©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of an Additional Member of the Cytochrome c Oxidase Subunit VIIa Family of Proteins (*)

(Received for publication, December 18, 1995; and in revised form, February 27, 1996)

Fernando Segade (§) Belén Hurlé(¶) Estefanía Claudio (**) Sofía Ramos Pedro S. Lazo

From the Departamento de Bioquímica y Biología Molecular, Universidad de Oviedo, 33006 Oviedo, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 (^1)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, (^2)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.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases and DNA modifying enzymes were from Boehringer Mannheim. [alpha-P]dCTP (3000 Ci/mmol) and [alpha-S]dATP (1000 Ci/mmol) were purchased from Amersham International. The GeneAmp PCR reagent kit was from Perkin-Elmer. All other chemicals were reagent or molecular biology grade and were obtained from Boehringer Mannheim, Merck, or Sigma.

SIG81 cDNA Cloning

Isolation of SIG81 cDNA by differential screening of a subtracted cDNA library from the murine macrophage cell line RAW 264.7 constructed in the ZAPII vector (Stratagene) has been described previously(2) . Independent SIG81-containing clones were isolated after three rounds of screening of 40 times 10^3 clones of a RAW 264.7 cDNA library in ZAPII (2) following standard procedures(19) . The cDNA insert of SIG81 was labeled by random priming using the ``RediPrime'' (Amersham) labeling system in the presence of [alpha-P]dCTP to be used as a probe. pBluescript SK phagemids containing SIG81 cDNAs were excised in vivo from the ZAPII vector by coinfection of Escherichia coli XL1-Blue cells with VCS M13 helper phage (Stratagene), according to the supplier's instructions.

DNA Sequencing and Analysis

Nucleotide sequences were determined by the dideoxy chain termination method (20) on single- and double-stranded template DNA using Sequenase 2.0 (USB Amersham) with custom-designed oligonucleotide primers. Sequences were read manually from both strands and in some cases sequence ambiguities were solved by substituting dITP for dGTP in the sequencing reactions. Sequence analysis and comparison were performed with the University of Wisconsin's Genetics Computer Group version 8.0 package of programs (21) . The mammalian COX7a sequences (human, bovine, mouse, and rat) were downloaded from the GenBank/EMBL data base (see accession numbers in legend to Fig. 3). Amino acid sequences were aligned using the PILEUP program, and refinements to the alignment were made manually. After sequences were optimally aligned, a matrix of pairwise distances expressed as the number of amino acid substitutions per 100 residues was generated with the Kimura method(22) . The phylogenetic tree was constructed by the Neighbor Joining Method(23) , with branch length proportional to calculated genetic distance.


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.



RNA Preparation and Northern Analysis

Total cellular RNA was extracted from Swiss mouse tissues by the guanidinium isothiocyanate phenol-acid method(24) . For Northern analysis, 15 µg of total RNA were denatured with 2.2 M formaldehyde, fractionated in 1.2% agarose, 2.2 M formaldehyde gels (19) , and transferred to Hybond N (Amersham) membranes. RNA blots were hybridized with the appropriate radiolabeled probe and washed in 0.2 times SSC, 0.2% SDS at 65 °C. In addition to the SIG81 insert, blots were hybridized with a mouse 28 S rRNA probe(2) , and a 221-bp COX7a probe spanning positions 61 to 281 of the mouse COX7a-L cDNA (GenBank accession number X58486) obtained by reverse transcription-PCR from RAW 264.7 poly(A) RNA. (^3)

