(Received for publication, December 18, 1995; and in revised form, February 27, 1996)
From the
We report the cloning, nucleotide sequence, evolutionary analysis, and intracellular localization of SIG81, a silica-induced cDNA from mouse macrophages. The cDNA encodes a 111-amino acid protein with extensive sequence identity with members of the mammalian cytochrome c oxidase subunit VIIa (COX7a) family. A human SIG81 sequence >80% identical with the mouse cDNA was deduced from homologous sequences in the human expressed tags data base. The deduced amino-terminal region shows features common to mitochondrial targeting sequences. A phylogenetic analysis of the carboxyl-terminal domain homologous to COX7a identifies SIG81 as a divergent member of the family with an ancient origin. Southern blot analysis showed that the mouse genome contains two to three copies of the SIG81 gene. Northern blot analysis revealed that the SIG81 transcript is approximately 1 kb and expressed in every tissue tested, with higher levels of expression observed in kidney and liver. Antibodies raised against a glutathione S-transferase SIG81 fusion protein detected a 13.5-kDa protein that co-fractionates with mitochondrial localized enzymatic activity. Taken together, our data suggest that SIG81 is a novel member of the COX7a family that is constitutively expressed in mouse cells.
The activation of macrophage cells characterized by an increase
in the secretion of cytokines and inflammatory mediators can be
achieved by silica particles in an in vitro model that mimics
the initial development of certain fibrosing lung diseases(1) .
Using mouse RAW 264.7 macrophages, we constructed a subtracted cDNA
library enriched in sequences overexpressed in silica-treated cells.
The differential screening of the library led to the isolation of nine
cDNAs corresponding to genes induced by silica(2) . SIG81
(silica-induced genes, clone 81) was expressed as a 1-kb ()transcript induced more than 3-fold by silica in RAW 264.7
cells(2) . When we studied the expression pattern of the SIG81
gene, we found that SIG81 responded to inflammatory mediators such as
interferon-
and bacterial lipopolysaccharide with modest increases
in mRNA levels and consistently showed a more than 2-fold induction in
response to the activation of protein kinase C or an increase in
intracellular calcium (2) . The analysis of a partial
nucleotide sequence from SIG81 revealed a significant similarity to the
liver isoform of cytochrome c oxidase subunit VIIa (COX7a).
COX (cytochrome-c oxidase, EC 1.9.3.1) is an essential
enzyme in the respiratory chain of eukaryotic organisms that catalyzes
the transfer of electrons from cytochrome c to molecular
oxygen in the terminal reaction of the mitochondrial electron transport
chain (for review, see (3) ). The COX enzyme is a
metalloprotein complex that, in mammalian cells, consists of 13
subunits: the three largest subunits are encoded in the mitochondrial
DNA, and the remaining 10 smaller subunits are coded by the nuclear
genome(4) . The mitochondria-encoded I, II, and III subunits
are the catalytic core of the enzyme, whereas it has been advanced that
the nuclear subunits are regulators of the enzymatic activity of
COX(5) , either as allosteric receptors (6) or
regulating proton translocation(7) . In mammals, three of the
nuclear subunits, among them COX7a, are present as tissue-specific
isoforms(3) , namely the liver (COX7a-L) and heart/muscle
(COX7a-H) isotypes, which differ both in amino acid sequence and in
expression patterns. Thus, in humans and bovines, COX7a-H is the
predominant isoform in heart and skeletal muscles (8, 9, 10, 11) , although low levels
are detected in smooth muscle(12) . The COX7a-L isoform is
widely expressed in many tissues, including heart and
muscle(7, 10, 11) . In several species such
as rat (9) and mouse (13) , COX7a-L is the only isoform
so far identified. As deduced from the corresponding cDNAs described
from human(14) , bovine(15) , rat (16) , and
mouse, ()COX7a-L is translated as an 83-amino acid precursor
protein. The amino-terminal domain containing the first 23 residues of
the protein is a mitochondrial targeting presequence that is cleaved
after mitochondrial import (10) to generate the mature form of
the protein. Although the function of COX7a in the COX complex is still
undefined, its presence is required for maintaining normal levels of
COX activity in human liver cells (17) and, in yeast, its
homolog COX7 is implicated in the assembly of the functional COX
complex(18) .
