Structure of the bc1 Complex from Seculamonas ecuadoriensis a Jakobid Flagellate with an Ancestral Mitochondrial Genome
* Institut für Angewandte Genetik, Universität Hannover, Hannover, Germany3r, http://www.100md.com
Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany3r, http://www.100md.com
Canadian Institute for Advanced Research, Département de Biochimie, Université de Montréal, Montréal, Québec, Canada3r, http://www.100md.com
Abstract3r, http://www.100md.com
In eubacteria, the respiratory bc1 complex (complex III) consists of three or four different subunits, whereas that of mitochondria, which have descended from an {alpha} -proteobacterial endosymbiont, contains about seven additional subunits. To understand better how mitochondrial protein complexes evolved from their simpler bacterial predecessors, we purified complex III of Seculamonas ecuadoriensis, a member of the jakobid protists, which possess the most bacteria-like mitochondrial genomes known. The S. ecuadoriensis complex III has an apparent molecular mass of 460 kDa and exhibits antimycin-sensitive quinol:cytochrome c oxidoreductase activity. It is composed of at least eight subunits between 6 and 46 kDa in size, including two large "core" subunits and the three "respiratory" subunits. The molecular mass of the S. ecuadoriensis bc1 complex is slightly lower than that reported for other eukaryotes, but about 2x as large as complex III in bacteria. This indicates that the departure from the small bacteria-like complex III took place at an early stage in mitochondrial evolution, prior to the divergence of jakobids. We posit that the recruitment of additional subunits in mitochondrial respiratory complexes is a consequence of the migration of originally {alpha} -proteobacterial genes to the nucleus.
Key Words: jakobid flagellates • Seculamonas ecuadoriensis • mitochondria • bc1 complex, evolutionxed, 百拇医药
Introductionxed, 百拇医药
The bc1 complex (also termed complex III or ubiquinol:cytochrome c reductase) and the structurally and functionally similar b6f complex, are integral components of respiratory and photosynthetic electron transfer chains across all domains of life. The bc1 complex of both mitochondria and bacteria is a dimeric enzyme and is present in aerobic as well as anaerobic energy-transducing respiratory chains. It catalyzes reduction of cytochrome c by oxidation of ubiquinol, with concomitant generation of a proton gradient that is utilized by the F0F1 ATP synthase to generate ATP. Electron transport is carried out by three redox center-bearing subunits: cytochromes b and c1, and the "Rieske" iron-sulfur protein . In most bacteria, the bc1 complex consists solely of these three so-called respiratory subunits and, in some cases, one additional low-molecular-mass subunit, which is noncatalytic .
The evolutionary origin of mitochondria has been traced back to the {alpha} -subdivision of Proteobacteria . Testifying to this ancestry are, among other features, three subunits of the mitochondrial bc1 complex that are clearly homologs of the {alpha} -proteobacterial respiratory subunits (for a review, see ). However, complex III from very different eukaryotes, i.e., potato, yeast, and bovine, contains seven additional subunits: two relatively large subunits designated "core" proteins, and five small polypeptides. The core proteins as well as the small subunits do not carry redox centers and are not directly involved in electron transfer . In bovine, and probably other animals as well, the presequence of the "Rieske" iron-sulfur protein is retained in the bc1 complex after proteolytic cleavage of the precursor protein, constituting an 11th subunit. To date, the bc1 complex of only three protists has been studied in detail, Euglena gracilis, Crithidia fasciculatum, and Leishmania tarentolae, which all belong to the Euglenozoa lineage. The subunit composition of the bc1 complexes from these organisms is much the same as in fungal, plant, and animal species .
For a long time, the functions of the seven additional subunits of mitochondrial complex III were unknown. Only the core subunits of the plant mitochondrial bc1 complex were found to possess processing peptidase activity . In contrast, the core subunits of animals and yeast are proteolytically inactive, and the mitochondrial processing peptidase (MPP) of these organisms is a soluble enzyme localized in the mitochondrial matrix. The core proteins of the bc1 complex and MPP exhibit sequence similarity and share typical features with metalloendoproteinases of the pitrilysin family, indicating a common phylogenetic origin . Gene deletions and complementation experiments suggest that the core subunits of yeast are involved in the assembly of the bc1 complex . The function and origin of the five small subunits of the bc1 complex remain unknown.?}, 百拇医药
To understand better how the mitochondrial bc1 complex evolved from its much simpler {alpha} -proteobacterial predecessor, and how it diversified in the various eukaryotic lineages, we characterized the bc1 complex from Seculamonas ecuadoriensis. This protist belongs to the jakobids, a group of unicellular, heterotrophic flagellates that comprise the better-known species Reclinomonas americana . Jakobids are assumed to have a very basal position in molecular phylogenies and include five aerobic genera: Seculamonas, Reclinomonas, Histiona, Jakoba, and Malawimonas. The four first genera share more morphological and ultrastructural features with one another than with Malawimonas and are therefore referred to as "core" jakobids ( O'Kelly, unpublished data). Up to now, six mitochondrial genomes of jakobids were completely sequenced, among these five core jakobids (R. americana NZ, R. americana 284, S. ecuadoriensis, Jakoba libera, and J. bahamensis) and the non-core jakobid, Malawimonas jakobiformis ). Core jakobid mitochondrial genomes display an astonishing number of bacterial features more closely resembling the genome of the ancestral -proteobacterial symbiont than any other mtDNA investigated today . Some of the 18 or so extra genes present in most jakobid mitochondrial genomes have apparently been lost during evolution in all other eukaryotic lineages and functionally replaced by genes of other origin (e.g., 2ß,ß' RNA polymerase, which has been replaced by a T3/T7-type enzyme;. Most of the extra genes, however, are believed to have migrated to the nucleus in more derived eukaryotes. Among these are mostly genes coding for ribosomal proteins, but respiratory chain components migrate as well. One well-documented example is succinate-ubiquinone oxidoreductase (respiratory complex II), whose subunits 2, 3, and 4 (Sdh2 to Sdh4) are mitochondrially encoded in core jakobids (and also some plants and some protists like Chondrus crispus, Porphyra purpurea, Rhodomonas salina). Nucleus-encoded genes specifying Sdh2 have been identified in a number of fungi and animals. Phylogenetic analysis of bacterial, mitochondrial, and nuclear DNA-encoded Sdh2 sequence strongly suggest that the nuclear sdh2 genes originated by transfer from a mitochondrial genome in which it was originally resident .
As former studies on the mitochondrial respiratory chain, and the bc1 complex in particular, have been conducted exclusively with derived eukaryotic taxa, jakobids are the organisms of choice to address the above evolutionary questions. Instead of R. americana, whose mtDNA sequence has been published previously , we have chosen the sister taxon S. ecuadoriensis for the protein-chemical experiments described here. The latter species is better amenable to biochemical studies that require a substantial amount of cell material, while it displays the same ancestral features and an almost identical mitochondrial gene set as R. americana (Burger and Lang, unpublished data).4p, 百拇医药
Materials and Methods4p, 百拇医药
Cell Culture4p, 百拇医药
Seculamonas ecuadoriensis ATCC 50688 was grown in 2.5-liter culture flasks with gentle shaking at 24°C in WCL medium . The protists were fed with live Enterobacter aerogenes (ATCC 13048). A 600 ml culture yielded about 0.5 g of S. ecuadoriensis cells after 8 days.