Bacterial Expression of GST-SIG81 Fusion Protein

On the basis of the SIG81 cDNA sequence, two oligonucleotide primers, OF26 5`-CGGATCCACATGTACTACAAG-3` (positions 26-39) and OF27 5`-CGGAATTCTCATTTGTTTCTG-3` (positions 364-350), were designed for a PCR amplification of the entire SIG81 coding region. Recognition sites for BamHI and EcoRI were included at the 5` ends of OF26 and OF27, respectively, to ensure a correct in-frame ligation to the expression vector. With SIG81 cDNA as a template, a 354-bp fragment was generated in a 30-cycle PCR that was cloned into pBluescript SK and sequenced from both ends. An insert with the expected sequence was then subcloned into the pGEX3X vector (Pharmacia Biotech Inc.) to construct pGEXSIG81. Large scale (500 ml of culture) production of GST-SIG81 was induced with 0.5 mM IPTG for 6 h. Bacterial lysis was carried out essentially as described (25) , except that lysozyme (100 µg/ml) was used. GST-SIG81 was purified from lysates by binding to 1 ml of glutathione-Sepharose beads (Pharmacia) for 2 h at 4 °C. After extensive washing, the bound protein was eluted with 1 ml of 75 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM reduced glutathione, 5 mM dithiothreitol, 2% N-octyl glucoside. We routinely obtained 600 µg of GST-SIG81 from a 500-ml culture.

Generation of Antibodies

For immunizations, bacterially expressed GST-SIG81 was injected intramuscularly into New Zealand White rabbits, and antisera were prepared as described(26) . For immunodetection experiments, antibodies were affinity-purified from the third bleeding serum using the SulfoLink Coupling Gel (Pierce). Coupling of GST-SIG81 fusion protein to the gel and affinity purification of the antibodies followed the manufacturer's instructions.

Subcellular Organelle Fractionation

Liver (0.25 g) from adult Swiss mice was homogenized in 40 volumes of ice-cold 10 mM HEPES, pH 7.4, 0.32 M sucrose, by using 20 strokes of a tight-fitting Potter-Elvehjem Teflon-glass homogenizer. To assess organelle intactness, organellar latency in the homogenate was determined (27) using a COX activity assay (see below) in the presence and in the absence of detergent. Latency values were around 82%, within the range for intact organelles (80-95%)(27) . Homogenates were then subfractionated into four subcellular compartments essentially as described(28) . A nuclear-enriched fraction was obtained by centrifugation of the homogenate at 750 times g for 5 min and washed by resuspension in HEPES/sucrose and recentrifugation. To remove contaminating organelles and whole cells(27) , the nuclear pellet was resuspended in 2 ml of SMTM buffer (10 mM Tris, pH 7.4, 0.25 M sucrose, 10 mM NaCl, 1.5 mM MgCl(2)), and 0.3 ml of a detergent solution (10% sodium deoxycholate, 10% Nonidet P-40, 1:2) was added. Washed nuclei were pelleted at 2300 times g for 5 min and finally resuspended in SMTM. A mitochondrial-enriched fraction was prepared by centrifugation of the 750 times g supernatants at 8000 times g for 10 min and washed by resuspension and recentrifugation. Mitochondria were separated from lysosomes and microsomal components by centrifugation in a Metrizamide discontinuous density gradient(27) . To prepare the gradient, the 8000 times g pellet was resuspended in HEPES/sucrose and mixed with a solution of 80% Metrizamide (Sigma) to obtain 2 ml of a suspension containing 35% Metrizamide, which was overlaid with 2 ml of 17% Metrizamide, and 2 ml of 5% Metrizamide. Metrizamide solutions were made in 10 mM Tris, pH 7.4, 0.25 M sucrose. The gradient was centrifuged at 100,000 times g for 2 h at 4 °C in a Kontron TST41.14 swinging bucket rotor. Gradients were fractionated from bottom to top into 50-µl aliquots. Finally, a pellet of microsomal membranes and a supernatant containing the cytosolic fraction were obtained by centrifugation of the 8,000 times g supernatant at 100,000 times g for 30 min.

Cytochrome c Oxidase Activity Assay

COX activity (27) was assayed by measuring the decrease in absorbance at 550 nm when subcellular extracts or gradient fractions were incubated with 50 µl of reduced horse heart cytochrome c (2.7 mg/ml in 40 mM potassium phosphate, pH 6.2, 0.12 mg/ml sodium hyposulfite) and 1 ml of 0.2% polyoxyethylene ether (detergent W1, Sigma), 40 mM potassium phosphate, pH 6.2, 0.25 M sucrose, at room temperature at 15-s intervals. For latency experiments, COX activity in intact organelles was determined without detergent added.