To gain some insight into the role of the SIG81 protein and its relationship to the bona fide COX nuclear subunits, we determined the complete sequence of the mouse SIG81 cDNA. In the present study, we demonstrate that the protein encoded in the SIG81 cDNA co-fractionates with mitochondria in subcellular fractionation experiments. We also report that SIG81 exhibits widespread expression and we present an evolutionary analysis showing that SIG81 is related to COX7a with a degree of divergence that points to an ancient origin. We propose that SIG81 may be a hitherto unrecognized member of the family of COX7a proteins.
Figure 3:
Phylogeny of the SIG81 genes. A,
alignment of mammalian SIG81 and COX7a protein sequences. Asterisks indicate conserved residues. Boldface indicates amino
acids present in a majority of the sequences. Dots indicate
gaps introduced during the alignment to maximize amino acid identity.
COX7a sequences were retrieved by GenBank accession
numbers: X15822 (human COX7a-L), M83186 (human COX7a-M), X15235 (bovine
COX7a-L), M83299 (bovine COX7a-M), X58486 (mouse COX7a-L), and X54080
(rat COX7a-L). B, phylogenetic distance tree based on the
SIG81 and COX7a protein sequences. Distances between sequences are
represented by the horizontal length of the branches. The scale bar indicates the distance corresponding to 10
differences in 100 positions.
For Western blot analysis, the polyvinylidene difluoride membranes were blocked with 3% nonfat dry milk in Tris-buffered saline at 4 °C overnight, then incubated with anti-SIG81 antiserum (1:200) or affinity-purified antibodies in blocking buffer for 2 h, and washed twice with Tris-buffered saline containing 0.1% Tween 20 for a total of 30 min. Membranes were incubated with a 1:10000 dilution of a peroxidase-linked goat anti-rabbit IgG (Sigma) in blocking buffer for 1 h, washed twice, and developed with the ECL system (Boehringer Mannheim) according to the manufacturer's instructions.
Figure 1:
Sequence of SIG81
cDNA and deduced amino acid sequence. A, the single open
reading frame is shown in uppercase letters; the 5`- and
3`-UTRs are shown in lowercase letters. The deduced amino acid
sequence of SIG81 in the one-letter code is indicated above the nucleotide sequence, aligned with the first nucleotide of each
codon. The numbers at the left and right sides indicate nucleotides and amino acids, respectively. Basic (+)
and hydroxylated () residues are shown above the
sequence corresponding to the putative mitochondrial targeting
presequence (positions 1-55). The arrow indicates the
amino terminus of the sequence with similarity to processed COX7a
proteins. The putative polyadenylation signal is shown in boldface
type. B, Northern analysis of total RNA extracted from
RAW 264.7 (lane M) and human T47D (lane H) cells.
Blots were hybridized with random-primed labeled mouse SIG81 (upper
panel) or 28 S rRNA (lower panel) probes. C,
schematic representation of the pairing of human ESTs with homology to
mouse SIG81. EST identification number in the data base is shown on the left, and identity values are shown on the right.
A FASTA search of the
GenBank/EMBL data bases with the entire SIG81 nucleotide
sequence as a query revealed a high similarity to 11 human ESTs (Fig. 1C) isolated from six independent cDNA libraries
(fetal liver spleen, fetal heart, infant brain, adult aorta, adult
blood, and adult lung). When the sequences of SIG81 and the 11 ESTs
were aligned, those ESTs encompassing the putative SIG81 coding region
(H09013, H60257, R10896, R10947, R58795, R91485, and T39299) showed a
higher degree of sequence conservation (77-83% identity) than the
ESTs pairing with the 3` UTR (D62561, H09580, H60563, and R98944).