Isolation of Membranes from S. ecuadoriensis^|, 百拇医药
The following steps were carried out at 4°C, unless specified otherwise. For isolation of membranes, cells were pelleted by centrifugation, suspended in 0.2 M Na-phosphate buffer, pH 7.2, and disrupted by sonication. Cellular debris was removed by centrifugation at 12,000xg for 8 min. Membranes were then separated from the supernatant by centrifugation through sucrose step gradients (60%, 32%, 15% sucrose in 1 mM EDTA, 1 mM PMSF, and 10 mM MOPS/KOH, pH 7.2) at 92,000xg and 2°C for 1 h. The membrane fraction from the 15%/32% interphase was collected and diluted with 1 mM EDTA, 1 mM PMSF, and 10 mM MOPS/KOH, pH 7.2. Membranes were pelleted by centrifugation at 100,000xg for 90 min. The enrichment of mitochondrial membranes was monitored by cytochrome c oxidase activity measurements according to .^|, 百拇医药
Isolation of Mitochondria from Solanum tuberosum^|, 百拇医药
Mitochondria from potato tubers were isolated as described by . The organelles were suspended in 0.4 M mannitol, 0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, and 10 mM KH2PO4, pH 7.2, at a concentration of 10 mg of mitochondrial protein per milliliter.
Cytochrome c Affinity Chromatographyi, 百拇医药
In preparation for affinity chromatography, 1.5 g membranes from S. ecuadoriensis were suspended in 2 ml ice-cold water and solubilized by slow addition of 10% Triton X100, to a final concentration of 3.3%. To remove membrane fragments and lipids, the suspension was centrifuged for 10 min at 60,000xg. A detailed protocol for the subsequent cytochrome c affinity chromatography is given in . Proteins bound to the cytochrome c column were eluted using a Tris-acetate gradient (20–200 mM Tris-acetate [pH 7.0]/0.04% Triton, 5% sucrose, 0.2 mM phenylmethylsulfonyl fluoride). Fractions containing subunits of the bc1 complex were identified by immunoblotting, pooled, and subsequently concentrated by ultrafiltration through filters with an exclusion limit of 300 kDa. Finally, the concentrate was analyzed by two-dimensional Blue-Native gel electrophoresis as described in the following section.i, 百拇医药
Separation of Mitochondrial Protein Complexes by Blue-Native and Tricine-SDS-PAGE
To determine the apparent molecular mass of mitochondrial protein complexes from S. ecuadoriensis, Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) was carried out . Protein complexes of potato mitochondria were loaded onto the Blue-Native gels as a size standard. The BN gels consisted of a separation gel (4.95% to 12.6% acrylamide) and a stacking gel (4% acrylamide). Sample preparation and electrophoresis was carried out as described by . To separate the subunits of the protein complexes resolved by BN-PAGE, entire stripes of the BN gel were transferred horizontally on Tricine-SDS-PAGE gels. A protocol for this second-dimension Tricine-SDS gel is given in . Tricine-SDS gels were either stained with Coomassie blue or silver nitrate or blotted onto filter membranes for immunological identification of proteins.60z?f#0, 百拇医药
Identification of Proteins by Amino Acid Sequencing and Immunostaining60z?f#0, 百拇医药
For internal sequence analysis, protein spots were cut from Tricine-SDS gels and digested with trypsin as outlined by . The resulting peptides were analyzed by Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS). For immunostaining, Tricine-SDS gels were blotted onto nitrocellulose membranes. Blots were incubated with antibodies directed against the core II protein from N. crassa (dilution 1:1000). Visualization of immunopositive bands was performed using the Vectastain ABC-Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions.
Quinol:Ferricytochrome c Activity Measurementstr, 百拇医药
The quinol:ferricytochrome c activity assay was essentially carried out as described by . Cytochrome c reduction was monitored at 24°C in a dual-wavelength photometer at 550 nm and 580 nm, using the extinction coefficient 20 mM-1 cm-1. The test solution contained 50 mM LiMOPS, pH 6.8, 100 mM K2SO4, 40 µM cytochrome c from horse heart (Sigma, type III), 40 µM KCN, and 100 µM decylquinone (kindly provided by Dr. U. Schulte, Düsseldorf University, Germany). Antimycin, an inhibitor of bc1 activity, was added to a final concentration of 2 µM. The turnover number is determined by extrapolating the rates of enzymatic reaction corrected for nonenzymatic rates to the infinite quinol concentration.str, 百拇医药
Sequence Similarity Searchesstr, 百拇医药
The peptide sequences determined in this study were searched against the following sequence repositories: the local jakobid database of the Organelle Genome Megasequencing Unit (OGMP), the nonredundant database (nrdb) of the National Centre for Biotechnology Information (NCBI); MITOP of the Munich Information Center for Protein Sequences (MIPS), a database for mitochondria-related genes, proteins, and diseases ; dbEST, the division of GenBank that contains sequence data and other information on "single pass" cDNA sequences and Expressed Sequence Tags (ESTs) from a number of organisms ; the motif databases Pfam and Prosite ; as well as the collection of HMM protein families . Downloaded and formatted for local searches were MITOP, dbEST, the Pfam families of Rieske (Fe-S) proteins, 14.5 kDa and 9.5 kDa subunits of complex III (UCR_14.5kD, UCR_9.5kD), and mitochondrial processing peptidases.
As search tools, we used FASTA, BLAST, PSI-BLAST and "Search for short sequences," available on the NCBI Blast homepage . A very lenient e-value (1000) was used to account for the short length of the input peptide sequences (<30 residues). The word size of 2 and PAM 30 matrix were used as advised by the BLAST "Search for short sequences." The search against the HMM protein families was performed using HMMER (online version) (). We also employed the online version of Mascot, which is a powerful search engine which uses mass spectrometry data to identify proteins from primary sequence databases .g, 百拇医药
Resultsg, 百拇医药
Purification of the bc1 Complex of S. ecuadoriensisg, 百拇医药
The bc1 complex of S. ecuadoriensis was purified using cytochrome c affinity chromatography as published for N. crassa and plants . According to the original protocols, the starting material for affinity chromatography should be isolated mitochondria. Owing to the slow growth of jakobid cultures, however, the preparation of pure mitochondria from quite limiting amounts of S. ecuadoriensis cells proved difficult. Therefore fractions of enriched mitochondrial membranes were generated from S. ecuadoriensis as described in the Experimental Procedures section. The fractions were fivefold to eightfold enriched in mitochondrial membrane proteins as monitored by cytochrome c oxidase measurements. The main purification step for the S. ecuadoriensis bc1 complex, cytochrome c affinity chromatography, takes advantage of the specific interaction of the cytochrome c1 subunit of the bc1 complex and cytochrome c, the natural binding partner during respiratory electron transport. Proteins bound to the cytochrome c column were eluted by a salt gradient of 20–200 mM Tris-acetate, and the fractions obtained were analyzed by Tricine-SDS-PAGE and by immunoblotting. A 46 kDa band of fractions 19–23 eluted by 90–100 mM Tris-acetate strongly cross reacted with an antiserum directed against the core II protein from N. crassa . These peak fractions were pooled and concentrated by ultrafiltration using filters with an exclusion limit of 300 kDa. Finally the concentrate was analyzed by two-dimensional BN-PAGE/Tricine-SDS-PAGE. One protein complex was visible on the one-dimensional gels that could be resolved into seven protein bands of 46 kDa, 33 kDa, 29 kDa, 28 kDa, 14.5 kDa, 10 kDa, and 6 kDa upon separation on a second gel dimension .
fig.ommittedn, 百拇医药
FIG. 1. Purification of the bc1 complex from Seculamonas ecuadoriensis by cytochrome c affinity chromatography. Fractions eluted from the affinity column were separated by SDS-PAGE, blotted, and probed with an antibody directed against the core II protein from N. crassa. The numbers of the fractions are indicated above the gel, and the molecular masses of standard proteins are shown to the left of the gel (in kDa). A graphical illustration of the Tris-acetate gradient (20–200 mM) used for elution of proteins bound to the cytochrome c column is given below the geln, 百拇医药
fig.ommittedn, 百拇医药
FIG. 2. Characterization of the purified bc1 complex from S. ecuadoriensis by BN-PAGE/Tricine-SDS-PAGE. Sizes of standard proteins are given on the right; protein spots of the bc1 complex are marked with arrows. The spot at 46 kDa cross reacts with an antiserum directed against the core II protein from N. crassa (Inset: Western blot)n, 百拇医药
That the isolated complex is indeed complex III of the respiratory chain was confirmed by two experiments. First, the native complex exhibits quinol:cytochrome c oxidoreductase activity (turnover number: 9.5 s-1), which is antimycin-sensitive. It should be noted that the electron transfer activity is relatively low compared to preparations from other eukaryotes , which might be due to the use of heterologous cytochrome c (from horse heart) as electron acceptor. Second, in an immunoblotting experiment, the 46 kDa spot on the 2D gels was shown to cross-react with the antiserum directed against the core II subunit of the bc1 complex from N. crassa (, inset).
