From the Departamento de Genética Molecular,
Instituto de Fisiología Celular, Universidad Nacional
Autónoma de México, Apartado Postal 70-243, México
04510, D.F. Mexico, § Department of Biochemistry and
Molecular Pharmacology, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107, ¶ Laboratoire de Microséquençage
des Protéines, Département des Biotechnologies, Institut
Pasteur, 75724 Paris Cedex 15, France, and
Departamento de
Biología Molecular y Bioquímica, Facultad de Ciencias,
Universidad de Málaga, E-29071 Málaga Spain
Received for publication, November 9, 2000
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ABSTRACT |
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The mitochondrial genomes of Chlamydomonad algae
lack the cox2 gene that encodes the essential subunit COX
II of cytochrome c oxidase. COX II is normally a single
polypeptide encoded by a single mitochondrial gene. In this work we
cloned two nuclear genes encoding COX II from both Chlamydomonas
reinhardtii and Polytomella sp. The cox2a
gene encodes a protein, COX IIA, corresponding to the N-terminal
portion of subunit II of cytochrome c oxidase, and the
cox2b gene encodes COX IIB, corresponding to the C-terminal region. The cox2a and cox2b genes are located
in the nucleus and are independently transcribed into mRNAs that
are translated into separate polypeptides. These two proteins assemble
with other cytochrome c oxidase subunits in the inner
mitochondrial membrane to form the mature multi-subunit complex. We
propose that during the evolution of the Chlorophyte algae, the
cox2 gene was divided into two mitochondrial genes that
were subsequently transferred to the nucleus. This event was
evolutionarily distinct from the transfer of an intact cox2
gene to the nucleus in some members the Leguminosae plant family.
Mitochondria are thought to descend from free-living
The transfer of mitochondrial genes to the nucleus is an ongoing
process, as shown by the presence of particular genes encoded in both
the mitochondrial and the nuclear genomes in certain species. These are
exemplified by COX II in some members of the family leguminosae (7-9)
and by subunit 9 of ATP synthetase in Neurospora crassa
(10).
The algae of the family Chlamydomonadaceae, including
Chlamydomonas reinhardtii and Polytomella sp.,
lack some of the genes that are typically found in mitochondrial
genomes, including nad3, nad4L, cox2,
cox3, atp6, and atp8
(11-13)1. We have shown
that, in at least two members of this family, C. reinhardtii
and Polytomella sp., the cox3 gene was
transferred to the nucleus, and the corresponding mitochondrial copy
has been lost (14).
Several mitochondrial respiratory chain complexes have been isolated
and characterized from the colorless alga Polytomella sp.
(15-17), a close relative of Chlamydomonas, including an
active, cyanide-sensitive cytochrome c oxidase preparation
(14). In this work, we show that COX II is present as a heterodimer in this complex. All COX II sequences that have been described to date are
single polypeptides encoded by one gene, normally in the mitochondrial
genome. In both Polytomella sp. and C. reinhardtii, COX II is encoded by two nuclear genes that were
named cox2a and cox2b. The cox2a gene
encodes a protein, COX IIA, corresponding to the N-terminal half of a
typical one-polypeptide COX II, and the cox2b gene encodes a
protein, COX IIB, equivalent to the C-terminal half of the same
subunit. We propose that these separate genes give rise to a
heterodimeric COX II that results from the noncovalent assembly of the
COX IIA and COX IIB polypeptides in cytochrome c oxidase of
the inner mitochondrial membrane.
Strain and Culture Conditions--
Polytomella
sp. (198.80, E. G. Pringsheim), from the Sammlung von
Algenkulturen (Gottingen, Germany), was grown as previously described
(15).
Protein Analysis--
Cytochrome c oxidase from
Polytomella sp. was obtained as previously described (14).
Polyacrylamide gel electrophoresis was performed as in Schägger
et al. (18) using 16% acrylamide gels. For tryptic
digestion analysis, gels were stained with Amido Black, and the
polypeptide of interest was excised from the gel. Polypeptides were
isolated as previously described (15) for N-terminal sequencing.
Amino-terminal Edman degradation was carried out on an Applied
Biosystems Sequencer at the Laboratoire de Microséquençage des Protéines, Institut Pasteur, Paris, France. An 18.6-kDa
polypeptide was isolated from polyacrylamide gels and subjected to
tryptic and endolysin-C digestion and separation on DEAE-C14 and
DEAE-C18 HPLC2 columns. Peaks
eluted from the columns were subjected to N-terminal sequence analysis.
Nucleic Acids Preparation--
Total DNA and total RNA from
Polytomella sp. and C. reinhardtii were isolated
as previously described (14). PCR fragments were cloned into pMOS
blue-T (Amersham Pharmacia Biotech) or pGEM-T easy (Promega). cDNA
was prepared from 1-2 µg of total RNA with Moloney murine leukemia
virus reverse transcriptase (Promega) or Superscript II reverse
transcriptase (Life Technologies). All standard molecular biology
techniques were as described (19). Sequencing was carried out by the
Kimmel Cancer Center DNA Sequencing Facility, Thomas Jefferson
University, and at the Unidad de Biología Molecular, Instituto
de Fisiología Celular, Universidad Nacional Autónoma de
México.
