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Originally published In Press as doi:10.1074/jbc.M510778200 on December 8, 2005

J. Biol. Chem., Vol. 281, Issue 6, 3743-3751, February 10, 2006
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COX24 Codes for a Mitochondrial Protein Required for Processing of the COX1 Transcript*

Mario H. Barros1, Alan M. Myers2, Sarah Van Driesche, and Alexander Tzagoloff3

From the Department of Biological Sciences, Columbia University, New York, New York 10027

Received for publication, October 3, 2005 , and in revised form, December 1, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In most strains of Saccharomyces cerevisiae the mitochondrial gene COX1, for subunit 1 of cytochrome oxidase, contains multiple exons and introns. Processing of COX1 primary transcript requires accessory proteins factors, some of which are encoded by nuclear genes and others by reading frames residing in some of the introns of the COX1 and COB genes. Here we show that the low molecular weight protein product of open reading frame YLR204W, for which we propose the name COX24, is also involved in processing of COX1 RNA intermediates. The growth defect of cox24 mutants is partially rescued in strains harboring mitochondrial DNA lacking introns. Northern blot analyses of mitochondrial transcripts indicate cox24 null mutants to be blocked in processing of introns aI2 and aI3. The dependence of intron aI3 excision on Cox24p is also supported by the growth properties of the cox24 mutant harboring mitochondrial DNA with different intron compositions. The intermediate phenotype of the cox24 mutant in the background of intronless mitochondrial DNA, however, suggests that in addition to its role in splicing of the COX1 pre-mRNA, Cox24p still has another function. Based on the analysis of a cox14-cox24 double mutant, we propose that the other function of Cox24p is related to translation of the COX1 mRNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytochrome c oxidase (COX)4 biogenesis is a complex process that requires the expression and interaction of subunits encoded by mitochondrial and nuclear genes. In Saccharomyces cerevisiae at least 20 nuclear gene products (1, 2) have been shown to assist COX assembly. These proteins promote steps, ranging from processing of mitochondrial COX-specific RNAs (3, 4) and their translation (5, 6) to recruitment, formation, and addition of the metal and heme prosthetic groups present in the catalytic subunits of the complex (7-9).

Cox1p (subunit 1) of COX is an important constituent containing the cytochrome a and a3 centers. This subunit is encoded by the mitochondrial COX1 gene, which is transcribed as a polycistronic precursor RNA containing COX1, ATP8, ATP6, and ENS2 (10). Most commonly used laboratory strains have a COX1 gene with variable but multiple introns (11). The aI3, aI4, aI5{alpha}, and aI5beta introns of COX1 are group I introns, whereas aI1, aI2, and aI5{gamma} belong to group II introns. Both types of introns have the ability to act as mobile elements with the difference that homing of group II introns depends on an RNA intermediate and homing of group I intron is a DNA-based process (12, 13). The mobility of group I introns is enhanced by endonucleases encoded in the introns themselves. aI3 encodes the I-SceIII endonuclease that cleaves the junction of the two flanking exons, needed for its homing but in addition appears to exert a positive effect in removal of the intron (14, 15).

Splicing of the COX1 primary transcript is assisted by protein factors referred to as maturases that are encoded by reading frames located within some of the introns of the COX1 and COB genes (16-20). Excision of some intervening sequences also depends on nuclear genes such as CBP2, and SUV3 and MSS116, the latter two coding for RNA helicases (3, 21, 22). Cbp2p interacts with and stabilizes a splicing competent secondary structure of the cytochrome b pre-mRNA (23). Suv3p has been implicated in stabilizing the COX1 transcript by regulating turnover of group I intronic RNAs (24, 25), whereas Mss116p has been shown to function in splicing of all mtDNA introns in S. cerevisiae and Neurospora crassa (26).