Analysis of Protein Complexes from S. ecuadoriensis by BN PAGE/Tricine-SDS-PAGE and Determination of the Molecular Mass of the bc1 Complex\7[, 百拇医药
To obtain further information on size and subunit composition of the bc1 complex from S. ecuadoriensis, protein complexes from fractions enriched in mitochondrial membranes were analyzed directly by Blue-Native and Tricine-SDS-PAGE. BN-PAGE is a very reliable method for molecular mass determination of protein complexes . Apparent molecular masses were estimated by co-electrophoresis of protein complexes from S. ecuadoriensis and mitochondrial protein complexes from potato, which served as size reference . The membrane fraction of S. ecuadoriensis contains five protein complexes in the size range of 100 to 700 kDa and additional minor bands. Analysis of the separated protein complexes on a second gel dimension by Tricine-SDS-PAGE allowed to identify the protein complexes from potato on the basis of subunit compositions . One of the separated protein complexes of S. ecuadoriensis comprises an identical subunit composition like the bc1 complex purified by affinity chromatography . Direct comparison of the protein complexes of potato mitochondria and S. ecuadoriensis mitochondrial fractions on Blue-Native gels revealed an apparent molecular mass of 460 kDa for the bc1 complex from S. ecuadoriensis . A further protein complex of S. ecuadoriensis runs at 550 kDa on Blue-Native gels and can be separated into 10 subunits upon analysis on a second gel dimension. The subunit composition of this 550 kDa complex resembles the one reported for mitochondrial ATP synthase complexes from other organisms . The identity of the dominant protein complex at about 150 kDa could not be determined on the basis of subunit composition.
fig.ommitted-jo, 百拇医药
FIG. 3. Determination of the molecular mass of the bc1 complex from S. ecuadoriensis by Blue-Native gel electrophoresis (BN-PAGE). A, One-dimensional resolution of protein complexes from potato (P) and S. ecuadoriensis (S) by BN-PAGE. The molecular masses of the mitochondrial protein complexes from potato are taken from (the band at 480 kDa corresponds to the potato bc1 complex). The bc1 complex from S. ecuadoriensis runs at about 460 kDa. B, Two-dimensional resolution of protein complexes from S. ecuadoriensis and mitochondrial protein complexes from potato by BN-PAGE/Tricine-SDS-PAGE. Sizes of standard proteins are given on the right. Protein complexes from potato were identified by their characteristic subunit composition ; protein complexes from S. ecuadoriensis were identified by sequence analysis of individual subunits by mass spectrometry (analyzed subunits are indicated by arrows and numbered according to ). I, NADH dehydrogenase; F0F1, F0F1 ATP synthase; bc1, bc1 complex; F1, F1 part of the F0F1 ATP synthase; FD, formate dehydrogenase; question marks indicate protein complexes that could not unambiguously be identified
Identification of Individual Subunits of Protein Complexes Separated by BN-PAGE/Tricine-SDS-PAGEwi, http://www.100md.com
To confirm the identity of the protein complexes and to obtain data on individual subunits, selected protein spots were subjected to peptide sequencing by Electrospray Ionization/Tandem Mass Spectrometry (ESI-MS/MS). We determined 1 to 3 peptide sequences of 7 different proteins , which form part of three different protein complexes and which are indicated and numbered on the gel in . The sequence of peptide 1 of the 46 kDa protein forming part of the bc1 complex exhibits significant similarity to a conserved stretch of the ß MPP/core I subunit of the bc1 complex from other eukaryotes. Notably, this peptide covers one of the few regions that distinguish {alpha} and ß MPP paralogs . Peptide 2 of the same protein spot exhibits some weak similarities to the core II proteins from N. crassa. As shown above, this protein spot also cross reacts with an antiserum directed against the core II protein from N. crassa . These data strongly suggest that the bc1 complex from S. ecuadoriensis comprises two core proteins with identical apparent molecular masses of 46 kDa.
fig.ommitted}u8sch+, http://www.100md.com
Table 1 Peptide Sequences of Subunits of Protein Complexes from Seculamonas ecuadoriensis}u8sch+, http://www.100md.com
fig.ommitted}u8sch+, http://www.100md.com
FIG. 4. Identification of the core I/ß MPP subunit of S. ecuadoriensis by sequence comparison with core I and ß MPP proteins of other organisms. Residues identical in at least six organisms are underlayed in black; other residues conserved in at least 4 organisms are underlayed in gray. Positions of the sequence stretches and accession numbers of the proteins are given on the right}u8sch+, http://www.100md.com
Peptides 1 and 3 of the 29 kDa protein of the S. ecuadoriensis bc1 complex exhibit low sequence identity to cytochrome c1 . However, the stretches of similarity represent unconserved regions of the protein and contain numerous insertions/deletions across the taxa, and therefore they did not withstand more rigorous statistical tests.}u8sch+, http://www.100md.com
Also, neither peptides of the 14.5 kDa subunit nor those of the 10 kDa subunit of the S. ecuadoriensis bc1 complex displayed significant sequence similarity to known proteins. Sequence conservation of the small subunits of the mitochondrial bc1 complex is notoriously low . Much longer peptide sequences would be required than those determined in this study to identify phylogenetically distant, and, even more so, weakly conserved homologs.
The 48 and 30 kDa proteins of the F0F1 ATP synthase complex were unambiguously identified as the ß and the subunits of this protein complex. Both peptide sequences obtained for 48 kDa protein exhibit high sequence identity to an internal sequence stretch of ß subunits of the F0F1 ATP synthase complex from other organisms, and the two peptide sequences of the 30 kDa protein correspond exactly to amino acid stretches of the mitochondrial encoded subunit of the F1 part of S. ecuadoriensis.(^6, 百拇医药
The identity of the dominant protein complex at 150 kDa could not be resolved on the basis of the peptide sequence of a 26 kDa subunit.(^6, 百拇医药
Discussion(^6, 百拇医药
This article reports the identification of two protein complexes of the respiratory chain from the jakobid flagellate S. ecuadoriensis, the F0F1 ATP synthase and the bc1 complex. The experiments described here focus on the purification and characterization of the bc1 complex. To our knowledge this is the first report on a biochemical preparation of an enzyme from jakobid flagellates. The S. ecuadoriensis bc1 complex was purified on the basis of its affinity to the natural binding partner during respiratory electron transport, cytochrome c. The purified bc1 complex retains both quinol:cytochrome c oxidoreductase activity and antimycin sensivity.