Cloning the cox2b Gene from Polytomella sp.--
A genomic
Polytomella sp. cox2b fragment was amplified by
PCR using two degenerate primers: F1 (5'- CA(A/G) GA(C/T) AG(C/T) GC(C/T) AC(A/T) AG(C/T) CA(G/A) GC(C/T) CA(A/G) G-3') based on internal
sequence (QDSATSQAQA) of COX II, and B1 (5'-TG (G/A)TT (C/T)AA (A/G)CG
(A/T)CC (A/T)GG (A/G)AT (A/G)GC (A/G)TC CAT-3') based on the internal
sequence MDAIPGRLNQ. The resulting 300-nt product was used to isolate
genomic cox2b clones from a library of
Polytomella DNA PstI fragments of ~2 kilobases
in length in pBluescript.
The cox2b cDNA sequence from Polytomella sp.
was obtained by PCR and 5'-RACE (20) using primers based on the genomic
sequence obtained above. A poly(dT) tail was added to the 5' end of the cDNA with a terminal transferase (Roche Molecular Biochemicals). The forward primer was oligo(dT)/adapter primer:
5'-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3', and the reverse primer (B2)
was 5'-AGCTGTTTAAGACCATGACTTC-3'.
Cloning the cox2a Gene from Polytomella sp.--
A
cox2a cDNA fragment was amplified using the degenerate
primers F2 (5'-GA(G/A) GC(T/C) CC(T/C) GT(T/C) GC(T/C) TGG CAG CT(T/G) GG-3'), based on the N-terminal protein sequence EAPVAWQLG, and B3
(5'-CCA (A/G)TA CCA CTG (A/G)TG (A/G/T)CC (A/G)AT (A/G)GC C-3'), based
on the internal conserved sequence KAIGHQWYW. Nested PCR was done with
degenerate primers F3 (5'-CAG GA(T/C) TC(T/C) GC(T/C) AC(T/C) TC(T/C)
CAG GC(T/C) CAG G-3'), based on the N-terminal sequence of the protein
QDSATSQAQA and B4 (5'-GA(A/G) TA(A/G) AG(A/G) AG(A/G) GC(A/G)
AA(A/G)GA(A/G) GG-3'), based on the internal conserved sequence
PSFALLYS. For 3'-end cDNA cloning, oligo(dT)/adapter primer and
primer F4 (5'-TCCTCTACCACATCGCCACCC-3') were used. For nested PCR,
adapter (5'-GACTCGAGTCGACATCGA-3') and primer F5
(5'-ACTACACTAAGCAAGCTCTCCCTG-3') were used. For 5'-RACE, primers B5
(5'-TCAGGGAGAGCTTGCTTAGTGTAG-3'), with oligo(dT)/adapter primer, and B6
(5'-TTGGTGGCGATGTGGTAGAGG-3'), with adapter for nested PCR, were used.
Primers F6 (5'-AATGCTCGCCCAGCGTATC-3') and B7 (5'-AAACCTTCACACACCCATAGGC-3'), derived from the 5'- and 3'-RACE products, were used to amplify the full-length cDNA of the
cox2a gene. The genomic sequence of the
Polytomella sp. cox2a gene was obtained by PCR
amplification of total Polytomella sp. DNA with the same primers.
Cloning the cox2a and cox2b Genes of C. reinhardtii--
The
cDNAs for cox2a and cox2b were obtained by
screening a C. reinhardtii cDNA library in
A bacterial artificial chromosome clone containing
cox2b was obtained from a BAC genomic library from C. reinhardtii (22) by Genome Systems using the C. reinhardtii
cox2b cDNA obtained above. BAC DNA was sequenced directly
using internal primers.
Protein Sequence Analysis--
Mitochondrial targeting sequences
were analyzed using MitoProt II (23, 24). The same program was used to
calculate the segments with high local hydrophobicity (<H>) in a
distance comprising 13 to 17 residues. The mesoH was
determined by scanning each sequence for a maximum average
hydrophobicity measured in windows from 60 to 80 residues and averaging
the values (24). More hydrophobicity scales were included to reduce the
possibility of bias. Protein transmembrane stretches were predicted
using the program TodPred II (25). Three-dimensional structure modeling
was carried out using SWISS-MODEL (26).
Data Base Accession Numbers--
The nucleotide sequences are in
the DDBJ/EMBL/GenBankTM nucleotide sequence data
base under the accession numbers AF305078 (Polytomella sp.
cox2a cDNA), AF305079 (Polytomella sp.
cox2b cDNA), AF305080 (C. reinhardtii cox2a
cDNA), AF305540 (C. reinhardtii cox2b
cDNA), AF305541 (Polytomella sp. genomic cox2a), AF305542 (Polytomella sp. genomic
cox2b), and AF305543 (C. reinhardtii genomic
cox2b).
Subunit II Is Present in the Cytochrome c Oxidase Complex from
Polytomella sp.--
An active cytochrome c oxidase from
Polytomella sp. was isolated as previously described (14).
The four largest polypeptides with apparent molecular masses of 54.6, 29.6, 18.6, and 14.5 kDa were subjected to Edman degradation. The
54.6-kDa polypeptide, not susceptible to Edman degradation, was
identified as subunit I of cytochrome c oxidase (COX I)
since its mass was similar to that predicted for the mitochondrial
cox1 gene sequence (54,781 Da (27)). The 29.6-kDa
polypeptide (N-terminal sequence: SSDAGHHLSPRERYLV) was previously
identified as subunit III (14). The N-terminal sequence of the 14.5-kDa
polypeptide, DANSSELVLEPTRKYKAGLATRELW, did not show similarity with
any sequences in GenBankTM.