SDS-PAGE and Western Blot Analysis

Proteins were resolved by SDS-PAGE in polyacrylamide gels with the Tris/Tricine buffer system (29) . Gels (0.75 mm thickness) were made with a cross-linking relation of 16.5% T/6% C (of the total 16.5% of acrylamide monomers, 6% of the original monomers corresponds to bisacrylamide) and a 4% T/3% C polyacrylamide stacking gel. Electrophoreses were performed at 100 V, and proteins were transferred from gels to Hybond polyvinylidene difluoride membranes (Amersham) in 48 mM Tris, 39 mM glycine, 20% methanol, 0.0375% SDS, using a Bio-Rad Transblot semidry transfer cell at 20 V for 1 h.

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.

Southern Analysis of Mouse Genomic DNA

Total Swiss mouse genomic DNA was prepared from thymus using standard procedures(30) . Genomic DNA (20 µg) was digested with BamHI, HindIII, PstI, or XbaI and electrophoresed on 0.6% agarose gels. After transfer to Hybond N membranes, blots were hybridized with the appropriate random primer-labeled probe at 42 °C for 16 h. Blots were washed at 65 °C in 0.5 times SSC, 0.1% SDS.


RESULTS

Sequence Analysis of the SIG81 cDNA

SIG81 is one of the nine silica-induced cDNAs isolated by differential screening of a subtracted RAW 264.7 macrophage cDNA library(2) . The comparison of a partial SIG81 sequence against the GenBank/EMBL nucleotide data bases using the FASTA sequence alignment algorithm (31) revealed that SIG81 cDNA corresponded to a novel transcript with a significant similarity to mammalian COX-7a at its 5` end. For full-length sequencing of the SIG81 cDNA and to circumvent the possible errors being introduced during the enzymatic manipulations performed on the cDNAs for the construction of the subtracted cDNA library(2) , 12 independent cDNA clones were isolated from a RAW 264.7 macrophage cDNA library by screening with the radiolabeled 1.0-kb SIG81 insert. In addition to the original SIG81 cDNA, the complete sequences of four of the additional clones and an average of 250 bp from the 5` end of the remaining eight cDNAs were determined. The 5` end of every cDNA sequence was located within the first 11 bp of the sequence depicted in Fig. 1A. Therefore, it is likely that this cDNA represents a near full-length copy of the SIG81 transcript. The mouse SIG81 cDNA spans 1035 bp followed by a poly(A) tail. The ATG at base 28 is probably the genuine initiator since it is located within a favorable environment for translation initiation with an A three bases upstream of the ATG(32) . The cDNA sequence contains a 5`-untranslated region (UTR) highly enriched in G + C (78% versus 50% for the overall sequence) that probably indicates a functionally significant mRNA leader region(32) . Following the TGA end codon at position 361-363, there is a 672-bp 3`-UTR with a consensus polyadenylation site located at position 1020.


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 (bullet) 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.



Phylogenetic Analysis of SIG81

As depicted in Fig. 3A, a multiple sequence alignment of the COX7a-homology domains in the deduced mouse and human SIG81 and the mature mammalian COX7a proteins available in GenBank reveals conserved clusters of short amino acid sequences. Within a species, SIG81 and COX7a proteins showed a degree of homology comparable with that between L- and H-isoforms. Thus, there is 55% identity between mouse SIG81 and COX7a-L, and 59% identity for human SIG81 and COX7a-L, while human COX7a-L and -H are just 51% identical. Hence, a phylogenetic distance analysis was performed to elucidate the evolutionary relationship between SIG81 and COX7a proteins. After aligning the carboxyl-terminal homologous regions of COX7a and SIG81 proteins, evolutionary distances between each pair of sequences were calculated by the Kimura protein distance method(22) . The topology of the resulting dendrogram indicates that SIG81 proteins clearly constitute a monophyletic group most closely related to the COX7a-H cluster (Fig. 3B). However, the rate of sequence divergence is significantly different not only among subfamilies but also within each subfamily of proteins as illustrated by the unequal length of the branches. Note that mouse SIG81 diverged from the other groups significantly further than its human homolog. For all these reasons there is no adequate molecular clock to confidently date the divergence of the proteins, and, therefore, it is not feasible to determine whether SIG81 or COX7a-H diverged first from the common ancestor to COX7a-L. It was suggested that the common ancestor to COX7a-H and COX7a-L genes contained a mitochondrial presequence(34) , but the divergence of SIG81 and COX7a proteins at their amino termini regions is too great to afford a phylogenetic relationship in this study.