The molecular mass of the bc1 complex from S. ecuadoriensis lies at 460 kDa as determined by BN-PAGE. Under denaturing electrophoresis conditions the complex was resolved into seven protein bands with apparent molecular masses of 46 kDa, 33 kDa, 29 kDa, 28 kDa, 14.5 kDa, 10 kDa, and 6 kDa . Three lines of evidence strongly suggest that the largest protein spot encompasses two proteins, the core I and core II subunits. First, the 46 kDa spot cross reacts with an antiserum directed against the core II protein of the bc1 complex from N. crassa, while a peptide derived from this spot also exhibits significant sequence identity to the core I/ß-MPP subunit from different eukaryotes. Second, the core subunits of all mitochondrial bc1 complexes characterized to date closely comigrate in gel electrophoresis in the size range of 45 to 55 kDa. Third, the apparent molecular mass of the S. ecuadoriensis bc1 complex (460 kDa) can only be explained by assuming a dimeric holoenzyme that includes two core subunits per monomer as further discussed below.
In all bacteria and mitochondria characterized up to now, quinol:cytochrome c oxidoreductase activity is based on electron transfer reactions between the prosthetic groups of cytochrome b, cytochrome c1, and the iron-sulfur protein. Given that the purified bc1 complex of S. ecuadoriensis displays electron transport activity, it must include these three subunits. The molecular masses of the respiratory subunits are quite conserved in potato, bovine, and yeast, with 42–44 kDa for cytochrome b, 27–28 kDa for cytochrome c1, and 20–23 kDa for the iron-sulfur protein . A considerable degree of conservation across three different eukaryotic phyla allowed us to assign the following three protein species of the purified bc1 complex of S. ecuadoriensis to respiratory subunits. First, the 33 kDa protein of S. ecuadoriensis is most likely cytochrome b. This is consistent with the fact that cytochrome b, because of its highly hydrophobic properties, typically displays a migration behavior that makes its molecular mass appear ~
25% smaller than it really is. Second, the 29 kDa protein of S. ecuadoriensis is probably cytochrome c1 and is thus only slightly larger than the proteins of its well-characterized counterparts. Finally, the 28 kDa subunit of S. ecuadoriensis represents, very likely, the iron-sulfur protein. Although the S. ecuadoriensis protein is approximately 30% larger than its homologs of the model organisms, such a size deviation is not without precedent. A comparatively large iron-sulfur subunit was also reported for the bc1 complex from L. tarentolae . summarizes the demonstrated and inferred subunit assignments of the bc1 complex from S. ecuadoriensis.:jqz9p, 百拇医药
fig.ommitted:jqz9p, 百拇医药
FIG. 5. Identities of the subunits of the bc1 from S. ecuadoriensis. A scheme of the gel is given on the right, and the sizes of standard proteins are given in the middle. Cyt, cytochrome; FeS, iron-sulfur protein; question marks indicate subunits of unknown identity
As already mentioned, bacterial and mitochondrial bc1 complexes alike are dimers. Assuming the presence of two core proteins, the molecular masses of the eight separated subunits of the S. ecuadoriensis bc1 complex sums up to 423 kDa for the dimeric complex, a value that is somewhat smaller than the experimentally determined molecular mass of the complex (460 kDa). The difference of 27 kDa could be due to the presence of one or two further low molecular mass subunits that may not have been detected in our experiments. These small proteins are difficult to spot because they migrate closely together and are poorly stainable./%8ri], 百拇医药
While the bacterial bc1 complex comprises three or four subunits at most, with a molecular mass of the dimeric complex of ~/%8ri], 百拇医药
220 kDa , the mitochondrial counterpart is at least twice as large. At present, complex III has been characterized from highly diverse eukaryotes, including plants, fungi, animals, and euglenoid and kinetoplastid protists . All these mitochondrial bc1 complexes have an apparent molecular mass of 470–495 kDa and consist of three respiratory subunits, two large core proteins and, at least in higher eukaryotes (but probably in all eukaryotic taxa investigated up to now), five small subunits. As we show here, the bc1 complex from the jakobid protistan S. ecuadoriensis has only a slightly smaller molecular mass (460 kDa) than that from fungi, mammals, and plants (470–495 kDa) and includes at least eight different subunits. Because jakobids are believed to be a primitive eukaryotic lineage, this finding was unexpected.
The view that jakobids are minimally derived eukaryotes is based on their ultrastructural similarities with the retortamonads, an amitochondriate group considered to have diverged close to the eukaryotic origin . S. ecuadoriensis is a typical member of jakobids and of the core jakobids in particular, as first established by analysis of the basal body ultrastructure and other cellular characters . Furthermore, phylogenetic analyses using mitochondrion-encoded protein genes clearly affiliate S. ecuadoriensis with R. americana (not shown). In global eukaryotic trees, however, available multiple mitochondrial protein data fail to place the jakobids relative to the other eukaryotic lineages with confidence, which is most likely a result of the low sampling of jakobid taxa . Similarly uncertain topologies are obtained with single nuclear genes (- and ß-tubulin) Nevertheless, and irrespective of their exact phylogenetic position relative to the other eukaryotes, mitochondrial genomes of core jakobids, such as R. americana and S. ecuadoriensis, display an astonishing number of bacterial features, more closely resembling the genome of the ancestral -proteobacterial symbiont than any other mtDNA investigated today .
Our initial hypothesis posited that jakobid mitochondrial complexes are evolutionary intermediates between those of -proteobacteria and mitochondria from higher eukaryotes. Although the finding of much the same complex III structure in S. ecuadoriensis and plants or fungi does not corroborate this view, the particular complex investigated may not be a suitable choice for detecting a more bacteria-like structure. Indeed, considering the number of mitochondrion-encoded subunits, complex III of the jakobids is as much derived as that of all other eukaryotes studied. Among the three genes coding for respiratory subunits of {alpha} -proteobacterial origin, only the apoprotein b gene still resides in mtDNA, whereas the other two, i.e., the cytochrome c1 and iron-sulfur protein, are nucleus-encoded.z:ll;1h, 百拇医药
It is conceivable that the number of originally -proteobacterial genes that have migrated to the nucleus is correlated with the number of secondarily acquired subunits in a respiratory complex. In fact, the migration of mitochondrial genes to the nucleus may actually be the underlying cause for the recruitment of additional subunits the crucial role of which may involve mediation of protein complex assembly from components that are now synthesized in different cellular compartments and synchronization of the expression of genes now located in different genomes.
If this hypothesis is correct, one should expect a more primitive structure in those mitochondrial membrane complexes that have retained a larger number of mitochondrion-encoded subunits in jakobids than in other eukaryotes. The mitochondrial NADH dehydrogenase (complex I of the respiratory chain) and the ATP synthase are two such cases. Additional subunits of these protein complexes are encoded by mitochondrial genes in R. americana (as well as S. ecuadoriensis, unpublished data) if compared to the mitochondrial genomes of other eukaryotes (). Like the situation in complex III, all mitochondrial ATP synthases characterized in eukaryotes other than jakobids have approximately five more subunits over and above the eight that are typically present in the bacterial enzyme (). To test the hypothesis that subunit recruitment is the consequence of gene migration from the mitochondrial genome to the nuclear genome, investigation of the complex composition of S. ecuadoriensis ATP synthase is under way.