The N-terminal sequence of the 18.6-kDa polypeptide,
EAPVAWQLGFQDSATSQAQA, was similar to COX II from other species,
allowing identification of this polypeptide as COX II. A second
sequence, MDAIPGR(R/L)NQIWLTINREG, was obtained during the Edman
degradation of the 18.6-kDa polypeptide region at a yield of less than
50% that of the yield of the first sequence. This sequence also was similar to COX II from other species and was identified as an internal
fragment of COX II that was obtained after partial cleavage of the
protein during Edman degradation.
Cloning of the cox2b Gene from Polytomella sp. and C. reinhardtii--
On the basis of the primary amino acid sequences
obtained for COX II, two degenerate oligodeoxynucleotide primers were
designed. Using these primers, a PCR amplification product of 300 nt
was obtained using total DNA from Polytomella sp. as a
template. This PCR product encoded the C-terminal portion of the
cox2 gene but lacked the region that encodes the N-terminal
sequence of COX II. The absence of an N-terminal sequence was
attributed to nonspecific annealing of the primer based on the
N-terminal amino acid sequence.
The 300-nt cox2 gene fragment hybridized to a 2-kilobase
PstI fragment of Polytomella sp. total DNA in
Southern analyses. To obtain a full-length gene, a mini-library was
constructed from PstI fragments of ~2 kilobases, and a
positive clone was isolated and sequenced. This genomic sequence
contained a 462-nt open reading frame that encoded a 153-amino acid
protein homologous to the C terminus of COX II. The gene was named
cox2b. There was no open reading frame corresponding to the
N-terminal portion of COX II in the 960 nt preceding the 462-nt open
reading frame.
Using primers based on the genomic sequence, a portion of a cDNA
corresponding to the cox2b open reading frame was amplified from Polytomella sp. total RNA using reverse
transcription-PCR. The remainder of the 5'-cDNA sequence was
obtained by 5'-RACE. The full-length cDNA obtained contained a
65-nt 5'-untranslated region and a 462-nt open reading frame identical
to that of the genomic clone. This showed that this gene encoded only
the C-terminal portion of COX II and did not contain introns. The
overall organization of the cox2b gene is shown in Fig.
1. The predicted protein contained the
MDAIPGRLNQIWLTINREG internal sequence that was obtained from direct
protein sequencing as well as the sequence GQCSEICG known to be the COX
II binding site for binuclear copper (28). The major N-terminal
sequence of COX II, determined by Edman degradation, was not present in
the deduced protein sequence. The N-terminal 43 amino acids lacked
homology to any COX II proteins. The remaining sequence was homologous
with the C-terminal half of many COX II proteins. The highest
similarity was with COX II from the alga Prototheca
wickerhamii (29). Altogether, these data suggested that the
cox2 gene had been split into two genes in
Polytomella sp.
The PCR amplification product of the cox2b gene of
Polytomella sp. was also used to isolate a cox2b
cDNA from a Cloning the cox2a Gene from Polytomella sp. and C. reinhardtii--
To clone the gene that encoded the N-terminal region
of COX II, nested PCR was performed with primers derived from the major N-terminal sequence obtained from the protein, and internal sequences (KAIGHQWYW and PSFALLYS) were conserved among COX II proteins. Using
Polytomella sp. cDNA as a template, a 250-nt PCR product was obtained that exhibited similarity with other cox2
genes. The full-length cDNA, obtained using 5'- and 3'-RACE (Fig.
1), contained an open reading frame of 816 nt predicted to encode a
protein of 271 amino acids including the sequence EAPVAWQLGF determined
for the N terminus of the 18.6-kDa polypeptide. This gene was named
cox2a, since it encoded the N-terminal portion of the COX II
protein (COX IIA).
The N terminus of the mature Polytomella sp. COX IIA
protein, derived from direct sequencing, corresponded to Glu-131 of the predicted sequence, indicating that this protein contains a
mitochondrial targeting sequence of 130 amino acids. The mature protein
is predicted to be 141 amino acids long and to contain two putative
transmembrane stretches, from Ile-28 to Thr-48 and from Val-69 to
Leu-89, and the highly conserved sequence GRQWYWSY present in all
sequences of COX II subunits known to date (Fig. 2). The sequence from
Glu-131 to Glu-246 was homologous with the N-terminal portion of many COX II proteins. This sequence was most similar to COX II from the alga
P. wickerhamii. The COX IIA protein contained a C-terminal 20-amino acid region, lacking similarity to conventional COX II proteins, that had a high density of charged amino acids.
Primers corresponding to the 5' and 3' ends of the cox2a
cDNA sequence of Polytomella sp. were used to amplify a
portion of the cox2a nuclear gene from total DNA. This
1773-nt PCR product was cloned and sequenced. The genomic
cox2a gene contained 6 introns, ranging in size from 84 to
136 nt.
The Polytomella sp. cox2a cDNA was used to
isolate a partial cDNA clone of cox2a from a C. reinhardtii cDNA library. The complete sequence of C. reinhardtii cox2a was obtained by 5'-RACE (Fig. 1). The C. reinhardtii cox2a cDNA contained a 5'- untranslated region of
30 nt, an open reading frame of 855 nt, encoding a protein of 284 amino
acids, and a 3'-untranslated region of 201 nt. The predicted C. reinhardtii COX IIA mature polypeptide exhibited 72% identity and
81% similarity with the sequence predicted for the COX IIA protein
from Polytomella sp. (Fig. 2). The gene sequence predicts an
extension of 21 residues at the C-terminal end that lacked homology to
COX II proteins but that was highly similar to the extension predicted
by the cox2a gene from Polytomella sp.
