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UPDATE ON CHLAMYDOMONAS OXIDATIVE PHOSPHORYLATION PROTEOME

The Mitochondrial Oxidative Phosphorylation Proteome of Chlamydomonas reinhardtii Deduced from the Genome Sequencing Project¹

Genetics of Microorganisms (P.C., R.F.M., C.R.) and Algology, Mycology and Experimental Systematics (D.B.), Institute of Plant Biology B22, University of Liege, B–4000 Liege, Belgium; and Departamento de Genética Molecular, Instituto de Fisiología Celular (D.G.-H.) and Instituto de Biología (A.R.-P.), Universidad Nacional Autónoma de Mexico, 04510 Mexico D.F., Mexico

Mitochondria originated from an endosymbiotic process that involved an -proteobacterium (Martin and Muller, 1998; Gray et al., 1999). The evolution of the organelle has been associated with the massive migration of the endosymbiont genes to the nucleus of the host and with the acquisition of new proteins that were originally present in the host (Karlberg et al., 2000; Andersson et al., 2003; Richly et al., 2003). Mitochondrial proteomes are thus a mosaic of mitochondria- and nucleus-encoded proteins, the latter having a combined eubacterial/eukaryotic origin. The increasing number of complete genome sequences has naturally led to an attempt to establish a relationship between these genomes and their corresponding transcriptomes and proteomes. Of particular interest for bioenergeticists are the efforts performed toward establishing the proteomes of chloroplasts and mitochondria.

Mitochondria are the site of oxidative phosphorylation (OXPHOS). This process comprises an electron-transfer chain that is driven by substrate oxidation and is coupled to the synthesis of ATP through an electrochemical transmembrane gradient. Therefore, the OXPHOS proteome (or more simply the OXPHOSome) will be the anatomical description of the protein components that participate in this process (complexes I–V and additional oxidoreductases).

	THE STANDARDS OF REFERENCE: THE BOVINE, YEAST, AND PLANT OXPHOSOMES

TOP THE STANDARDS OF REFERENCE:... THE CHLAMYDOMONAS OXPHOSOME DATA COMPLEX I COMPLEX II COMPLEX III OR BC1... CYTOCHROME C COMPLEX IV COMPLEX V ADDITIONAL ENZYMES CONCLUDING REMARKS LITERATURE CITED

 
Historically, the bovine OXPHOS complexes were the first ones to be isolated and characterized (Hatefi et al., 1979), and three-dimensional structures of the complexes III (Xia et al., 1997; Zhang et al., 1998), IV (Tsukihara et al., 1996), and V (Abrahams et al., 1994) are now available. The genes coding for the subunits of the complexes have also been identified, and the N-terminal sequences of the corresponding mature proteins have been determined (Yanamura et al., 1988; Collinson et al., 1994; Iwata et al., 1998; Hirst et al., 2003). Currently, the organization of the different bovine OXPHOS complexes into supercomplexes is being actively explored (Schägger and Pfeiffer, 2000). The bovine system constitutes, therefore, an obliged reference for comparative studies. In other animal organisms, including human (http://www.sanger.ac.uk/HGP/) and the nematode Caenorhabditis elegans (Tsang and Lemire, 2003), the availability of genome sequences has allowed an immediate identification of most of the respiratory-chain components by comparison to bovine sequences.

A considerable amount of information is also available in the yeast Saccharomyces cerevisiae. Both its mitochondrial and nuclear genomes have been completely sequenced (The Saccharomyces Genome Database at http://www.yeastgenome.org/; Goffeau et al., 1996), and an inventory of the yeast gene products that are present in mitochondria has been drawn up (Schon, 2001). Furthermore, the OXPHOS complexes II (Lemire and Oyedotun, 2002), III (Ljungdahl et al., 1987), IV (Poyton et al., 1995), and V (Velours and Arselin, 2000) have been isolated and characterized. In addition, the crystallographic structures of complex III (Lange and Hunte, 2002) and of a complex V subcomplex (Stock et al., 1999) have been obtained, and supramolecular structures associating complexes III and IV have been described (Schägger and Pfeiffer, 2000). S. cerevisiae cannot, however, be considered as the reference simply because its mitochondria are deprived of complex I. Nevertheless, mitochondria of other fungi such as Neurospora crassa (Friedrich et al., 1998) and Yarrowia lipolytica (Kerscher et al., 2001) that possess a complex I have been successfully used to study the structure and function of this enzyme.