Acknowledgements:3, http://www.100md.com
We thank Dr. U. Schulte, Düsseldorf, for instructing us on quinol:cytochrome c reductase activity measurements in his laboratory. This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and the Canadian Institute for Health Research (CIHR). G.B. is a Canadian National Associate, and B.F.L is an Imasco fellow in the program of Evolutionary Biology of the Canadian Institute of Advanced Research (CIAR), which we thank for salary and interaction support.:3, http://www.100md.com
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Accepted for publication September 24, 2002.(Stefanie Marx Maja Baumgärtner Sivakumar Kunnan Hans-Peter Braun B. Franz Lang and Gertraud Bur)
Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany3r, http://www.100md.com
Canadian Institute for Advanced Research, Département de Biochimie, Université de Montréal, Montréal, Québec, Canada3r, http://www.100md.com
Abstract3r, http://www.100md.com
In eubacteria, the respiratory bc1 complex (complex III) consists of three or four different subunits, whereas that of mitochondria, which have descended from an {alpha} -proteobacterial endosymbiont, contains about seven additional subunits. To understand better how mitochondrial protein complexes evolved from their simpler bacterial predecessors, we purified complex III of Seculamonas ecuadoriensis, a member of the jakobid protists, which possess the most bacteria-like mitochondrial genomes known. The S. ecuadoriensis complex III has an apparent molecular mass of 460 kDa and exhibits antimycin-sensitive quinol:cytochrome c oxidoreductase activity. It is composed of at least eight subunits between 6 and 46 kDa in size, including two large "core" subunits and the three "respiratory" subunits. The molecular mass of the S. ecuadoriensis bc1 complex is slightly lower than that reported for other eukaryotes, but about 2x as large as complex III in bacteria. This indicates that the departure from the small bacteria-like complex III took place at an early stage in mitochondrial evolution, prior to the divergence of jakobids. We posit that the recruitment of additional subunits in mitochondrial respiratory complexes is a consequence of the migration of originally {alpha} -proteobacterial genes to the nucleus.
Key Words: jakobid flagellates • Seculamonas ecuadoriensis • mitochondria • bc1 complex, evolutionxed, 百拇医药
Introductionxed, 百拇医药
The bc1 complex (also termed complex III or ubiquinol:cytochrome c reductase) and the structurally and functionally similar b6f complex, are integral components of respiratory and photosynthetic electron transfer chains across all domains of life. The bc1 complex of both mitochondria and bacteria is a dimeric enzyme and is present in aerobic as well as anaerobic energy-transducing respiratory chains. It catalyzes reduction of cytochrome c by oxidation of ubiquinol, with concomitant generation of a proton gradient that is utilized by the F0F1 ATP synthase to generate ATP. Electron transport is carried out by three redox center-bearing subunits: cytochromes b and c1, and the "Rieske" iron-sulfur protein . In most bacteria, the bc1 complex consists solely of these three so-called respiratory subunits and, in some cases, one additional low-molecular-mass subunit, which is noncatalytic .
The evolutionary origin of mitochondria has been traced back to the {alpha} -subdivision of Proteobacteria . Testifying to this ancestry are, among other features, three subunits of the mitochondrial bc1 complex that are clearly homologs of the {alpha} -proteobacterial respiratory subunits (for a review, see ). However, complex III from very different eukaryotes, i.e., potato, yeast, and bovine, contains seven additional subunits: two relatively large subunits designated "core" proteins, and five small polypeptides. The core proteins as well as the small subunits do not carry redox centers and are not directly involved in electron transfer . In bovine, and probably other animals as well, the presequence of the "Rieske" iron-sulfur protein is retained in the bc1 complex after proteolytic cleavage of the precursor protein, constituting an 11th subunit. To date, the bc1 complex of only three protists has been studied in detail, Euglena gracilis, Crithidia fasciculatum, and Leishmania tarentolae, which all belong to the Euglenozoa lineage. The subunit composition of the bc1 complexes from these organisms is much the same as in fungal, plant, and animal species .
For a long time, the functions of the seven additional subunits of mitochondrial complex III were unknown. Only the core subunits of the plant mitochondrial bc1 complex were found to possess processing peptidase activity . In contrast, the core subunits of animals and yeast are proteolytically inactive, and the mitochondrial processing peptidase (MPP) of these organisms is a soluble enzyme localized in the mitochondrial matrix. The core proteins of the bc1 complex and MPP exhibit sequence similarity and share typical features with metalloendoproteinases of the pitrilysin family, indicating a common phylogenetic origin . Gene deletions and complementation experiments suggest that the core subunits of yeast are involved in the assembly of the bc1 complex . The function and origin of the five small subunits of the bc1 complex remain unknown.?}, 百拇医药
To understand better how the mitochondrial bc1 complex evolved from its much simpler {alpha} -proteobacterial predecessor, and how it diversified in the various eukaryotic lineages, we characterized the bc1 complex from Seculamonas ecuadoriensis. This protist belongs to the jakobids, a group of unicellular, heterotrophic flagellates that comprise the better-known species Reclinomonas americana . Jakobids are assumed to have a very basal position in molecular phylogenies and include five aerobic genera: Seculamonas, Reclinomonas, Histiona, Jakoba, and Malawimonas. The four first genera share more morphological and ultrastructural features with one another than with Malawimonas and are therefore referred to as "core" jakobids ( O'Kelly, unpublished data). Up to now, six mitochondrial genomes of jakobids were completely sequenced, among these five core jakobids (R. americana NZ, R. americana 284, S. ecuadoriensis, Jakoba libera, and J. bahamensis) and the non-core jakobid, Malawimonas jakobiformis ). Core jakobid mitochondrial genomes display an astonishing number of bacterial features more closely resembling the genome of the ancestral -proteobacterial symbiont than any other mtDNA investigated today . Some of the 18 or so extra genes present in most jakobid mitochondrial genomes have apparently been lost during evolution in all other eukaryotic lineages and functionally replaced by genes of other origin (e.g., 2ß,ß' RNA polymerase, which has been replaced by a T3/T7-type enzyme;. Most of the extra genes, however, are believed to have migrated to the nucleus in more derived eukaryotes. Among these are mostly genes coding for ribosomal proteins, but respiratory chain components migrate as well. One well-documented example is succinate-ubiquinone oxidoreductase (respiratory complex II), whose subunits 2, 3, and 4 (Sdh2 to Sdh4) are mitochondrially encoded in core jakobids (and also some plants and some protists like Chondrus crispus, Porphyra purpurea, Rhodomonas salina). Nucleus-encoded genes specifying Sdh2 have been identified in a number of fungi and animals. Phylogenetic analysis of bacterial, mitochondrial, and nuclear DNA-encoded Sdh2 sequence strongly suggest that the nuclear sdh2 genes originated by transfer from a mitochondrial genome in which it was originally resident .
As former studies on the mitochondrial respiratory chain, and the bc1 complex in particular, have been conducted exclusively with derived eukaryotic taxa, jakobids are the organisms of choice to address the above evolutionary questions. Instead of R. americana, whose mtDNA sequence has been published previously , we have chosen the sister taxon S. ecuadoriensis for the protein-chemical experiments described here. The latter species is better amenable to biochemical studies that require a substantial amount of cell material, while it displays the same ancestral features and an almost identical mitochondrial gene set as R. americana (Burger and Lang, unpublished data).4p, 百拇医药
Materials and Methods4p, 百拇医药
Cell Culture4p, 百拇医药
Seculamonas ecuadoriensis ATCC 50688 was grown in 2.5-liter culture flasks with gentle shaking at 24°C in WCL medium . The protists were fed with live Enterobacter aerogenes (ATCC 13048). A 600 ml culture yielded about 0.5 g of S. ecuadoriensis cells after 8 days.