Thus, the 3` UTRs, although they exhibit 66-72% identity are more
divergent that would be expected for sequences with few evolutionary
constraints. A putative human SIG81 cDNA sequence was assembled from
EST and, by analogy to mouse SIG81, results in a cDNA with an estimated
size of 900 pb comparable to that of mouse cDNA (data not shown). The
sizes of mouse and human SIG81 mRNAs were estimated by Northern blot
analysis of total RNA prepared from mouse RAW 264.7 macrophages and
human T47D cells, respectively. Using mouse SIG81 as a probe, we found
a unique 1-kb hybridizing band in both RNA preparations, thus
confirming the size estimated from the cDNA sequences (Fig. 1B).
The conceptual translation of the open reading frame in the mouse SIG81 cDNA resulted in a 111-amino acid protein with a calculated molecular mass of 12,385 Da. Although the inferred human homolog translated from the assembled ESTs contains two extra residues, both species share >91% identical amino acids. A hydropathy plot obtained by the Kyte-Doolittle method (33) predicted a largely hydrophilic peptide with two hydrophobic regions that might represent potential membrane-associated regions at amino acids 34-48 and 85-105 (Fig. 2). A search for protein motifs using the ProSite data base (Release 12.2) revealed pattern matches for two protein kinase C phosphorylation sites (amino acids 9 and 54), one casein kinase II phosphorylation site (amino acid 49), one tyrosine kinase phosphorylation site (amino acid 84), and two N-myristoylation sites (amino acids 14 and 94). Remarkably, a similar search of the human SIG81 protein translated from the EST-assembled cDNA revealed that only the protein kinase phosphorylation site at position 54 is not present in human SIG81, thus implying that at least several of the other motifs may be functionally critical. FASTA comparison between mouse SIG81 protein and sequences in the SwissProt and NBRF protein data bases found that SIG81 is very similar (>54% identity) to the nuclear-encoded COX7a. Interestingly, their similarity is nevertheless restricted to a 57-residue carboxyl-terminal stretch (hereafter termed the COX7a-homology domain), whereas the amino-terminal domains differ both in size (55 residues in SIG81, 23 residues in COX7a-L, and 21 residues in COX7a-H) and sequence (see below).
Figure 2: Hydrophobicity plot of mouse SIG81 protein. The hydropathy plot was obtained with the method of Kyte and Doolittle (33) with a moving window of 7 amino acids. The amino acid positions are indicated on the x-axis; the hydrophobic (+) or hydrophilic(-) indices are shown on the y-axis.
Figure 4: Expression of SIG81 mRNA in mouse tissues. Northern blot analyses were performed with 15 µg of total cellular RNA extracted from six adult Swiss mouse tissues: heart, liver, skeletal muscle, kidney, brain, and testes. Blots were subsequently hybridized to radiolabeled mouse SIG81 (A), COX7a-L (B), and 28 S rRNA (C) probes.
Figure 5:
Southern analysis of mouse genomic DNA.
Twenty µg of mouse thymus DNA were digested with the indicated
restriction enzymes, fractionated by electrophoresis, blotted, and
probed with the entire radiolabeled SIG81 cDNA probe. The scale at the left side shows the sizes in kilobases of HindIII-digested -DNA.
Figure 6:
Western blot detection of SIG81 protein. Lanes 1, eluate (1 µl, 300 µg/ml) from
glutathione-Sepharose gel obtained from pGEX3X-transformed bacteria
after a 6-h induction with IPTG; lanes 2, postnuclear
supernatant (100 µg of total protein) from mouse liver obtained
after a 750 g centrifugation as described under
``Experimental Procedures.'' Proteins were electrophoresed in
a high resolution SDS-PAGE gel (29) and electrophoretically
transferred to Hybond polyvinylidene difluoride membrane without
staining, and then detected with rabbit preimmune serum (A) or
affinity-purified anti-GST-SIG81 antibodies (B). The scale at the left shows the sizes in kDa of molecular mass
standards.