The cox2a and cox2b Genes of Polytomella sp. and C. reinhardtii Are
Nuclear-localized, Single-copy Genes--
The nuclear location of both
cox2a and cox2b was confirmed by Southern
analysis. After electrophoresis of Polytomella sp. total DNA
through agarose gels, the mtDNA was detected as a discrete band below
the bulk of the nuclear DNA (Fig.
3A, left panel). This was confirmed with a cox1 gene probe (27) that
hybridized with the mtDNA and a
To determine whether cox2a and cox2b were present
as single-copy genes in the genomes of Polytomella sp. and
C. reinhardtii, additional Southern blot analyses were
performed. Single hybridization bands were obtained for the
cox2a and cox2b genes of Polytomella sp. and C. reinhardtii with several restriction enzymes
(Fig. 3, panel B and data not shown). This suggested that
both genes were present in only one copy in their respective genomes.
The expression of cox2a and cox2b of
Polytomella sp. and C. reinhardtii was examined
by Northern analyses. Probes derived from the cox2a and
cox2b genes hybridized to independent transcripts of sizes
consistent with the corresponding cDNA sequences for both algae
(Fig. 3, panel C). The presence of a larger, mature transcript that could suggest a transpliced product was not observed in
any case. The cox2b gene of Polytomella sp.
exhibited a double band. Since the genomic sequence of this
cox2b gene contained three putative polyadenylation sites in
the 3'-noncoding region, it is possible that these bands correspond to
mRNAs that have different sites of polyadenylation.
There is a significant bias in codon usage in these genera of algae
(30), and this bias differs between mitochondrial and nuclear genes
(14). Analysis of the codon usage for the cox2a and
cox2b genes of Polytomella sp. and C. reinhardtii indicated that the codon usage was consistent with
their nuclear localization (data not shown). In addition, the conserved
polyadenylation signals TGTAA (33), present in the vast majority of
nuclear genes in the Chlamydomonad family, were present at the 3' ends
of the cDNA sequences of cox2a and cox2b for
both algae.
Two COX II Polypeptides Are Present in Polytomella sp. Cytochrome c
Oxidase--
Since the primary protein sequences derived from the
18.6-kDa region of a polyacrylamide gel corresponded to the predicted amino acid sequences of both COX IIA and COX IIB from
Polytomella sp., it is likely that this region of the gel
contained both subunits. To confirm that Polytomella sp.
contained two independent COX II polypeptides, the purified cytochrome
c oxidase complex of this alga was subjected to
matrix-assisted laser desorption-time of flight mass spectrometry
analysis. The complex contained two major polypeptides in the expected
mass range for COX IIA and COX IIB, one of 15,984 Da (theoretical
16,222 Da for mature COX IIA) and one of 17,169 Da (theoretical 17,219 Da for full-length COX IIB). The differences between the predicted and
observed masses are likely due to post-translational modifications of
the proteins. The 17,169-Da polypeptide was isolated by reverse
phase-HPLC, trypsinized, and analyzed by matrix-assisted laser
desorption-time of flight mass spectrometry analysis. The trypsin
digestion products exhibited molecular masses that were consistent with
the theoretical molecular masses expected for COX IIB peptides (Table
I).
We wished to determine whether COX IIB contained a cleavable
mitochondrial-targeting sequence or if the novel N-terminal charged domain was present in the mature protein. The observed band of 18.6 kDa
(containing COX IIA and COX IIB) was purified from polyacrylamide gels
and subjected to digestion by trypsin or endolysin followed by HPLC
separation and Edman degradation (Fig.
4). Amino-terminal sequences were
obtained for all the endolysin C-derived fragments, except for the
16.0-kDa polypeptide, which exhibited a blocked N terminus. The
sequence was obtained from COX IIB starting at Asp-5 of the full-length
sequence (tryptic fragment DQLK). That the mass spectrometry analysis
of the mature protein gave a mass consistent with an intact N terminus
suggests mature COX IIB is the full-length protein. Several overlapping
sequences were obtained for the tryptic products and for the endolysin
C fragments of the protein. A total of 154 amino acids out of the 294 residues predicted by the cox2a and cox2b genes
were identified in the 18.6-kDa region of the gel by direct amino acid
sequence analysis. This analysis, using a different cytochrome
c oxidase preparation from that first used for Edman
degradation, confirmed that Glu-131 was the N terminus of the mature
COX IIA. No difference was found between the predicted sequences and
the sequences obtained by Edman degradation.
COX II Is Encoded by Two Distinct Nuclear Genes in the Algae of the
Family Chlamydomonadaceae--
COX II is conserved in cytochrome
c oxidases throughout all phyla. It constitutes the main
core of the enzyme along with COX I and COX III (34). Although the
cox2 gene is absent from the mtDNA of Polytomella
sp.,1 the presence of COX II in cytochrome c
oxidase from Polytomella sp. was confirmed by N-terminal and
internal amino acid sequence analysis of polypeptides with an apparent
molecular mass of 18.6 kDa from the purified complex. This size is
consistent with a 14.5-kDa polypeptide that was identified
immunochemically as COX II in cytochrome c oxidase isolated
from C. reinhardtii (35).