Some of the accessory factors appear to have several functions in mitochondrial RNA metabolism. PET309, first isolated as a COX1 translation facilitator, also affects the stability of intron-containing COX1 RNA (4) and was recently found to be associated with CBP1, a cytochrome b RNA stabilization and translation factor (27). NAM2/MSL1, the mitochondrial leucyl-tRNA synthetase, is required for excision of the aI4 and bI4 introns of COB (28, 29). Excision of the aI5beta intron of COX1 depends on several nuclear gene products. Already mentioned are the helicases Suv3p and Mss116p. Other factors include Pet54p, Mss18p, and Mrs1p. Pet54p was first characterized as a translational factor for COX3 mRNA (6), but was subsequently shown to also play a role in aI5beta excision (30). Mrs1p is required for processing of introns bI3 of COB, and aI5beta and aI5{gamma} of COX1 (31). At present the only proteins known to function in processing of a single intron are Mss18p (3) and Cpb2p (23), which target aI5beta and bI5, respectively.

In the course of analyzing the biochemical defects of the respiratory deficient pet mutant of S. cerevisiae, we identified a new gene, which when mutated causes the accumulation of COX1 intermediates transcripts. This gene has been designated COX24 in keeping with our previous convention for naming genes involved in expression of cytochrome oxidase. COX24 corresponds to reading frame YLR204W and was previously named QRI5 (32). The phenotype of cox24 mutants and characterization of their mitochondrial RNAs lead us to propose that Cox24p plays an important role in processing of the COX1 primary transcript, but like the other aforementioned processing factors, may have another function as well.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Media—The strains of yeast used in this study are listed in Table 1. The respiratory deficient mutants of complementation group G82 were derived from S. cerevisiae D273-10B/A1 by mutagenesis with nitrosoguanidine or ethyl methanesulfonate (1). The following media were used routinely to grow yeast: YPD (2% glucose, 1% yeast extract, 2% peptone), YPGal (2% galactose, 1% yeast extract, 2% peptone), and YEPG (2% ethanol, 3% glycerol, 1% yeast extract, 2% peptone).


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  		TABLE 1
Genotypes and sources of yeast strains

 
Cloning of COX24—pG82/T1, a recombinant plasmid containing COX24, was isolated by transformation of C149/UL1 with a yeast genomic library consisting of partial Sau3A fragments of yeast nuclear DNA cloned in the yeast/Escherichia coli shuttle plasmid YEp13 (37). Approximately 5 x 108 cells were transformed with 50 µg of the plasmid library by the method of Beggs (38).

Disruption of COX24—A BamHI-BglII fragment of pG82/T1 containing COX24 was transferred to pUC19. The resultant plasmid (pG82/ST10) was used to replace the COX24 coding sequence with the yeast HIS3 gene. Amplification of pG82/ST10 with bi-directional primers 5-GGCAGATCTGGGCTGTAAAAACCTCTCAC and 5'-GGCAGATCTGTTTATCTCGTTCTGGTGTC resulted in a clean deletion of COX24. The linear product containing COX24 5'- and 3'-flanking regions in pUC19 was digested with BglII and ligated to HIS3 on a 1-kb BamHI fragment. The cox24::HIS3 null allele was recovered from this plasmid as a BamHI-XbaI fragment and was substituted by homologous recombination (39) for the wild-type gene in respiratory competent strains W303-1B and W303-1A.

Construction of a Hybrid Gene Expressing Cox24p Tagged with a Hemagglutin Epitope at the Carboxyl Terminus—pG82/T1 was used as a PCR template for amplification of the COX24-HA gene coding for Cox24p with 12 carboxyl-terminal residues consisting of three glycine residues as spacers and the hemagglutinin tag (HA). The PCR primers used for amplification were: 5'-GGCGGATC-CGTCCACCTTCGCAATATCTAC and 5'-GGCAAGCTTTCAAGCGTAGTCTGGGACGTCGTATGGGTACCCTCCTCCTCTACCCTGAGATAGCTTTCT. The PCR product was digested with a combination of BamHI and HindIII, and was cloned in the yeast/E. coli shuttle plasmids YEp352 and YIp352 (40) yielding pG82/ST16 and pG82/ST17, respectively. W303{Delta}COX24 was transformed either with uncut pG82/ST16 or with pG82/ST17 linearized at the NcoI site of the URA3 marker for integration at the homologous site in chromosomal DNA (39).