The higher plant Arabidopsis (Arabidopsis thaliana) can also be considered as a model system. Its three genomes have been sequenced (The Arabidopsis Information Resource at http://arabidopsis.org/info/agi.jsp), and the different respiratory complexes have been isolated by Blue-Native PAGE (BN-PAGE; Jansch et al., 1996). The presence of OXPHOS supercomplexes in plant mitochondria has also been detected (Eubel et al., 2003). In addition, 416 proteins of the mitochondrial proteome have been identified by liquid chromatography-tandem mass spectrometry (Heazlewood et al., 2004), and the constructed database was made available to the public at http://www.mitoz.bcs.uwa.edu.au.

	THE CHLAMYDOMONAS OXPHOSOME DATA

TOP THE STANDARDS OF REFERENCE:... THE CHLAMYDOMONAS OXPHOSOME DATA COMPLEX I COMPLEX II COMPLEX III OR BC1... CYTOCHROME C COMPLEX IV COMPLEX V ADDITIONAL ENZYMES CONCLUDING REMARKS LITERATURE CITED

 
The green alga Chlamydomonas reinhardtii allows the study of the OXPHOSome of a photosynthetic, unicellular organism. The sequence of its 15.8-kb linear mitochondrial genome is known (Michaelis et al., 1990), and a draft of the complete sequence of its nuclear genome is now available (http://www.jgi.doe.gov; Shrager et al., 2003). Moreover, the recent development of procedures to isolate Chlamydomonas mitochondria almost free of thylakoid contaminants (Eriksson et al., 1995; Nurani and Franzen, 1996; Cardol et al., 2002), to separate the major mitochondrial complexes in BN-PAGE (van Lis et al., 2003; Cardol et al., 2004), and to analyze by bioinformatics the algal-nucleic acid sequences (http://www.chlamy.org/) allowed us to initiate the characterization of the algal OXPHOSome (van Lis et al., 2003; Cardol et al., 2004).

In this review, using a genomic approach and taking into account previous published data, we present a compilation of the proteins that could be components of the Chlamydomonas OXPHOSome or could participate in its biogenesis. We found that, among polypeptidic sequences identified, the large majority have counterparts in mammals, fungi, and higher plants, whereas the remaining proteins are unique to C. reinhardtii or only common to two or three lineages.

	COMPLEX I

TOP THE STANDARDS OF REFERENCE:... THE CHLAMYDOMONAS OXPHOSOME DATA COMPLEX I COMPLEX II COMPLEX III OR BC1... CYTOCHROME C COMPLEX IV COMPLEX V ADDITIONAL ENZYMES CONCLUDING REMARKS LITERATURE CITED

 
Complex I (rotenone-sensitive NADH:ubiquinone oxidoreductase; EC 1.6.5.3) is the largest and most complicated enzyme of the mitochondrial respiratory chain. In the bovine complex I, 45 different subunits have been characterized and build up into a membrane-bound assembly with a molecular mass of approximately 980 kD (Hirst et al., 2003). The detailed dissection of complex I from the higher plants Arabidopsis and rice (Oryza sativa; Heazlewood et al., 2003a), and the fungi N. crassa (Videira and Duarte, 2002) and Y. lipolytica (Abdrakhmanova et al., 2004), have confirmed the complexity of the enzyme. A simpler membrane-associated type-I NADH dehydrogenase has been characterized in bacteria. It comprises 14 subunits, all being conserved among eukaryotes. These subunits bind the FMN and the eight iron-sulfur clusters of the enzyme. Seven of these subunits are the homologs of the hydrophobic ND1, 2, 3, 4, 4L, 5, and 6 subunits encoded in the mitochondrial genome of eukaryotes (Dupuis et al., 1998; Friedrich et al., 1998). The additional subunits present in mitochondrial complex I, the so-called supernumerary subunits, are considered to participate in the assembly, the stability, or the regulation of the enzyme (Friedrich et al., 1998; Heazlewood et al., 2003a).