Polytomella sp. and C. reinhardtii each contain
two distinct nuclear genes that encode two COX II homologs.
cox2a encodes a protein, COX IIA, corresponding to the
N-terminal half of a canonical COX II. cox2b encodes a
protein, COX IIB, corresponding to the C-terminal half of a canonical
COX II. The cox2a and cox2b genes from
Polytomella sp. and C. reinhardtii were shown to
be nuclear-localized by Southern hybridization analyses. These genes also displayed other features typical of nuclear genes in Chlamydomonad algae: a biased codon usage, introns in the genomic sequences, and a
TGTAA polyadenylation signal.
COX IIA and IIB were unresolved by SDS-polyacrylamide gel
electrophoresis and migrated with an apparent molecular mass of 18.6 kDa. Mass spectrometry and reverse phase HPLC analyses confirmed that
distinct COX IIA and IIB proteins existed in the purified cytochrome
c oxidase of Polytomella sp. The COX IIB
polypeptide may have a blocked N terminus (MSADKDQL), since only the
major N-terminal sequence EAPVAWQLGF of COX IIA was detected by direct N-terminal sequence analysis of the 18.6-kDa polypeptide.
COX IIA and IIB Have Been Adapted to Facilitate Import into
Mitochondria--
Most nuclear-encoded mitochondrial proteins contain
a mitochondrial targeting sequence that directs the import of the
protein into mitochondria. Such sequences have been identified in the nuclear-encoded cox2 genes from cowpea (7) and soybean (8). The predicted full-length COX IIA proteins each has a mitochondrial targeting sequence of 130 amino acids, since the amino acid sequence obtained for the mature Polytomella sp. COX IIA commences at
Glu-131 of the predicted polypeptide. These sequences are 37%
identical (Fig. 2). The COX III proteins from these organisms also
contain unusually long (>100 amino acids) mitochondrial-targeting
sequences (14). It is possible that during import into mitochondria the targeting sequences may act as chaperones for the import of the COX
subunits and the assembly of the enzyme complex. Alternatively, after
cleavage they may be maintained as components of the cytochrome c oxidase complex, as was observed with the targeting
sequence of the Rieske subunit of beef heart mitochondrial cytochrome
bc1 complex (36).
In contrast to COX IIA, the COX IIB proteins lack a canonical,
cleavable, mitochondrial-targeting sequence, since the COX IIB sequence
was obtained starting at Asp-5 of the full-length sequence (Fig. 4).
Additionally, mass spectrometry analysis of the mature protein gave a
mass consistent with an intact N terminus. However, the N-terminal 43 amino acids in both predicted COX IIB proteins show no similarity with
other known COX II sequences and contains a high density of charged
residues. The numerous negatively charged amino acids are not
consistent with a mitochondrial-targeting sequence, normally
characterized by a paucity of acidic residues and abundance of basic
residues (37). It is possible that the 43-amino acid stretch may
function as a noncleavable mitochondrial-targeting sequence. The
N-terminal region of Polytomella sp. COX IIB possesses a
positively charged amphiphilic
The highest average hydrophobicity over 60-80 amino acids of a
polypeptide chain (termed mesohydrophobicity, or mesoH)
along with the maximum local hydrophobicity (<H>) of likely
transmembrane segments are useful indicators of the likelihood that a
protein could be imported into the mitochondrion (23). Both
Polytomella sp. and C. reinhardtii mature COX IIA
proteins, despite their two transmembrane domains, exhibited <H> and
mesoH patterns significantly lower than mtDNA-encoded COX II
polypeptides from other organisms but still higher than most proteins
imported into mitochondria (data not shown). The nuclear-encoded COX II
proteins from legumes were also less hydrophobic than their
mtDNA-encoded counterparts and more similar to the COX IIA proteins of
the Chlamydomonad algae.
The low hydrophobicity parameters exhibited by the COX IIB proteins may
also facilitate their import into mitochondria. These proteins contain
no transmembrane stretches, exhibit very low <H> and mesoH
values, and are predicted to be imported readily into mitochondria
(data not shown).
Assembly of COX IIA and COX IIB in Cytochrome c Oxidase--
The
normal COX II subunit exhibits two transmembrane stretches and a
hydrophilic region containing the consensus copper binding site. We
predict that the two mature Chlamydomonad COX II polypeptides would
assemble to give a heterodimeric COX II with an overall structure
similar to that determined by x-ray crystallography of the bacterial
and mammalian monomeric COX II subunits (28, 40).
Hydropathy profile analysis predicted two transmembrane stretches for
the COX IIA polypeptides from both algae (data not shown), similar to
conventional COX II proteins. In addition, both contain a C-terminal
20-amino acid region that has a high density of charged amino acids and
is not homologous to known COX II proteins. The two COX IIB
polypeptides contain a 42-amino acid extension at their N terminus that
also exhibits a high density of charged amino acids and that is also
not homologous to known COX II proteins. We propose that the C-terminal
extension of COX IIA interacts with the N-terminal extension of the COX
IIB protein. This interaction may stabilize the two COX II subunits in
the cytochrome c oxidase complex (Fig.
5). According to this model, the extra
loop formed by the interaction of the N-terminal and C-terminal
extensions is topologically distant from the site of interaction of
soluble cytochrome c with the COX IIB subunit.
Evolutionary Considerations--
The transfer of cox2
genes from the mitochondria to the nucleus in the Chlamydomonad algae
satisfies many of the criteria proposed by Brennicke et al.