Isolation of Membranes from S. ecuadoriensis^|, 百拇医药
The following steps were carried out at 4°C, unless specified otherwise. For isolation of membranes, cells were pelleted by centrifugation, suspended in 0.2 M Na-phosphate buffer, pH 7.2, and disrupted by sonication. Cellular debris was removed by centrifugation at 12,000xg for 8 min. Membranes were then separated from the supernatant by centrifugation through sucrose step gradients (60%, 32%, 15% sucrose in 1 mM EDTA, 1 mM PMSF, and 10 mM MOPS/KOH, pH 7.2) at 92,000xg and 2°C for 1 h. The membrane fraction from the 15%/32% interphase was collected and diluted with 1 mM EDTA, 1 mM PMSF, and 10 mM MOPS/KOH, pH 7.2. Membranes were pelleted by centrifugation at 100,000xg for 90 min. The enrichment of mitochondrial membranes was monitored by cytochrome c oxidase activity measurements according to .^|, 百拇医药
Isolation of Mitochondria from Solanum tuberosum^|, 百拇医药
Mitochondria from potato tubers were isolated as described by . The organelles were suspended in 0.4 M mannitol, 0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, and 10 mM KH2PO4, pH 7.2, at a concentration of 10 mg of mitochondrial protein per milliliter.
Cytochrome c Affinity Chromatographyi, 百拇医药
In preparation for affinity chromatography, 1.5 g membranes from S. ecuadoriensis were suspended in 2 ml ice-cold water and solubilized by slow addition of 10% Triton X100, to a final concentration of 3.3%. To remove membrane fragments and lipids, the suspension was centrifuged for 10 min at 60,000xg. A detailed protocol for the subsequent cytochrome c affinity chromatography is given in . Proteins bound to the cytochrome c column were eluted using a Tris-acetate gradient (20–200 mM Tris-acetate [pH 7.0]/0.04% Triton, 5% sucrose, 0.2 mM phenylmethylsulfonyl fluoride). Fractions containing subunits of the bc1 complex were identified by immunoblotting, pooled, and subsequently concentrated by ultrafiltration through filters with an exclusion limit of 300 kDa. Finally, the concentrate was analyzed by two-dimensional Blue-Native gel electrophoresis as described in the following section.i, 百拇医药
Separation of Mitochondrial Protein Complexes by Blue-Native and Tricine-SDS-PAGE
To determine the apparent molecular mass of mitochondrial protein complexes from S. ecuadoriensis, Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) was carried out . Protein complexes of potato mitochondria were loaded onto the Blue-Native gels as a size standard. The BN gels consisted of a separation gel (4.95% to 12.6% acrylamide) and a stacking gel (4% acrylamide). Sample preparation and electrophoresis was carried out as described by . To separate the subunits of the protein complexes resolved by BN-PAGE, entire stripes of the BN gel were transferred horizontally on Tricine-SDS-PAGE gels. A protocol for this second-dimension Tricine-SDS gel is given in . Tricine-SDS gels were either stained with Coomassie blue or silver nitrate or blotted onto filter membranes for immunological identification of proteins.60z?f#0, 百拇医药
Identification of Proteins by Amino Acid Sequencing and Immunostaining60z?f#0, 百拇医药
For internal sequence analysis, protein spots were cut from Tricine-SDS gels and digested with trypsin as outlined by . The resulting peptides were analyzed by Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS). For immunostaining, Tricine-SDS gels were blotted onto nitrocellulose membranes. Blots were incubated with antibodies directed against the core II protein from N. crassa (dilution 1:1000). Visualization of immunopositive bands was performed using the Vectastain ABC-Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions.
Quinol:Ferricytochrome c Activity Measurementstr, 百拇医药
The quinol:ferricytochrome c activity assay was essentially carried out as described by . Cytochrome c reduction was monitored at 24°C in a dual-wavelength photometer at 550 nm and 580 nm, using the extinction coefficient 20 mM-1 cm-1. The test solution contained 50 mM LiMOPS, pH 6.8, 100 mM K2SO4, 40 µM cytochrome c from horse heart (Sigma, type III), 40 µM KCN, and 100 µM decylquinone (kindly provided by Dr. U. Schulte, Düsseldorf University, Germany). Antimycin, an inhibitor of bc1 activity, was added to a final concentration of 2 µM. The turnover number is determined by extrapolating the rates of enzymatic reaction corrected for nonenzymatic rates to the infinite quinol concentration.str, 百拇医药
Sequence Similarity Searchesstr, 百拇医药
The peptide sequences determined in this study were searched against the following sequence repositories: the local jakobid database of the Organelle Genome Megasequencing Unit (OGMP), the nonredundant database (nrdb) of the National Centre for Biotechnology Information (NCBI); MITOP of the Munich Information Center for Protein Sequences (MIPS), a database for mitochondria-related genes, proteins, and diseases ; dbEST, the division of GenBank that contains sequence data and other information on "single pass" cDNA sequences and Expressed Sequence Tags (ESTs) from a number of organisms ; the motif databases Pfam and Prosite ; as well as the collection of HMM protein families . Downloaded and formatted for local searches were MITOP, dbEST, the Pfam families of Rieske (Fe-S) proteins, 14.5 kDa and 9.5 kDa subunits of complex III (UCR_14.5kD, UCR_9.5kD), and mitochondrial processing peptidases.
As search tools, we used FASTA, BLAST, PSI-BLAST and "Search for short sequences," available on the NCBI Blast homepage . A very lenient e-value (1000) was used to account for the short length of the input peptide sequences (<30 residues). The word size of 2 and PAM 30 matrix were used as advised by the BLAST "Search for short sequences." The search against the HMM protein families was performed using HMMER (online version) (). We also employed the online version of Mascot, which is a powerful search engine which uses mass spectrometry data to identify proteins from primary sequence databases .g, 百拇医药
Resultsg, 百拇医药
Purification of the bc1 Complex of S. ecuadoriensisg, 百拇医药
The bc1 complex of S. ecuadoriensis was purified using cytochrome c affinity chromatography as published for N. crassa and plants . According to the original protocols, the starting material for affinity chromatography should be isolated mitochondria. Owing to the slow growth of jakobid cultures, however, the preparation of pure mitochondria from quite limiting amounts of S. ecuadoriensis cells proved difficult. Therefore fractions of enriched mitochondrial membranes were generated from S. ecuadoriensis as described in the Experimental Procedures section. The fractions were fivefold to eightfold enriched in mitochondrial membrane proteins as monitored by cytochrome c oxidase measurements. The main purification step for the S. ecuadoriensis bc1 complex, cytochrome c affinity chromatography, takes advantage of the specific interaction of the cytochrome c1 subunit of the bc1 complex and cytochrome c, the natural binding partner during respiratory electron transport. Proteins bound to the cytochrome c column were eluted by a salt gradient of 20–200 mM Tris-acetate, and the fractions obtained were analyzed by Tricine-SDS-PAGE and by immunoblotting. A 46 kDa band of fractions 19–23 eluted by 90–100 mM Tris-acetate strongly cross reacted with an antiserum directed against the core II protein from N. crassa . These peak fractions were pooled and concentrated by ultrafiltration using filters with an exclusion limit of 300 kDa. Finally the concentrate was analyzed by two-dimensional BN-PAGE/Tricine-SDS-PAGE. One protein complex was visible on the one-dimensional gels that could be resolved into seven protein bands of 46 kDa, 33 kDa, 29 kDa, 28 kDa, 14.5 kDa, 10 kDa, and 6 kDa upon separation on a second gel dimension .
fig.ommittedn, 百拇医药
FIG. 1. Purification of the bc1 complex from Seculamonas ecuadoriensis by cytochrome c affinity chromatography. Fractions eluted from the affinity column were separated by SDS-PAGE, blotted, and probed with an antibody directed against the core II protein from N. crassa. The numbers of the fractions are indicated above the gel, and the molecular masses of standard proteins are shown to the left of the gel (in kDa). A graphical illustration of the Tris-acetate gradient (20–200 mM) used for elution of proteins bound to the cytochrome c column is given below the geln, 百拇医药
fig.ommittedn, 百拇医药
FIG. 2. Characterization of the purified bc1 complex from S. ecuadoriensis by BN-PAGE/Tricine-SDS-PAGE. Sizes of standard proteins are given on the right; protein spots of the bc1 complex are marked with arrows. The spot at 46 kDa cross reacts with an antiserum directed against the core II protein from N. crassa (Inset: Western blot)n, 百拇医药
That the isolated complex is indeed complex III of the respiratory chain was confirmed by two experiments. First, the native complex exhibits quinol:cytochrome c oxidoreductase activity (turnover number: 9.5 s-1), which is antimycin-sensitive. It should be noted that the electron transfer activity is relatively low compared to preparations from other eukaryotes , which might be due to the use of heterologous cytochrome c (from horse heart) as electron acceptor. Second, in an immunoblotting experiment, the 46 kDa spot on the 2D gels was shown to cross-react with the antiserum directed against the core II subunit of the bc1 complex from N. crassa (, inset).