Tissue Distribution of SIG81 mRNA Expression

Northern blot analysis of total cellular RNA extracted from a variety of mouse tissues was performed to assess the range of SIG81 gene expression. As shown in Fig. 4A, every tissue tested contained a single transcript that hybridized with the SIG81 cDNA probe. Although the highest steady state levels of SIG81 mRNA were found in kidney and liver (Fig. 4A), the transcript was also very abundant in skeletal muscle and heart (Fig. 4C). There seems to be no direct relation between SIG81 mRNA abundance and the specific metabolism of a tissue. Rehybridization of the same blot with a probe for COX7a-L revealed a strong hybridization signal in kidney whereas substantially lower levels of COX7a-L mRNA were detected in the remaining tissues (Fig. 4B). COX7a-L is the only COX7a isoform heretofore known in mouse (13) so it is not surprising to find significant levels of the transcript in heart and skeletal muscle.


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.



Genomic Analysis of Mouse DNA

As a preliminary study on the SIG81 gene(s) present in the genome, we performed a Southern blot analysis of total mouse genomic DNA cleaved with four restriction enzymes (BamHI, HindIII, PstI, and XbaI). When probed with the radiolabeled full-length SIG81 cDNA, 2-3 strongly and 1-2 more weakly hybridizing bands were revealed in each lane (Fig. 5). The result suggests that at least two SIG81 gene copies may be present in the mouse genome that could correspond to a small family of genes and/or pseudogenes.


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.



Protein Expression and Subcellular Localization of SIG81

To establish whether the SIG81 transcript is in fact translated in mouse cells as well as to determine the subcellular location of the protein, rabbit polyclonal antibodies were raised against a GST-SIG81 fusion protein overproduced in E. coli. In a Western blot analysis of extracts prepared from IPTG-induced bacteria transformed with the pGEXSIG81 plasmid and purified through glutathione-Sepharose, the antibodies reacted strongly against the 40-kDa GST-SIG81 fusion protein (Fig. 6B, lane 1). In mouse liver postnuclear supernatant, an anti-GST-SIG81 reactive band with an apparent molecular mass of 13.5 kDa was present (Fig. 6B, lane 2) that was not detected when the blot was probed with a preimmune serum from the same rabbit (Fig. 6A, lane 2). We therefore concluded that the 13.5-kDa band does correspond to the protein product encoded in SIG81. The observed size is in close agreement with that of the 12.4 kDa calculated from the SIG81 cDNA sequence indicating that the protein might not be processed to a lower molecular mass form.


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 times 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 times g and the 100,000 times g supernatant. The 8,000 times 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 times g pellets and the 100,000 times 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.




DISCUSSION

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. (^4)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.^3 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.^4

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.


FOOTNOTES

*
This work was supported in part by Comisión Interministerial de Ciencia y Tecnología Grant SAF94-0389. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) X80899[GenBank].

§
To whom correspondence should be addressed. Tel.: 34-8510-4213; Fax: 34-8510-3534; PSL{at}dwarf1.quimica.uniovi.es.

Supported by a Ministerio de Educación y Ciencia predoctoral fellowship.

**
Supported by a Fundación para la Investigación Científica y Técnica predoctoral fellowship.

(^1)
The abbreviations used are: kb, kilobase(s); bp, base pair(s); COX, cytochrome c oxidase; COX7a, cytochrome c oxidase subunit VIIa; COX7a-H and -L, heart and liver isoform of cytochrome c oxidase subunit VIIa, respectively; PCR, polymerase chain reaction; EST, expressed sequence tag; GST, glutathione S-transferase; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; UTR, untranslated region; PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hydroxymethyl)methylglycine.

(^2)
M. Starborg and C. Höög, unpublished sequence.

(^3)
F. Segade, unpublished data.

(^4)
B. Hurlé, unpublished data.


ACKNOWLEDGEMENTS

We sincerely appreciate the expert technical advice of Dr. M. Uriarte on Tris/Tricine gels. We are grateful to Dr. C. López-Otín for the synthesis of primers and to Dr. Y. S. López-Boado for her excellent advice and critical review of the manuscript.


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