(41) and Claros et al. (42) for this event: acquisition of a
region encoding a mitochondrial-targeting sequence (at least for
cox2a), altered codon usage, acquisition of a
polyadenylation signal, and diminished <H> and
mesoH of the protein products. In addition, any
mitochondrial cox2 genes that presumably existed have been
eliminated, suggesting that this transfer occurred early in evolution.
There are several precedents for mitochondrial genes being split into
two. The mitochondrial nad1 genes of Tetrahymena
pyriformis and Paramecium aurelia (43) and rapeseed
mitochondrial gene encoding a homologue of the bacterial protein Ccl1
(44) have also been divided into two independently transcribed reading
frames, although retained on the mtDNA.
It is possible that in some organisms the transfer of cox2a
and cox2b to the nucleus is ongoing, as in some legumes, or
has been arrested at an intermediate stage. cox1,
cox2, and cox3 were present in the mtDNA of the
Chlorophycean alga Scenedesmus obliquus (45, 46). However,
the cox2 gene was truncated and was predicted to be a
pseudogene. The putative Scenedesmus COX II protein exhibits high similarity with COX IIA from Polytomella sp. and
C. reinhardtii (Fig. 2), although it lacks a
mitochondrial-targeting sequence, as would be expected for a
mtDNA-encoded protein. The C-terminal region of the
Scenedesmus COX II is also similar to the unique C-terminal
regions of COX IIA of Polytomella sp. and C. reinhardtii. We suggest that the mitochondrial cox2
gene from S. obliquus is likely to be an active
cox2a gene and that the missing fragment of the gene
(i.e. cox2b) has been transferred to the nucleus.
The gene content and fragmentation pattern of the ribosomal RNA genes
on the mtDNA of S. obliquus suggest that this mitochondrial genome represents an intermediate stage between the
Prototheca-like green algae and the
Chlamydomonas-like green algae (45). S. obliquus
may represent a stage of green algal evolution in which the
cox2b gene (encoding for a highly hydrophilic polypeptide) has been transferred to the nucleus, whereas the cox2a gene
(encoding a more hydrophobic polypeptide) is retained in the
mitochondrial genome. Since the mitochondrial genetic code of S. obliquus has diverged from the standard genetic code (45, 46), it
is likely that the cox2a gene has been retained on the mtDNA
since it could not be functionally transferred to the nucleus in its
current form. We hypothesize that two independent cox2a and
cox2b genes existed in the mitochondrial genome of the
common ancestor of Chlamydomonad algae and that these genes were
transferred from the mitochondria to the nucleus before the separation
of the colorless genera of Polytomella from the main
photosynthetic lineage of Chlamydomonas.
The functional transfer of intact cox2 genes to the nucleus
has been reported in detail for members of the legume subfamily Papilionoideae (7, 8, 9). Our analysis of the Chlamydomonad cox2a and cox2b genes suggests that the division
of the mitochondrial cox2 gene into two nuclear genes that
encode two independent proteins is an evolutionary event unrelated to
the functional gene transfer of intact mitochondrial cox2
genes to the nucleus in some legumes.
A number of organisms, particularly protists, have mitochondrial
genomes that lack the cox2 gene, including the
apicomplexan protozoan Plasmodium falciparum (47) and
the Chlorophyte alga Pedinomonas minor (1), whose mtDNA has
been sequenced. It is likely that these organisms have transferred
their cox2 genes to the nucleus and that, in the green alga
P. minor, this has also involved the transfer of
cox2a and cox2b to the nucleus. Further analyses
of such organisms will determine whether the process outlined here has
an evolutionary lineage outside of the Chlorophyta.
The present work highlights a novel strategy utilized by some
Chlorophyte algae that allowed functional transfer of a mitochondrial gene to the nucleus. This strategy has allowed the functional transfer
to the nucleus of a mitochondrial gene encoding an essential, hydrophobic subunit of a crucial respiratory complex. It is remarkable that a protein with such specific functional requirements
(e.g. a cytochrome c docking site, binding site
for metals involved in catalysis) should have evolved into two
components containing added domains that maintain the ability of
the protein to perform its roles.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-proteobacteria (1), whose closest extants are bacteria of the genus Rickettsia (2). After the endosymbiotic event, there was a transfer of genes from the protomitochondrion to the nucleus such that
few genes now remain in mitochondrial genomes (3). Those genes that
remain encode only a sub-set of the mitochondrial proteins needed for
oxidative phosphorylation and a portion of the factors necessary for
their expression (4). The mtDNA-encoded respiratory chain subunits are
highly hydrophobic polytopic proteins that contain two or more
transmembrane stretches (5, 6). The genes for nad1,
nad2, nad3, nad4, nad4L,
nad5, and nad6 (encoding subunits 1, 2, 3, 4, 4L,
5, and 6 of NADH:ubiquinone oxidoreductase), cob (encoding
cytochrome b of the bc1 complex),
cox1, cox2, and cox3 (encoding
subunits COX I, COX II, and COX III of cytochrome c
oxidase), and atp6 and atp8 (encoding subunits
a and A6L of the F0 portion of ATP synthetase)
are found in the mitochondrial genomes of most organisms.