Analysis of Protein Complexes from S. ecuadoriensis by BN PAGE/Tricine-SDS-PAGE and Determination of the Molecular Mass of the bc1 Complex\7[, 百拇医药
To obtain further information on size and subunit composition of the bc1 complex from S. ecuadoriensis, protein complexes from fractions enriched in mitochondrial membranes were analyzed directly by Blue-Native and Tricine-SDS-PAGE. BN-PAGE is a very reliable method for molecular mass determination of protein complexes . Apparent molecular masses were estimated by co-electrophoresis of protein complexes from S. ecuadoriensis and mitochondrial protein complexes from potato, which served as size reference . The membrane fraction of S. ecuadoriensis contains five protein complexes in the size range of 100 to 700 kDa and additional minor bands. Analysis of the separated protein complexes on a second gel dimension by Tricine-SDS-PAGE allowed to identify the protein complexes from potato on the basis of subunit compositions . One of the separated protein complexes of S. ecuadoriensis comprises an identical subunit composition like the bc1 complex purified by affinity chromatography . Direct comparison of the protein complexes of potato mitochondria and S. ecuadoriensis mitochondrial fractions on Blue-Native gels revealed an apparent molecular mass of 460 kDa for the bc1 complex from S. ecuadoriensis . A further protein complex of S. ecuadoriensis runs at 550 kDa on Blue-Native gels and can be separated into 10 subunits upon analysis on a second gel dimension. The subunit composition of this 550 kDa complex resembles the one reported for mitochondrial ATP synthase complexes from other organisms . The identity of the dominant protein complex at about 150 kDa could not be determined on the basis of subunit composition.
fig.ommitted-jo, 百拇医药
FIG. 3. Determination of the molecular mass of the bc1 complex from S. ecuadoriensis by Blue-Native gel electrophoresis (BN-PAGE). A, One-dimensional resolution of protein complexes from potato (P) and S. ecuadoriensis (S) by BN-PAGE. The molecular masses of the mitochondrial protein complexes from potato are taken from (the band at 480 kDa corresponds to the potato bc1 complex). The bc1 complex from S. ecuadoriensis runs at about 460 kDa. B, Two-dimensional resolution of protein complexes from S. ecuadoriensis and mitochondrial protein complexes from potato by BN-PAGE/Tricine-SDS-PAGE. Sizes of standard proteins are given on the right. Protein complexes from potato were identified by their characteristic subunit composition ; protein complexes from S. ecuadoriensis were identified by sequence analysis of individual subunits by mass spectrometry (analyzed subunits are indicated by arrows and numbered according to ). I, NADH dehydrogenase; F0F1, F0F1 ATP synthase; bc1, bc1 complex; F1, F1 part of the F0F1 ATP synthase; FD, formate dehydrogenase; question marks indicate protein complexes that could not unambiguously be identified
Identification of Individual Subunits of Protein Complexes Separated by BN-PAGE/Tricine-SDS-PAGEwi, http://www.100md.com
To confirm the identity of the protein complexes and to obtain data on individual subunits, selected protein spots were subjected to peptide sequencing by Electrospray Ionization/Tandem Mass Spectrometry (ESI-MS/MS). We determined 1 to 3 peptide sequences of 7 different proteins , which form part of three different protein complexes and which are indicated and numbered on the gel in . The sequence of peptide 1 of the 46 kDa protein forming part of the bc1 complex exhibits significant similarity to a conserved stretch of the ß MPP/core I subunit of the bc1 complex from other eukaryotes. Notably, this peptide covers one of the few regions that distinguish {alpha} and ß MPP paralogs . Peptide 2 of the same protein spot exhibits some weak similarities to the core II proteins from N. crassa. As shown above, this protein spot also cross reacts with an antiserum directed against the core II protein from N. crassa . These data strongly suggest that the bc1 complex from S. ecuadoriensis comprises two core proteins with identical apparent molecular masses of 46 kDa.
fig.ommitted}u8sch+, http://www.100md.com
Table 1 Peptide Sequences of Subunits of Protein Complexes from Seculamonas ecuadoriensis}u8sch+, http://www.100md.com
fig.ommitted}u8sch+, http://www.100md.com
FIG. 4. Identification of the core I/ß MPP subunit of S. ecuadoriensis by sequence comparison with core I and ß MPP proteins of other organisms. Residues identical in at least six organisms are underlayed in black; other residues conserved in at least 4 organisms are underlayed in gray. Positions of the sequence stretches and accession numbers of the proteins are given on the right}u8sch+, http://www.100md.com
Peptides 1 and 3 of the 29 kDa protein of the S. ecuadoriensis bc1 complex exhibit low sequence identity to cytochrome c1 . However, the stretches of similarity represent unconserved regions of the protein and contain numerous insertions/deletions across the taxa, and therefore they did not withstand more rigorous statistical tests.}u8sch+, http://www.100md.com
Also, neither peptides of the 14.5 kDa subunit nor those of the 10 kDa subunit of the S. ecuadoriensis bc1 complex displayed significant sequence similarity to known proteins. Sequence conservation of the small subunits of the mitochondrial bc1 complex is notoriously low . Much longer peptide sequences would be required than those determined in this study to identify phylogenetically distant, and, even more so, weakly conserved homologs.
The 48 and 30 kDa proteins of the F0F1 ATP synthase complex were unambiguously identified as the ß and the subunits of this protein complex. Both peptide sequences obtained for 48 kDa protein exhibit high sequence identity to an internal sequence stretch of ß subunits of the F0F1 ATP synthase complex from other organisms, and the two peptide sequences of the 30 kDa protein correspond exactly to amino acid stretches of the mitochondrial encoded subunit of the F1 part of S. ecuadoriensis.(^6, 百拇医药
The identity of the dominant protein complex at 150 kDa could not be resolved on the basis of the peptide sequence of a 26 kDa subunit.(^6, 百拇医药
Discussion(^6, 百拇医药
This article reports the identification of two protein complexes of the respiratory chain from the jakobid flagellate S. ecuadoriensis, the F0F1 ATP synthase and the bc1 complex. The experiments described here focus on the purification and characterization of the bc1 complex. To our knowledge this is the first report on a biochemical preparation of an enzyme from jakobid flagellates. The S. ecuadoriensis bc1 complex was purified on the basis of its affinity to the natural binding partner during respiratory electron transport, cytochrome c. The purified bc1 complex retains both quinol:cytochrome c oxidoreductase activity and antimycin sensivity.