Recently, the composition of the C. reinhardtii complex I has been analyzed (Cardol et al., 2004). Combining proteomic and genomic approaches, 42 proteins of molecular masses ranging from 7 to 77 kD were identified for a molecular mass totaling 950 to 1,000 kD (Table I). Comparison of complex I subunit compositions from C. reinhardtii, mammals, fungi, and higher plants revealed that all eukaryotic enzymes contained 31 common components, including the 14 highly conserved subunits homologous to the prokaryotic type-I NADH dehydrogenase enzyme (Cardol et al., 2004).

Among the 43 identified or putative subunits of the C. reinhardtii complex I (including NUOS4b, which is closely related to NUOS4), one (NUO21) is present in higher plants and fungi but has no counterpart in the bovine enzyme (Table I). Five subunits seem to be common to higher plants (Heazlewood et al., 2003a) and Chlamydomonas only (Cardol et al., 2004): NUOP1 (GenBank/AAS58501), NUOP3 (GenBank/AAS58502), and the three so-called ferripyochelin-binding proteins FBP1, FBP2, and CAH9 (GenBank/AAS48195, AAS48196, and AAS48197) that are related to the carbonic anhydrase (Parisi et al., 2004). Furthermore, three subunits, NUOP4 (GenBank/AAS58498), NUOP5 (GenBank/AAS58503), and NUOP6 (GenBank/AAS58500), could be specific of the green alga lineage (Cardol et al., 2004).

The presence of chaperones involved in the assembly of complex I has also been investigated. To date, only two chaperones, the CIA30 and CIA84 proteins, have been described in N. crassa (Kuffner et al., 1998; Table I). The homologous gene of the CIA30 chaperone is present in human (Janssen et al., 2002), higher plants (Arabidopsis/At1g72420), and Chlamydomonas (Chlamy v2.0/C_50184), whereas we did not find any homolog of CIA84 in other organisms (Table I).

	COMPLEX II

TOP THE STANDARDS OF REFERENCE:... THE CHLAMYDOMONAS OXPHOSOME DATA COMPLEX I COMPLEX II COMPLEX III OR BC1... CYTOCHROME C COMPLEX IV COMPLEX V ADDITIONAL ENZYMES CONCLUDING REMARKS LITERATURE CITED

 
Complex II, or succinate:ubiquinone oxidoreductase (EC 1.3.99.1), is the respiratory-chain complex enzyme with the lowest molecular mass. This complex is considered as a bifunctional enzyme that participates both in the mitochondrial electron-transport chain and in the Krebs cycle. Classically, it contains only four polypeptides, all encoded in the nucleus: the flavoprotein SDH1 subunit, the iron-sulfur SDH2 subunit, and two hydrophobic membrane anchors, the SDH3 and SDH4 subunits. The corresponding genes are found in the Chlamydomonas genome database (Table I).

As judged by BN-PAGE, complex II from Arabidopsis contains four additional subunits of unknown function. Two of these subunits have been identified (Arabidopsis/At1g47420 and At1g08480; Eubel et al., 2003). Whereas no homolog of At1g08480 gene product has been found in the Chlamydomonas genome, the Arabidopsis/At1g47420 gene product shares 50% and 31% identities with the Arabidopsis/At1g47260 and the Chlamy v2.0/C_160093 gene products, respectively. Surprisingly, these two proteins are complex I components (Heazlewood et al., 2003a; Cardol et al., 2004) and are structurally related to carbonic anhydrases (Parisi et al., 2004).

Only one complex II chaperone, the Tcm62 protein related to the Hsp60 chaperone, has been identified in mitochondria of S. cerevisiae (Dibrov et al., 1998). This chaperone could be involved in mitochondrial gene expression at elevated temperatures (Klanner et al., 2000). We did not find any counterpart of Tcm62 in the databases of animals, algae, or plants.