EXPERIMENTAL PROCEDURES
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DISCUSSION
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gt10 (21)
using the Polytomella sp. cox2a cDNA or
cox2b genomic DNA as probes. A total of 17 positive clones
were obtained from 5 × 104 plaque-forming units
screened. PCR using two primers based on
gt10 sequences was used to
identify the positive clones with the largest inserts. Phage DNA from
these clones was isolated with the Qiagen Lambda mini kit. The
5' end of the cox2a cDNA was completed by RACE using
primers based on the cDNA sequence obtained.
RESULTS
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ABSTRACT
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DISCUSSION
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View larger version (23K):
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Fig. 1.
Organization of the cox2a and cox2b genes of
Chlamydomonad algae. The coding regions of cox2a
and cox2b cDNAs from Polytomella sp. and
C. reinhardtii are indicated by a box. The
regions that are conserved with other COX II proteins are
white, the putative mitochondrial-targeting sequences are
striped, and the highly charged sequences unique to the
Chlamydomonad cox2 genes are stippled.
Nontranslated regions are shown as a black line. Putative
polyadenylation signals are indicated as vertical black
bars. The locations of the highly conserved COX II sequences
GRQWYWSY and GQCSE(I/L)CG are indicated above the boxes. The single
cox2 mitochondrial gene of P. wickerhamii,
representing a canonical cox2 gene, is included for
comparison.
gt10 cDNA library of C. reinhardtii.
A comparison of this cox2b cDNA with the genomic
sequence (see below) showed that this cox2b cDNA
sequence lacked the first four codons of the cox2b gene and
a 5'-untranslated region. It exhibited 85% identity with the
Polytomella sp. cox2b (Fig.
2). The cox2b cDNA from
C. reinhardtii was used to isolate cox2b from a
C. reinhardtii BAC genomic library. The genomic
cox2b sequence contained the complete coding region and was
identical to the cDNA except for the presence of one intron of 187 nt, located 21 nucleotides upstream of the stop codon. There was no
open reading frame encoding a known protein in the 1.7 kilobases
preceding the start codon for the C. reinhardtii cox2b gene.
The predicted COX IIB protein was 153 amino acids long and was 85%
identical and 92% similar to COX IIB of Polytomella sp.
Both C. reinhardtii and Polytomella sp. are
therefore likely to use two genes to code for COX II.
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Fig. 2.
Sequence comparison of COX II, COX IIA, and
COX IIB polypeptides. Shown are the sequences of COX II of
P. wickerhamii (Pw), Vigna
unguiculata (Vu), and S. obliquus
(So). Also shown are COX IIA and COX IIB of C. reinhardtii (Cr), and Polytomella sp.
(Ps). A black bar indicates the end of the COX
IIA protein of C. reinhardtii and Polytomella sp.
and the N terminus of the COX IIB protein. An asterisk
denotes an identical amino acid, and a colon indicates a
similar amino acid. The 20-amino acid extension at their C terminus of
COX IIA and the 42-amino acid extension at the N terminus of COX IIB
are boxed. These unique extensions of COX IIA and COX IIB
are hypothesized to interact for the assembly of the two COX II
subunits in the cytochrome c oxidase complex. The putative
transmembrane stretches are indicated within gray boxes. The
black triangle indicates the N terminus of the mature COX
IIA that was determined for Polytomella sp. The highly
conserved COX II regions G(R/H)QWYWSY present within COX IIA and
GQCSE(I/L)CG present within COX IIB are underlined.
Numbers indicate the last amino acid for each protein.
-tubulin gene probe (30) that
hybridized with the nuclear DNA (Fig. 3A, left
panel). The cox2a and cox2b genes of
Polytomella sp. hybridized with the major DNA fraction and
not with the mtDNA band, confirming their nuclear localization. A
similar analysis was carried out with total DNA from C. reinhardtii using a cox1 gene probe that hybridized
with the mtDNA (31) and a cytochrome c gene probe (32) that
hybridized with the nuclear DNA (Fig. 3A, left
panel). Those results confirmed that the cox2a and
cox2b genes were also nuclear-encoded in C. reinhardtii.
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Fig. 3.
The cox2a and
cox2b genes are single-copy, nuclear-localized, and
functionally expressed in Chlamydomonad algae. A,
nuclear localization of the cox2a and cox2b
genes. Thirty µg of total DNA from Polytomella sp.
(left panel) or C. reinhardtii (right
panel) was electrophoresed through a 0.7% agarose gel. The first
lanes (DNA) of both panels are photographs
of the ethidium bromide-stained sample after electrophoresis. The
positions of the nuclear DNA (nu) and mtDNA (mt)
are shown by arrows. The remaining lanes show
autoradiograms after Southern analysis. The left panel shows
Polytomella sp. DNA hybridized with probes for mtDNA-encoded
Polytomella sp. cox1 (second lane),
nuclear DNA-encoded Polytomella agilis -tubulin
(TubB1, third lane), and Polytomella
sp. cox2a (fourth lane) and cox2b
(cox2b, fifth lane) genes. The right
panel shows Southern analyses of the C. reinhardtii DNA
hybridized with mtDNA-encoded C. reinhardtii cox1 (second
lane), nuclear DNA-encoded cytochrome c of
C. reinhardtii (Cyc, third lane), and
C. reinhardtii cox2a (fourth lane) and
cox2b (cox2b, fifth lane) genes.