The molecular mass of the bc1 complex from S. ecuadoriensis lies at 460 kDa as determined by BN-PAGE. Under denaturing electrophoresis conditions the complex was resolved into seven protein bands with apparent molecular masses of 46 kDa, 33 kDa, 29 kDa, 28 kDa, 14.5 kDa, 10 kDa, and 6 kDa . Three lines of evidence strongly suggest that the largest protein spot encompasses two proteins, the core I and core II subunits. First, the 46 kDa spot cross reacts with an antiserum directed against the core II protein of the bc1 complex from N. crassa, while a peptide derived from this spot also exhibits significant sequence identity to the core I/ß-MPP subunit from different eukaryotes. Second, the core subunits of all mitochondrial bc1 complexes characterized to date closely comigrate in gel electrophoresis in the size range of 45 to 55 kDa. Third, the apparent molecular mass of the S. ecuadoriensis bc1 complex (460 kDa) can only be explained by assuming a dimeric holoenzyme that includes two core subunits per monomer as further discussed below.
In all bacteria and mitochondria characterized up to now, quinol:cytochrome c oxidoreductase activity is based on electron transfer reactions between the prosthetic groups of cytochrome b, cytochrome c1, and the iron-sulfur protein. Given that the purified bc1 complex of S. ecuadoriensis displays electron transport activity, it must include these three subunits. The molecular masses of the respiratory subunits are quite conserved in potato, bovine, and yeast, with 42–44 kDa for cytochrome b, 27–28 kDa for cytochrome c1, and 20–23 kDa for the iron-sulfur protein . A considerable degree of conservation across three different eukaryotic phyla allowed us to assign the following three protein species of the purified bc1 complex of S. ecuadoriensis to respiratory subunits. First, the 33 kDa protein of S. ecuadoriensis is most likely cytochrome b. This is consistent with the fact that cytochrome b, because of its highly hydrophobic properties, typically displays a migration behavior that makes its molecular mass appear ~
25% smaller than it really is. Second, the 29 kDa protein of S. ecuadoriensis is probably cytochrome c1 and is thus only slightly larger than the proteins of its well-characterized counterparts. Finally, the 28 kDa subunit of S. ecuadoriensis represents, very likely, the iron-sulfur protein. Although the S. ecuadoriensis protein is approximately 30% larger than its homologs of the model organisms, such a size deviation is not without precedent. A comparatively large iron-sulfur subunit was also reported for the bc1 complex from L. tarentolae . summarizes the demonstrated and inferred subunit assignments of the bc1 complex from S. ecuadoriensis.:jqz9p, 百拇医药
fig.ommitted:jqz9p, 百拇医药
FIG. 5. Identities of the subunits of the bc1 from S. ecuadoriensis. A scheme of the gel is given on the right, and the sizes of standard proteins are given in the middle. Cyt, cytochrome; FeS, iron-sulfur protein; question marks indicate subunits of unknown identity
As already mentioned, bacterial and mitochondrial bc1 complexes alike are dimers. Assuming the presence of two core proteins, the molecular masses of the eight separated subunits of the S. ecuadoriensis bc1 complex sums up to 423 kDa for the dimeric complex, a value that is somewhat smaller than the experimentally determined molecular mass of the complex (460 kDa). The difference of 27 kDa could be due to the presence of one or two further low molecular mass subunits that may not have been detected in our experiments. These small proteins are difficult to spot because they migrate closely together and are poorly stainable./%8ri], 百拇医药
While the bacterial bc1 complex comprises three or four subunits at most, with a molecular mass of the dimeric complex of ~/%8ri], 百拇医药
220 kDa , the mitochondrial counterpart is at least twice as large. At present, complex III has been characterized from highly diverse eukaryotes, including plants, fungi, animals, and euglenoid and kinetoplastid protists . All these mitochondrial bc1 complexes have an apparent molecular mass of 470–495 kDa and consist of three respiratory subunits, two large core proteins and, at least in higher eukaryotes (but probably in all eukaryotic taxa investigated up to now), five small subunits. As we show here, the bc1 complex from the jakobid protistan S. ecuadoriensis has only a slightly smaller molecular mass (460 kDa) than that from fungi, mammals, and plants (470–495 kDa) and includes at least eight different subunits. Because jakobids are believed to be a primitive eukaryotic lineage, this finding was unexpected.
The view that jakobids are minimally derived eukaryotes is based on their ultrastructural similarities with the retortamonads, an amitochondriate group considered to have diverged close to the eukaryotic origin . S. ecuadoriensis is a typical member of jakobids and of the core jakobids in particular, as first established by analysis of the basal body ultrastructure and other cellular characters . Furthermore, phylogenetic analyses using mitochondrion-encoded protein genes clearly affiliate S. ecuadoriensis with R. americana (not shown). In global eukaryotic trees, however, available multiple mitochondrial protein data fail to place the jakobids relative to the other eukaryotic lineages with confidence, which is most likely a result of the low sampling of jakobid taxa . Similarly uncertain topologies are obtained with single nuclear genes (- and ß-tubulin) Nevertheless, and irrespective of their exact phylogenetic position relative to the other eukaryotes, mitochondrial genomes of core jakobids, such as R. americana and S. ecuadoriensis, display an astonishing number of bacterial features, more closely resembling the genome of the ancestral -proteobacterial symbiont than any other mtDNA investigated today .
Our initial hypothesis posited that jakobid mitochondrial complexes are evolutionary intermediates between those of -proteobacteria and mitochondria from higher eukaryotes. Although the finding of much the same complex III structure in S. ecuadoriensis and plants or fungi does not corroborate this view, the particular complex investigated may not be a suitable choice for detecting a more bacteria-like structure. Indeed, considering the number of mitochondrion-encoded subunits, complex III of the jakobids is as much derived as that of all other eukaryotes studied. Among the three genes coding for respiratory subunits of {alpha} -proteobacterial origin, only the apoprotein b gene still resides in mtDNA, whereas the other two, i.e., the cytochrome c1 and iron-sulfur protein, are nucleus-encoded.z:ll;1h, 百拇医药
It is conceivable that the number of originally -proteobacterial genes that have migrated to the nucleus is correlated with the number of secondarily acquired subunits in a respiratory complex. In fact, the migration of mitochondrial genes to the nucleus may actually be the underlying cause for the recruitment of additional subunits the crucial role of which may involve mediation of protein complex assembly from components that are now synthesized in different cellular compartments and synchronization of the expression of genes now located in different genomes.
If this hypothesis is correct, one should expect a more primitive structure in those mitochondrial membrane complexes that have retained a larger number of mitochondrion-encoded subunits in jakobids than in other eukaryotes. The mitochondrial NADH dehydrogenase (complex I of the respiratory chain) and the ATP synthase are two such cases. Additional subunits of these protein complexes are encoded by mitochondrial genes in R. americana (as well as S. ecuadoriensis, unpublished data) if compared to the mitochondrial genomes of other eukaryotes (). Like the situation in complex III, all mitochondrial ATP synthases characterized in eukaryotes other than jakobids have approximately five more subunits over and above the eight that are typically present in the bacterial enzyme (). To test the hypothesis that subunit recruitment is the consequence of gene migration from the mitochondrial genome to the nuclear genome, investigation of the complex composition of S. ecuadoriensis ATP synthase is under way.
Acknowledgements:3, http://www.100md.com
We thank Dr. U. Schulte, Düsseldorf, for instructing us on quinol:cytochrome c reductase activity measurements in his laboratory. This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and the Canadian Institute for Health Research (CIHR). G.B. is a Canadian National Associate, and B.F.L is an Imasco fellow in the program of Evolutionary Biology of the Canadian Institute of Advanced Research (CIAR), which we thank for salary and interaction support.:3, http://www.100md.com
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Accepted for publication September 24, 2002.(Stefanie Marx Maja Baumgärtner Sivakumar Kunnan Hans-Peter Braun B. Franz Lang and Gertraud Bur)