	COMPLEX III OR BC1 COMPLEX

TOP THE STANDARDS OF REFERENCE:... THE CHLAMYDOMONAS OXPHOSOME DATA COMPLEX I COMPLEX II COMPLEX III OR BC1... CYTOCHROME C COMPLEX IV COMPLEX V ADDITIONAL ENZYMES CONCLUDING REMARKS LITERATURE CITED

 
The mitochondrial complex III (ubiquinol-cytochrome c oxidoreductase; EC 1.10.2.2) from mammals, yeast, and higher plants is an oligomeric membrane protein complex made of 10 highly conserved subunits (Braun and Schmitz, 1995; Iwata et al., 1998; Eubel et al., 2003).

The C. reinhardtii bc₁ complex has been isolated and found to be composed of 9 subunits, with molecular masses ranging from 10 to 50 kD (Atteia, 1994; van Lis et al., 2003). Among these polypeptides, subunit core I (53 kD) has been identified by N-terminal sequencing and corresponds to the Chlamy v2.0/C_1290016 sequence (Table I). Before the genomic approach, the sequences of the mitochondrial cytochrome b gene (GenBank/NC_001638; Gray and Boer, 1988), the nuclear genes encoding the Rieske iron-sulfur protein (RIP1;GenBank/AJ320239), and the cytochrome c₁ (GenBank/AJ417788) have also been characterized (Atteia et al., 2003). The search of the genome sequences of C. reinhardtii allowed us to find the 10 classical subunits of complex III: core I and core II proteins (QCR1 and QCR2), cytochrome b, cytochrome c₁, RIP1, and the five additional subunits: QCR7 (equivalent to the bovine 14-kD subunit VI or QP-C), QCR8 (equivalent to bovine 9.5-kD subunit VII or ubiquinol-binding protein), QCR6 (equivalent to the bovine 7.8-kD subunit VIII or hinge protein), QCR9 (equivalent to the bovine 7.2-kD subunit X), and QCR10 (equivalent to bovine 6.4-kD subunit XI; Table I). The C. reinhardtii putative QCR8 and QCR10 protein sequences present a very low identity with the corresponding sequences of the other eukaryotes, but using a window of seven residues (Kyte and Doolittle, 1982), we have found good similarities between the hydropathy profiles (data not shown), which supports the idea that all these proteins are orthologs. Thus, on the basis of this putative subunit composition, the complex III of C. reinhardtii would be very similar to the complex III of the other eukaryotes.

The QCR1 and QCR2 homologs from higher plants possess a proteolytic activity (mitochondrial processing peptidase, or MPP) that cleaves the transit peptide of preproteins when imported into mitochondria (Glaser and Dessi, 1999). In Chlamydomonas, QCR1 exhibits consensus sequences typical of -MPP proteins, while QCR2 does not share consensus sequences for -MPP activity (van Lis et al., 2003). Nevertheless, more than two polypeptide bands are resolved in the core region of Chlamydomonas complex III in two-dimensional SDS-PAGE after BN-PAGE (R. van Lis, A. Atteia, and D. González-Halphen, unpublished data). Therefore, a putative isoform of QCR2 (Chlamy v2.0/C_10187) could be responsible for catalyzing the -MPP activity.

Homologs of proteins ABC1, CBP3, and BCS1 that act in yeast as chaperones essential for proper conformation and functioning of the complex (Bousquet et al., 1991; Nobrega et al., 1992; Cruciat et al., 1999; Shi et al., 2001) are present in the Chlamydomonas sequence database (Table I). Besides these chaperones, several proteins of S. cerevisiae were found to be required for proper expression, maturation, assembly, and stability of cytochrome b (CBP1, CBP2, CBP6, CBS1, and CBS2; Dieckmann et al., 1982; McGraw and Tzagoloff, 1983; Dieckmann and Tzagoloff, 1985; Rodel, 1986) or would be involved in the assembly of complex III (CBP4; Crivellone, 1994). None of these proteins was found to possess counterparts in animals or plants (Table I).

	CYTOCHROME C

TOP THE STANDARDS OF REFERENCE:... THE CHLAMYDOMONAS OXPHOSOME DATA COMPLEX I COMPLEX II COMPLEX III OR BC1... CYTOCHROME C COMPLEX IV COMPLEX V ADDITIONAL ENZYMES CONCLUDING REMARKS LITERATURE CITED

 
The mitochondrial cytochrome c from C. reinhardtii is encoded by a single nuclear gene (Swissprot/S29514), and its structure is very similar to that of higher plant cytochromes c (Amati et al., 1988).