Miscellaneous Procedures—Standard methods were used for plasmid manipulations (41). The COX24 gene and flanking regions were sequenced by the method of Maxam and Gilbert (42). Yeast mitochondria were prepared by the method of Faye et al. (43) except that Zymolyase 20T (ICN Laboratories, Aurora, OH) instead of glusulase was used to obtain spheroplasts. Spectral analyses of mitochondrial cytochromes were performed as described previously (44). Protein concentrations were determined by the method of Lowry et al. (45).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of COX24—C149 is one of three independent respiratory deficient strains assigned to complementation group G82 of a pet mutant collection (1). Transformation of the derivative strain C149/L1 with a yeast genomic library yielded a leucine-independent and respiratory competent transformant (C149/L1/T1) that was used to isolate the recombinant plasmid pG82/T1. Complementation tests with subclones containing different regions of the nuclear DNA insert in this plasmid revealed that the gene responsible for restoring respiration in C149/L1 corresponds to reading frame YLR204W on chromosome XII (data not shown). The mutant W303{Delta}COX24 harboring a null allele of the gene, henceforth referred to as COX24, was obtained by homologous recombination with a linear fragment of nuclear DNA in which the entire reading frame was replaced with HIS3. The identity of COX24 as the gene responsible for the respiratory defect of C149/L1 was confirmed by the lack of complementation of the point mutant by the null mutant W303{Delta}COX24 and the sequence of the gene in C149/L1, which was found to have a single base (guanine) deletion in the codon corresponding to lysine at position 87 of the polypeptide chain. The deletion of this nucleotide creates a stop codon immediately after the mutation, thereby shortening the protein by 24 amino acids.


Figure 1
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  		FIGURE 1.
Spectra, mitochondrial translation, and steady-state levels of COX subunits in cox24 mutants. A, mitochondria were isolated from the respiratory competent parental strain W303-1A and from the cox24 null mutant W303{Delta}COX24 grown on rich 2% galactose media (YPGal). The mitochondria were extracted at a protein concentration of 5 mg/ml in the presence of 1% potassium deoxycholate and 1 M KCl (44). The spectra of the extracts were obtained at room temperature following oxidation of the sample in the reference cuvette with potassium ferricyanide and reduction of the sample cuvette with sodium dithionite. The {alpha} absorption bands corresponding to cytochromes c, c1, b, a, and a3 are indicated. B, detail of the shift from 605 to 595 nm of the cytochromes a and a3 {alpha} absorption band in W303{Delta}COX24. C, mitochondrial gene products were labeled in vivo with [35S]methionine (36) in W303-1A (a wild-type strain with a normal mitochondrial genome), W303/I0 (wild-type with intronless mtDNA), aW303{Delta}COX24 (a cox24 null mutant with mtDNA containing introns), and W303{Delta}COX24/I0 (a cox24 null mutant with intronless mtDNA). Total proteins from an equivalent number of cells were separated by SDS-PAGE on a 12.5% polyacrylamide gel containing 6 M urea and 6% glycerol. The mitochondrial translation products identified in the margin are: Var1 (Var1 ribosomal protein), Cox1 (subunit 1 of COX), Cox2 (subunit 2 of COX), Cox3 (subunit 3 of COX), Cyt.b (cytochrome b), Atp6 (subunit 6 of ATPase), Atp8 (subunit 8 of ATPase), and Atp9 (subunit 9 of ATPase). D, mitochondria from the wild-type strain W303-1A, the cox24 null mutant aW303{Delta}COX24, and the revertant W303{Delta}COX24/R2 were separated by SDS-PAGE, transferred to nitrocellulose, and reacted with monoclonal antibodies against Cox1p and Cox3p and with polyclonal antibodies against Cox2p, Cox4p, and Cox5p. The blots were then treated either with peroxidase-conjugated anti-mouse or anti-rabbit IgG. The proteins were visualized with the Super Signal chemiluminescent substrate kit (Pierce).

