removed the first 22 residues of the mature alpha-subunit. Subunit beta was also identified by N-terminal sequencing of 31 residues. This subunit of 63 kDa exhibited a higher apparent molecular mass than alpha, as judged by its mobility on denaturing polyacrylamide gel electrophoresis. This beta-subunit is 7-8 kDa larger than the beta-subunits of other mitochondrial ATPases. It is suggested that the beta-subunit from Polytomella spp. may have a C-terminal extension similar to that described for the green alga C. reinhardtii (Franzén, L.-G. and Falk, G.(1992) Plant Mol. Biol. 19, 771-780). In addition, it was found that the C-terminal extension of the beta-subunit of C. reinhardtii showed homology with the endogenous ATPase inhibitors from various sources and with the epsilon-subunit from the F0F1-ATP synthase from Escherichia coli, which is considered to be a functional homolog of the inhibitor proteins. The data reported here provide the first biochemical evidence for a close relationship between the colorless alga Polytomella spp. and its photosynthetic counterpart C. reinhardtii. It is also suggested that the C-terminal extensions of the beta-subunits of the ATP synthases from these algae, may play a regulatory role in these enzymes.

Identification, cDNA sequence and deduced amino acid sequence of the mitochondrial Rieske iron-sulfur protein from the green alga Chlamydomonas reinhardtii. Implications for protein targeting and subunit interaction.

Specific oligonucleotide probes were used to isolate a cDNA clone for the mitochondrial Rieske iron-sulfur protein of the green alga Chlamydomonas reinhardtii. The protein is synthesized as a longer precursor with a cleavable N-terminal presequence of 54 amino acids but without a C-terminal extension. Comparison of the predicted secondary structure of this N-terminal sequence with that of the targeting signal of the chloroplast Rieske protein from C. reinhardtii [de Vitry (1994) J. Biol. Chem. 269, 7603-7609] indicates that, although they both have the potential to form amphiphilic alpha helices, the mito-chondrial presequence may form a more hydrophobic helix that could penetrate deeper into the membrane. The N-terminal part of the mature mitochondrial Rieske protein is characterized by a long, strongly hydrophilic N-terminal domain and by a positive charge in the middle of the hydrophobic stretch that is presumed to interact with the bc1 complex. Thus, the protein from C. reinhardtii differs from the Rieske proteins from mammals or fungi.

Etiolated cells of Chlamydomonas reinhardtii: choice material for characterization of mitochondrial membrane polypeptides.

We have investigated a light-conditional mutant of Chlamydomonas reinhardtii (J12) that is unable to synthesize chlorophyll in the dark with the aim of characterizing the mitochondrial membrane polypeptides of this alga. A crude membrane fraction derived from etiolated cells was analyzed by gel electrophoresis, immunoblot analysis, and pulse-labeling in the presence of specific protein synthesis inhibitors. This fraction contained both mitochondrial and etioplast membranes, and the latter contained appreciable amounts of subunits of the cytochrome b6f complex. The mitochondria-encoded subunit 1 of cytochrome-c oxidase called COX1 was identified, and its synthesis was detected in this membrane fraction. The redox-difference spectra of mitochondrial cytochromes were studied in whole cells and membrane fractions, in both respiratory-competent and -deficient strains. Mitochondrial membranes could be further purified after sucrose gradient centrifugation. The use of etiolated cells and their membrane extracts, in association with appropriate methodologies, opens ways to study the molecular genetics of mitochondria in C. reinhardtii and allows us to address the question of the cooperation established between the three genetic compartments of a plant cell.

Identification of mitochondrial respiratory proteins from the green alga Chlamydomonas reinhardtii.

Membrane preparations from the green alga Chlamydomonas reinhardtii contain both thylakoid and mitochondrial membranes [1]. These preparations have been intensely used to study the structure, function and biogenesis of protein complexes involved in the photosynthetic pathway. We show here that these preparations are also suitable for studying protein complexes of the mitochondrial respiratory chain of the alga. The respiratory complexes, fractionated on a sucrose gradient in the presence of Triton X-100, were identified by their catalytic properties and their polypeptide content. From the bottom to the top of the sucrose gradient, we identified the NADH: ubiquinone oxidoreductase (complex I), the mitochondrial ATP synthase (F0F1-ATPase), the cytochrome bc1 complex and the cytochrome c oxidase. At the top of the gradient, another enzyme was detected which displayed an NADH: menaquinone oxidoreductase activity.

Identification of mitochondrial proteins in membrane preparations from Chlamydomonas reinhardtii.

Preparations enriched in Chlamydomonas reinhardtii thylakoids have proven useful in the study of photosynthesis. Many of their polypeptides however remain unidentified. We report here on three of those, h1 (34 kDa), h2 (11 kDa), and P3 (63 kDa). h1, h2, and P3 are present in all tested mutants of C. reinhardtii lacking either one or several of the photosynthetic chain complexes or depleted in thylakoid membranes. h2 is an ascorbate-reducible, soluble c550-type cytochrome encoded in the nucleus. It cross-reacts immunologically with mitochondrial cytochromes c from various sources and contains a hexapeptide encoded in C. reinhardtii cytochrome c cDNA. P3, a nuclear-encoded peripheral protein, cross-reacts with various ATP synthase beta subunits. Its N-terminal sequence is encoded in C. reinhardtii mitochondrial beta subunit cDNA. h1 behaves as an integral hemoprotein; it is absent in a mitochondrial mutant that carries a deletion in apocytochrome b gene. We conclude that C. reinhardtii mitochondrial membranes copurify with thylakoid membranes. h1 is part of the cytochrome bc1 complex, h2 is cytochrome c, and P3 is the beta subunit of mitochondrial ATP synthase.