Three distinct pathways of cytochrome c biogenesis have been described (Kranz et al., 1998). System I occurs in mitochondria from higher plants and protozoa. System II takes place in many gram-positive bacteria, in cyanobacteria, and in chloroplasts from higher plants and green algae. System III involves heme lyase enzymes in yeasts (CYC3 and CYT2) and animals (CCHL). Whereas no system I counterparts could be identified, three putative homologous enzymes of system III were found to be encoded in the Chlamydomonas genome (Table I).

	COMPLEX IV

TOP THE STANDARDS OF REFERENCE:... THE CHLAMYDOMONAS OXPHOSOME DATA COMPLEX I COMPLEX II COMPLEX III OR BC1... CYTOCHROME C COMPLEX IV COMPLEX V ADDITIONAL ENZYMES CONCLUDING REMARKS LITERATURE CITED

 
In yeast (Taanman and Capaldi, 1992) and mammals (Yanamura et al., 1988), complex IV (cytochrome c oxidase; EC 1.9.3.1) is composed of 12 or 13 well-characterized subunits. In Arabidopsis, the enzyme is made of 10 to 12 subunits, but only 7 have been identified (Eubel et al., 2003; Table I).

Most generally, COX1 (binding heme a, heme a₃, and Cu_B), COX2 (binding Cu_A), and COX3 subunits, which form the catalytic core of the enzyme, are encoded in the mitochondrial genome, whereas the other subunits, which are regulatory proteins, are encoded in the nucleus. Exceptions to this situation exist, however: in some legumes only subunits COX1 and COX3 are mitochondria encoded (Daley et al., 2002), whereas in C. reinhardtii (Michaelis et al., 1990) and in Polytomella parva (Fan and Lee, 2002), COX1 is the only subunit encoded in the mitochondrial genome.

The Chlamydomonas cytochrome c oxidase complex was resolved by BN-PAGE into 10 polypeptides of molecular masses ranging from 8 to 40 kD (van Lis et al., 2003). However, only eight coding sequences were found in the Chlamydomonas sequence database (Table I). Six encode homologs of proteins found in other eukaryotic complexes IV (orthologs of bovine subunits COX1, 2, 3, 5b, 6a, and 6b), whereas a seventh one (Chlamy v2.0/C_110052) encodes the homolog of the plant-specific COX5c subunit (Jansch et al., 1996; Hamanaka et al., 1999). We have also found the sequence corresponding to COX90 (GenBank/AAM88388), a Chlamydomonas-specific protein said to be essential for complex IV assembly and considered to belong to the enzyme complex (Lown et al., 2001).

The subunit composition of Chlamydomonas complex IV deduced from the genome sequencing project is thus quite similar to the one of complex IV from Arabidopsis (Table I). This raises the question whether sequences homologous to bovine COX4, 5a, 6c, 7a, 7b, 7c, and COX8 subunits (fungal COX5–9 subunits) are present in Chlamydomonas and Arabidopsis but are too divergent to be identified by sequence analysis, or whether the cytochrome c oxidase composition is actually different in photosynthetic organisms.

Chlamydomonas complex IV additional subunits show some unusual characteristics (R. van Lis, A. Atteia, and D. González-Halphen, unpublished data): (1) the first 60 residues of the mature subunit COX6b are highly hydrophobic and have counterparts in plant sequences, but not in animal or yeast sequences; (2) subunit COX5b lacks the 3 conserved cysteins that are known to bind a zinc atom in cytochrome c oxidase from other organisms (Rizzuto et al., 1991); and (3) subunit COX6a shows an N-terminal region with high similarity with COX5a from mammals, while the C-terminal region is more similar to the COX6a subunit.