The amino terminus of the peptide product coded by SIG81 is enriched in basic (three lysines) and hydroxylated (three phenylalanines, four serines, and three threonines) amino acids (Fig. 1) reminiscent of mitochondrial targeting sequences(35) . Because of the remarkable homologies between the carboxyl-terminal regions of SIG81 and the mitochondrial COX7a, we addressed the question of whether SIG81 is also localized in the mitochondria. We performed a subcellular fractionation of mouse liver into four crude subfractions enriched in nuclear, mitochondrial, microsomal, and cytosolic compartments(28) . The mitochondrial-enriched subfraction was further purified by differential gradient centrifugation. Aliquots from the initial four subfractions and from the gradient fractions were analyzed by Western blotting using anti-GST-SIG81 antibodies. To position the mitochondria within the gradient, aliquots were also assayed for COX activity. As shown in Fig. 7, the distribution of the 13.5-kDa protein along the gradient fractions parallels that of the COX enzymatic activity, and it is not detected in subfractions corresponding to any other subcellular compartment. Thus, the correlation of the distribution of the anti-SIG81 reactive protein with the COX activity strongly supports the notion of a mitochondrial localization for SIG81.
Figure 7:
Subcellular localization of SIG81 protein.
A mouse liver homogenate was fractionated into subfractions sedimenting
at 750, 8,000, and 100,000 g and the 100,000
g supernatant. The 8,000
g pellet was
separated on a discontinuous density gradient (see ``Experimental
Procedures''). Sixty 50-µl fractions were collected from bottom to top. Upper panel, aliquots from the 750 and
the 100,000
g pellets and the 100,000
g supernatant, indicated as N, P
,
and S
, respectively, and from the indicated
gradient fractions (10-50) were resolved by a
Tris/Tricine SDS-polyacrylamide gel electrophoresis, processed for a
Western blot analysis, and probed with the anti-GST-SIG81 antibodies. Lower panel, COX activity in gradient fractions normalized to
percent maximum levels.
We report herein the isolation and characterization of the cDNA encoding mouse SIG81, a novel gene whose product appears to be closely related to the nuclear subunit COX7a of the mitochondrial COX complex. Remarkably, the average 91% identity between the inferred mouse and human SIG81 protein sequences is significantly higher than the homologies between mouse and human COX7a-L (69% identity), or human and bovine COX7a-H (61% identity). For the carboxyl-terminal region of SIG81, which we have termed the COX7a-homology domain, the similarity between SIG81 and the COX7a isoforms is comparable to the values found between the isoforms themselves. Close examination of the SIG81 sequences confirmed that the COX7a-homology domain includes 11 amino acids that are present in the mature COX7a peptides from yeast to mammals (36) located in human SIG81 in identical positions, and one highly conserved proline is missing in mouse SIG81 due to a two-codon deletion. SIG81 proteins also contain the motif ELQKFFQKAD (amino acids 61-70) homologous to the sequence EKQKLFQED proposed as a functional core domain in mammalian COX7a(10) . However, in nonmammalian vertebrates such as rainbow trout, COX7a-L contains the slightly modified sequence EKQKLFQAX(37) whereas in yeast COX7a, homologies in this region are reduced just to the amino acid pairs QK and FQ(10, 36) , thus suggesting that a great deal of variation can be accommodated into the sequence of the core motif while retaining the functionality of the protein. Thus, the variant motif of SIG81 might exert a very similar (if not identical) function to the sequence in COX7a.
The evolutionary analysis of the COX7a-homology domain suggests that SIG81 proteins are a monophyletic group that could reasonably represent a hitherto unrecognized branch derived from a gene ancestral to both SIG81 and COX7a. Uncertainty in the divergence matrix and the absence of SIG81 sequences from other vertebrates prevented the validation and dating of the putative duplications leading to the three main clusters of sequences showed in the dendrogram. Attending just to the COX7a-homology domain, it is reasonable to consider SIG81 as a member of a novel subfamily of COX7a isogenes.