B, cox2a and cox 2b are single-copy
genes. Total DNA from Polytomella sp. (left) was
digested with EcoRV (lane1) or PvuII
(lane 2) for cox2a and EcoRV
(lane1) or EcoRI (lane 2) for
cox2b. Total DNA from C. reinhardtii
(right) was digested with KpnI (lane1)
or XbaI (lane2) for cox2a and
KpnI (lane 1) or HindIII
(lane2) for cox2b. Shown are autoradiograms of
the Southern blot analyses of the restricted DNAs hybridized with
probes for cox2a or cox2b. C, Northern
blot analysis. Shown are autoradiograms of Northern blots of total RNA
isolated from Polytomella sp. (left) or C. reinhardtii (right) hybridized with probes specific for
cox2a and cox2b genes from Polytomella
sp. and C. reinhardtii. The sizes calculated for the
hybridizing RNAs are indicated to the left of the autoradiograms.
bp, base pairs.
Mass spectrometry analysis of the tryptic fragments obtained from the
COX IIB subunit of Polytomella sp
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Fig. 4.
Protein sequence analysis of the COX IIA and
COX IIB polypeptides of Polytomella sp. Shown are
the amino acid sequences of COX IIA and COX IIB. The polypeptides
present in the region of the polyacrylamide gel (containing COX IIA and
COX IIB) were isolated and subjected to digestion by trypsin or
endolysin, HPLC separation, and Edman degradation. The N-terminal
sequences were obtained for the endolysin C-derived fragments and
tryptic digestion products of the proteins. The amino acids directly
identified by these amino acid sequence analyses are in bold
italic characters. The regions identified by N-terminal Edman
degradation analysis of the 18.6-kDa region of the polyacrylamide gel
are boxed. A total of 154 amino acids out of the 294 amino
acids were identified in this analysis. No difference was found between
the predicted sequences and the sequences obtained by Edman
degradation.
DISCUSSION
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DISCUSSION
REFERENCES
-helix from position 19 to 44 with a
high hydrophobic moment (µH ranging from 5.98 to 7.42) and a very
hydrophobic face (maximum hydrophobicity ranging from 3.55 to 6.35).
Similarly, the N-terminal region of C. reinhardtii COX IIB
possesses a putative amphiphilic helix from residues 14 to 32, with
µH ranging from 7.93 to 8.92 and a hydrophobic face (maximum
hydrophobicity ranging from 4.70 to 6.44). The amphiphilicity is
essential for the function of a mitochondrial targeting sequence (38).
Alternatively, there may be no conventional N-terminal, cleavable
mitochondrial targeting sequence, as in eukaryotic cytochrome c. Like COX IIB, cytochrome c is imported into
the inner membrane space, but a minimum length of N-terminal region
rather than a specific sequence is necessary for efficient
mitochondrial import (39).
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Fig. 5.
Model of the interaction of COX IIA with COX
IIB to form the heterodimeric COX II of Chlamydomonad algae. A
three-dimensional structure of Polytomella sp. COX IIA and
COX IIB was modeled with SWISS-MODEL based upon the three-dimensional
structure of COX II from the bovine enzyme (28). This model proposes an
interaction between the unique COX IIA C-terminal domain and the highly
charged COX IIB N-terminal domain. We hypothesize that the loop formed
by the interaction of the N-terminal and C-terminal extensions is
important for the proper assembly of the two COX II subunits in
cytochrome c oxidase in the mitochondrial inner
membrane.
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ACKNOWLEDGEMENTS |
---|
We thank Yun-Lu and T. A. Neubert (New York University) for mass spectroscopy analysis, M. W. Gray (Dalhousie University) and J. D. Palmer (Indiana University) for encouragement at the starting point of this project, L.-G. Franzén (Goteborg University) for the kind gift of the cDNA library of C. reinhardtii, L. Ongay, G. Codiz, and M. Sosa (Unidad de Biología Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México) for the synthesis of various oligonucleotides, and several colleagues and researchers who kindly provided DNA probes: T. D. Fox (Cornell University), M. Goldschmidt-Clermont (University of Geneva), E. H. Harris (Duke University), J. D. Palmer (Indiana University), and C. D. Silflow (University of Minnesota). We are also indebted to A. Atteia and A. Gómez-Puyou (Instituto de Fisiología Celular, Universidad Nacional Autónoma de México) and D. W. Krogmann (Purdue University) for critical review of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants TW01176 and HL59646, Consejo Nacional de Ciencia y Tecnologia, Mexico Grant 27754N, and Dirección General de Apoyo al Personal Académico, Universidad Nacional Autónoma de México Grant IN202598.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF305078 (Polytomella sp. cox2a cDNA), AF305079 (Polytomella sp. cox2b cDNA), AF305080 (C. reinhardtii cox2a cDNA), AF305540 (C. reinhardtii cox2b cDNA), AF305541 (Polytomella sp. genomic cox2a), AF305542 (Polytomella sp. genomic cox2b), and AF305543 (C. reinhardtii genomic cox2b).
** To whom correspondence should be addressed. Tel.: 011-525-622-5620; Fax: 011-525-622-5611 or 011-525-548-0387; E-mail: dhalphen@ifisiol.unam.mx.
Published, JBC Papers in Press, November 27, 2000, DOI 10.1074/jbc.M010244200
1 S. Funes, A. Antaramian, and D. González-Halphen, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HPLC, high precision liquid chromatography; mesoH mesohydrophobicity, mtDNA mitochondrial DNA; nt, nucleotide(s); PCR, polymerase chain reaction; <H>, maximum local hydrophobicity of a sequence segment; µH, hydrophobic moment; RACE, rapid amplification of cDNA ends; BAC, bacterial artificial chromosome.
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