In S. cerevisiae, more than 20 complex IV chaperones have been identified. The COX11, COX17, COX19, COX23, SCO1, and SCO2 proteins play a role in copper delivery to cytochrome c oxidase (Nobrega et al., 2002; Barros et al., 2004; Horng et al., 2004; Leary et al., 2004), whereas COX10 and COX15 are required for heme a biosynthesis (Glerum and Tzagoloff, 1994; Barros and Tzagoloff, 2002). Homologs of all these proteins are present in C. reinhardtii, mammals, and plants (Table I). A number of yeast proteins, such as SHY1, Pet54, Pet111, Pet122, Pet309, Pet494, Mss2, Mss51, COX14, and COX20, are supposed to act as translational activators of COX1, COX2, or COX3 mRNAs (Hell et al., 2000; Broadley et al., 2001; Barrientos et al., 2002, 2004; Naithani et al., 2003; Xu et al., 2004). Except for SUR1, the ortholog of SHY1, and for a protein presenting an Leu-rich pentatricopeptide repeat cassette motif found in Pet309, no homolog of these polypeptides has been identified in C. reinhardtii and Arabidopsis sequence databases. Concerning COX16, Pet100, and Pet191, three chaperones with no identified function in yeast (McEwen et al., 1993; Forsha et al., 2001; Carlson et al., 2003), two (COX16 and Pet191) have counterparts in human, Arabidopsis, and Chlamydomonas (Table I). Finally, a partial amino acid sequence that shares similarities with COX18 and OXA1 chaperones has also been found (Table I). These two proteins play a critical role in the biogenesis of yeast cytochrome c oxidase and are required for the insertion of various hydrophobic proteins in the inner mitochondrial membrane (He and Fox, 1997; Saracco and Fox, 2002; Funes et al., 2004).

	COMPLEX V

TOP THE STANDARDS OF REFERENCE:... THE CHLAMYDOMONAS OXPHOSOME DATA COMPLEX I COMPLEX II COMPLEX III OR BC1... CYTOCHROME C COMPLEX IV COMPLEX V ADDITIONAL ENZYMES CONCLUDING REMARKS LITERATURE CITED

 
The mitochondrial complex V (FoF₁-ATP synthase; EC 3.6.1.3) catalyzes the phosphorylation of ADP by inorganic phosphate using the proton motive force generated by the electron transport chain. The protein complex possesses two domains, the membrane-bound sector Fo involved in proton translocation and the extrinsic domain F₁ that catalyses ATP synthesis. In bacteria, while the Fo sector is composed of three subunits in a ratio ab₂c_10–14, the F₁-ATPase contains five subunits in a ₃₃ stoichiometry (Weber and Senior, 2003). The two sectors are connected by two stalks: a central stalk () that couples proton translocation with the catalytic region and a lateral stalk (b₂), which is considered to be part of the stator of the enzyme. The eukaryotic complex V contains homologous components but possesses additional subunits: in mammals, ATPase is made of 16 subunits (Collinson et al., 1994), whereas the yeast enzyme comprises 20 components, 13 being essential for the structure of the complex (Velours et al., 1998; Velours and Arselin, 2000; Table I).

Surprisingly, whereas almost all the mammal ATPase proteins are conserved in Arabidopsis (Heazlewood et al., 2003b; Table I), our search in the Chlamydomonas database led to the identification of only seven homologs. They correspond to subunits ATP5, 6, and 9 of the Fo domain and to subunits , , , and of the F₁ domain (Table I). In other respects, the complex V of Chlamydomonas was found to be composed of 14 subunits of molecular masses ranging from 7 to 60 kD (van Lis et al., 2003). Eight of these subunits were identified (Funes et al., 2002; van Lis et al., 2003, P. Cardol, unpublished data): the seven typical subunits ATP5, ATP6, ATP9, , , , and , and the so-called P60 subunit (61.0 kD; Chlamy v2.0/C_420010) or mitochondrial ATP synthase-associated protein (ASA1), which is also present in Polytomella sp. (GenBank/AJ558193). ASA1 protein is believed to be responsible for the extraordinary stability of the chlorophycean complex V dimer in BN-PAGE (Atteia, 1994; van Lis et al., 2003). Besides these components, six N-terminal amino acid sequences were determined by Edman degradation (Funes et al., 2002). These partial-polypeptide sequences allowed us to identify the corresponding sequences in the Chlamydomonas sequence database: EESSVANLVKS matches to Chlamyv2.0/C_50224, MKLLPESLQQEAA to Chlamyv2.0/C_10209, LSTLVEKFTFGSAAD to Chlamyv2.0/C_710036, ATGAAPSKK to Chlamyv2.0/C_230150, GAPAGSDHDHP to Chlamyv2.0/C_750022, and ATATFVPGVSGDASG to Chlamyv2.0/C_710028 (Table I). Surprisingly, none of these mitochondrial ATP synthase-associated proteins has a counterpart in other eukaryotic genomes. Since some of the classical subunits missing in Chlamydomonas are associated to the structure of the stator (Velours and Arselin, 2000), one may speculate that these six constituents unique to Chlamydomonas could also play a role as a stator in a structure quite different from the one present in the conventional ATP synthase.