A remarkable feature of the SIG81 sequence is the long
amino-terminal domain with no discernible homology to the amino termini
of COX7a that precluded an evolutionary analysis. To explain the lack
of homology observed between the mitochondrial presequences in COX7a-H
and -L isoforms, two alternative hypotheses have been advanced that can
be reasonably extended to the origin of the amino terminus of SIG81. It
is possible that the acquisition of COX7a presequences were independent
evolutionary events subsequent to the gene duplication that originated
the mature peptides(10) . Alternatively, the ancestral COX7a
gene contained a mitochondrial presequence (34) which evolved
very rapidly due to lower evolutionary constraints(10) . An
extended analysis with additional vertebrate SIG81 sequences is
necessary to assess how evolution of the amino-terminal region of SIG81
corresponds with that of the COX7a-homology domains. Nonetheless, the
amino-terminal region of SIG81 shows features typical of mitochondrial
targeting sequences. By analogy to COX7a, a processing of the precursor
SIG81 protein into a mature peptide containing only the COX7a-homology
domain might, but SIG81 protein is present in liver mitochondria only
as a 13.5-kDa product. We did not detect lower molecular mass proteins
that could correspond to a processed form of SIG81. Although an
extensive post-translational modification of a mature peptide might
account for a retarded migration of a processed peptide, we think it is
unlikely in the case of SIG81 given that only one tyrosine
phosphorylation site and one N-myristoylation motif are
present in the carboxyl-terminal region of SIG81. In fact, the
hydrophobic stretch (amino acids 35-44 in mouse SIG81) found
within the putative mitochondrial targeting sequence is reminiscent of
the so-called ``stop-transfer'' domain present in proteins to
be localized to the mitochondrial membranes(35) . In many
cases, targeting sequences containing stop-transfer domains are not
cleaved(35) . It is tempting to speculate that the divergence
in the amino-terminal domains of SIG81 and COX7a reflect distinct
intramitochondrial localization and thus justify the occurrence of two
highly homologous proteins being coincidentally expressed in the same
tissue. It is peculiar that, if SIG81 arose from the duplication of an
ancestral gene, it has maintained a widespread expression in various
tissues instead of exhibiting the more specialized pattern such as that
for COX7a-H (10, 36) and by many other genes that
arise by duplication. The tight regulation of SIG81 gene expression is
exemplified by the fact that no measurable differences were found in
mRNA levels in RAW 264.7 and J774A.1 macrophages and NIH 3T3
fibroblasts that were serum-starved and then growth-stimulated by
addition of serum to increase their metabolic rates. ()Likewise, SIG81 expression was unaltered in RAW 264.7
cells cultured in the presence of insulin or thyroid hormone, subjected
to stress provoked by heat shock, or altered in their redox
state.
It is noteworthy that a similar lack of response has
also been reported for several nuclear-encoded COX subunits, including
COX7a(38, 39, 40) . Alternatively, SIG81
expression may be regulated through a post-transcriptional mechanism.
SIG81 mRNA contains a GC-rich 5`-UTR indicative of a highly structured
transcript that could function in translational
regulation(32) . In fact, preliminary data on SIG81 protein
levels estimated by Western blotting with anti GST-SIG81 antibodies
suggest that the levels of SIG81 protein may be lower than expected
from such an abundant mRNA.
Why has SIG81 not been identified previously as a member of the COX7a family of proteins? Since nucleotide sequence homology between SIG81 and COX7a cDNAs from the same species is low (61% identity in a 177-bp stretch between mouse SIG81 and COX7a-L), it is unlikely that a standard screening of a cDNA library with a COX7a probe would identify SIG81 sequences, even at low stringency hybridization conditions. At the protein level, an identification failure may be even more complicated. One possibility is that SIG81 is associated with the COX complex as a weakly bound subunit which could be removed easily by the detergent treatments usually employed in COX isolation, as reported for other COX subunits such as III, VIa, VIb, and VIIa, that sometimes are missing totally or in part from COX preparations(3, 41) . Alternatively, SIG81 is simply very homologous to one COX subunit but it is not a bona fide COX subunit and, as suggested above, might not necessarily be located in the same mitochondrial compartment. Further studies are in progress to elucidate the role of SIG81 in the mitochondria and its putative functional relation to the COX7a family of proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X80899[GenBank].