 
Phenotype of the cox24 Null Mutant—C149/L1 and W303{Delta}COX24 exhibit a clear growth defect on non-fermentable carbon sources (ethanol/glycerol). The spectrum of mitochondrial cytochromes in the null mutant indicated the presence of a very low level of an "a" type cytochrome with an absorption maximum of the {alpha} peak at 595 nm instead of 605 nm, the normal positions of cytochrome a and a3 bands in cytochrome oxidase absorb normally (Fig. 1, A and B). The {alpha} absorption band of cytochrome b is also reduced in the mutant but is located at the expected wavelength (560 nm). Despite the reduction of spectrally detectable cytochrome b, in vivo labeling of mitochondrial products indicated normal translation of cytochrome b (Fig. 1C). A partial deficiency of cytochrome b is a common property of COX mutants.


Figure 2
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  		FIGURE 2.
Growth properties and steady-state concentrations of Cox1p in wild-type and cox24 mutants with normal and intronless mtDNA. A, serial dilutions of the intronless wild-type W303/I0, the cox24 null mutant W303{Delta}COX24, and the intronless cox24 mutants aW303{Delta}COX24/I0 and {alpha}W303{Delta}COX24/I0 were spotted on YPD (rich glucose) and YPEG (rich medium containing glycerol plus ethanol). The photograph was taken after the plates had been incubated at 30 °C for 2 days. B, the steady-state concentration of Cox1p in W303-1A, W303/I0, and the cox24 mutants aW303{Delta}COX24/I0 and W303{Delta}COX24/I0 were determined by Western blot analysis as in Fig. 1D with a monoclonal or polyclonal antibody against Cox1p. Mitochondrial proteins loaded on the gel were 20 µg in the top two panels and 30 µg in the bottom panel.

 
In contrast to cytochrome b, translation of Cox1p, Cox2p, and Cox3p, the three mitochondrially encoded subunits of cytochrome oxidase, was reduced. This was most evident for Cox1p, which was present in only trace amounts in the mutant. Subunit 6 (Atp6p) of the ATPase was normally translated (Fig. 1C), ruling out a defect in transcription or processing of the large polycistronic precursor RNA containing COX1, ATP8, and ATP6 (10).

The steady-state concentrations of COX subunits in the cox24 null mutant were examined by Western blot analysis with antibodies against Cox1p, Cox2p, and Cox3p, and the nuclear encoded subunits Cox4p and Cox5p. Of these five constituents, Cox1p and Cox2p were reduced to barely detectable levels in the mutant. A very substantial decrease was also seen in Cox3p. Both Cox4p and Cox5p were reduced but to a lesser extent (Fig. 1D). This pattern is characteristic of most mutants that fail to form functional COX, independent of whether the block occurs at a pre- or post-translation step of the assembly pathway (46).

W303{Delta}COX24 gives rise to revertants with growth properties on non-fermentable substrates intermediate between wild-type and the mutant. Although the suppressor mutation(s) has been ascertained to be in mtDNA, for unknown reasons we have not been able to map the mutations by deletion analysis with cytoplasmic petites obtained from several such revertants. The partial restoration of growth of one such revertant (W303{Delta}COX24/R2) correlates with an increase in the mitochondrial steady-state concentration of Cox1p (Fig. 1D).