Finally, the Chlamydomonas complex V exhibits two additional peculiar features: (1) in contrast to other organisms, ATP6 is nucleus encoded (Funes et al., 2002); and (2) it could include two isoforms of ATP9 (subunit c), since two genes are predicted to encode a c-like subunit (Table I). These genes are located adjacent to each other in the genome (scaffold_108:57,000–61,000) and seem to be divergently transcribed (P. Cardol, unpublished data). The N-terminus sequence of the ATP9 subunit (SVLAASKMVGA; van Lis et al., 2003) does not allow us to predict whether only one polypeptide or both of them are present in the mature complex.

To our knowledge, eight assembly factors for complex V have been identified in yeast. Only two of them, ATP11 and ATP12, widely conserved chaperones for the F₁ domain (Ackerman and Tzagoloff, 1990b; Wang et al., 2001), are present in the Chlamydomonas genome (Chlamy 2.0/C_150089, C_10143; Table I). In contrast, FMC1, which is required for the assembly or stability of F₁ domain in heat stress conditions (Lefebvre-Legendre et al., 2001); ATP10 and ATP22, two proteins required for the assembly of the Fo sector (Ackerman and Tzagoloff, 1990a; Helfenbein et al., 2003; Tzagoloff et al., 2004); AEP1 and AEP2, both required for correct expression of ATP9 (Payne et al., 1991; Finnegan et al., 1995); and AEP3, which stabilizes mitochondrial bicistronic mRNA-encoding subunits 6 and 8 (Ellis et al., 2004), do not seem to have counterparts in photosynthetic organisms and animals. ATP10 homologous sequences have, however, been identified in higher plants (Arabidopsis/AAF18252, rice/AAT77344).

	ADDITIONAL ENZYMES

TOP THE STANDARDS OF REFERENCE:... THE CHLAMYDOMONAS OXPHOSOME DATA COMPLEX I COMPLEX II COMPLEX III OR BC1... CYTOCHROME C COMPLEX IV COMPLEX V ADDITIONAL ENZYMES CONCLUDING REMARKS LITERATURE CITED

 
In addition to the proton-pumping complex I, plant and fungal mitochondria contain several type-II NAD(P)H dehydrogenases. These additional enzymes, located at the surface of the mitochondrial inner membrane and facing either the intermembrane space or the matrix, allow electron transfer from NAD(P)H to ubiquinone. They are single, low-molecular-weight polypeptides that are insensitive to rotenone (Moller et al., 1993). In S. cerevisiae, three enzymes have been well characterized (Marres et al., 1991; Luttik et al., 1998), whereas in Arabidopsis, three gene families (nda, ndb, and ndc) with different evolutionary origins encode NADH dehydrogenases that are targeted to mitochondria (Michalecka et al., 2003).

Our searches in the Chlamydomonas sequence database revealed that seven amino acid sequences share similarities with known type-II NAD(P)H dehydrogenases (Table I). Since several genomic sequences present gaps (NDA2, 4, and 5) and since some of the gene models seem to be erroneously predicted (NDA6 and 7), it is difficult to clearly associate these sequences to any of the NADH dehydrogenase families.

Mitochondria from plants, several fungi, and several protists also possess an alternative oxidase (AOX) that drives the electrons from the ubiquinol pool directly to molecular oxygen. This nonphosphorylating enzyme is thought to regulate the mitochondrial respiratory electron flow and to protect plant cells from oxidative damage (Maxwell et al., 1999). In C. reinhardtii, evidence for the presence of an AOX has been provided (Matagne et al., 1989; Eriksson et al., 1995) and the expression of two genes, AOX1 and AOX2, has been reported (Dinant et al., 2001). The AOX1 gene is more actively transcribed than AOX2 (Dinant et al., 2001) and its expression is strongly dependent on the nitrogen source, being down-regulated by ammonium and stimulated by nitrate (Baurain et al., 2003). In contrast to Chlamydomonas, Polytomella sp. seems to lack an AOX, maybe in relation to the loss of photosynthesis in this colorless alga (Reyes-Prieto et al., 2002).

	CONCLUDING REMARKS

TOP THE STANDARDS OF REFERENCE:... THE CHLAMYDOMONAS OXPHOSOME DATA COMPLEX I COMPLEX II COMPLEX III OR BC1... CYTOCHROME C COMPLEX IV COMPLEX V ADDITIONAL ENZYMES CONCLUDING REMARKS LITERATURE CITED

 
Generally speaking, the OXPHOS components of eukaryotes can be classified into two categories: the core and the supernumerary subunits. The core subunits are the conserved components that usually bind redox components and prosthetic groups and seem to constitute the minimal functional unit of each complex. In general, they have counterparts in the bacterial OXPHOS complexes. The so-called supernumerary subunits may have structural or regulatory functions or a transient role during the biogenesis of each complex. These subunits could additionally be classified into conserved and lineage-specific to distinguish those subunits that are unique to certain species.

Out of 156 protein families that constitute the OXPHOSome or are involved in its biogenesis in eukaryotes, 106 were found to be encoded in Chlamydomonas. Of these algal sequences, 87 have counterparts in mammals, fungi, and higher plants: 65 are subunits of mitochondrial complexes I, II, III, IV, and V, and cytochrome c, while 22 correspond to biogenesis factors. The 19 remaining subunits (including additional enzymes) were found in two or three lineages only. Finally, 10 constituents of OXPHOS complexes seem to be unique to Chlamydomonas (Table I). In particular, seven of these algal-specific subunits pertain to the ATP synthase, which makes this enzyme the most divergent and intriguing OXPHOS complex of the algal-respiratory chain.

The recent release of the complete genome sequence of C. reinhardtii has thus allowed the construction of a comprehensive catalog of the OXPHOS components of the green alga, comprising 116 proteins. At this stage, however, the exhaustive reconstruction of the Chlamydomonas OXPHOSome is necessarily incomplete due to the following factors: (1) the presence of incomplete sequences, assembly, and sequencing errors in the ongoing C. reinhardtii genome sequencing project; (2) the possible presence of other lineage-specific mitochondrial components in the OXPHOSome of Chlamydomonas that escaped identification in database searches; and (3) the very partial biochemical characterization of the OXPHOS complexes in chlorophycean algae. Future biochemical studies will be necessary to get a better view of the OXPHOSome of photosynthetic organisms and of Chlamydomonas in particular.

Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers given in Table I.

	ACKNOWLEDGMENTS

 
P.C. and D.B. are scientific research worker and postdoctoral researcher, respectively, of the Fonds de la Recherche Scientifique, Belgium. We acknowledge the efforts that led to the construction of version 2.0 of the Chlamydomonas database and especially the public access of the information made available by the Department of Energy Joint Genome Institute (U.S. Department of Energy's Office of Science).

Received October 1, 2004; returned for revision November 25, 2004; accepted November 25, 2004.

	FOOTNOTES

 
¹ This work was supported by the Fonds National de la Recherche Scientifique, Belgium (grant nos. 2.4587.04 and 2.4552.01); by the Fonds Spéciaux of the University of Liege; by the National Institutes of Health (grant no. TW01176); by Consejo Nacional de Ciencia y Tecnológia, Mexico (grant no. 27754N); and by Dirección General de Asuntas para el Personal Académico, Universidad Nacional Autónoma de México, Mexico (grant no. IN202598).

² These authors contributed equally to the paper.

www.plantphysiol.org/cgi/doi/10.1104/pp.104.054148.

^* Corresponding author; e-mail c.remacle{at}ulg.ac.be; fax 32–43663840.

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