Phenotype of a cox24 Mutant with Intronless Mitochondrial DNA—The lesion in the cox24 mutant appeared most likely to be in processing and/or translation of Cox1p. The parental W303 strains into which the cox24 null allele was introduced have mitochondrial genomes with COX1 introns aI1-aI4 and aI5{gamma} (47). To probe the possible involvement of Cox24p in intron processing, the cox24 null mutation was transferred to a strain lacking mitochondrial introns by a cross of W303{Delta}COX24/{rho}0 (a cox24 null mutant lacking mitochondrial DNA) to the intronless strain W303/I0. Diploid cells issued from the cross yielded meiotic progeny with the cox24 null allele in an intronless mitochondrial background (aW303{Delta}COX24/I0 and W303{Delta}COX24/I0). These strains were able to grow on non-fermentable substrates (YPEG), albeit not as well as the wild-type strains W303-1A or W303-1A/I0 (Fig. 2A). Growth of W303{Delta}COX24/I0 on glycerol/ethanol correlated with a partial restoration of Cox1p translation (Fig. 1C). Western analysis of mitochondria with a polyclonal antibody against Cox1p indicated a substantial increase in the steady-state concentration of this subunit in the mutant cells (Fig. 2B). Paradoxically, a Cox1p monoclonal antibody detected only trace amounts of Cox1p, even at very high loading of mitochondrial proteins (Fig. 2B). This suggests that the Cox1p produced in W303{Delta}COX24/I0 may be different from the normal protein.


Figure 3
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  		FIGURE 3.
Northern blot analysis of the mitochondrial COX1 and COB transcripts. Mitochondrial RNAs were extracted from mitochondria (200 µg of protein) of wild-type strains W303-1A and W303/I0 (intronless mtDNA), and the two cox24 mutants aW303{Delta}COX24/I0 and W303{Delta}COX24/I0, both of which also contained intronless mtDNA. Total mitochondrial RNAs were separated on a 1% agarose gel. The gel was stained with ethidium bromide, photographed, and the RNAs blotted to a nylon membrane (Nytran®, SuPerCharge, Schleicher and Schuell). Following cross-linking with UV light, the nylon membrane was pre-hybridized at 43 °C with 125 µg of salmon sperm DNA in 5x SSC, 5x Denhardts, 0.5% SDS. The blotted RNA were hybridized overnight at 43 °C with probes from exon 4 of COX1 (panel A) and exon 1 of COB (panel B). Both probes were labeled with [{alpha}-32P]dATP by random priming (48). Panel C shows the ethidium bromide-stained gel used for the Northern transfer. The 15 S and 21 S mitochondrial rRNAs are indicated.

 
COX1 Transcript Processing in cox24 Mutants—The partial rescue of the cox24 null mutation in a strain with intronless mtDNA pointed to a role of Cox24p in processing of the COX1 pre-mRNA. This is supported by the mitochondrial COX1 transcripts present in wild-type and mutant strains harboring mtDNAs with or without introns (Fig. 3A). Northern blots of total mitochondrial RNA hybridized to a probe from exon aE4 of COX1 indicated the accumulation of partially processed COX1 RNA intermediates in the cox24 null mutant with the original intron-containing mtDNA. In this background the mature COX1 mRNA was not detected. As expected, COX1 processing intermediates were absent in the wild-type or cox24 mutant with the intronless genome. In this background the only COX1 transcript detected by the probe had a size corresponding to the mature mRNA. This reinforces the conclusion that the cox24 mutation does not affect endonucleolytic cleavage of the primary polycistronic transcript. Northern blots of the same RNAs with an exon probe from the cytochrome b (COB) gene revealed the presence in the cox24 mutant of the mature size of COB mRNA. Although there was some accumulation of COB precursor transcripts in the mutant, the mRNA concentration was not appreciably less than in the wild type (Fig. 3B). The presence of the processed COB mRNA in the mutant is consistent with the in vivo labeling results showing normal translation of cytochrome b (Fig. 1C) and support a role of Cox24p confined to processing of the COX1 pre-mRNA.


Figure 4
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  		FIGURE 4.
Northern analysis of COX1 introns in the cox24 null mutant. Northern blots of mitochondrial RNA of the wild-type W303-1A, the cox14 mutant W303{Delta}COX24, and the revertant W303{Delta}COX24/R2 were hybridized with probes from COX1 introns aI1, aI2, aI3, aI4, and aI5{gamma} as described in the legend to Fig. 3. The processed introns are identified by the arrows in the margins. The uppermost left panel s hows the pattern of ethidium bromide-stained RNAs. The positions of the 15 S and 21 S mitochondrial rRNAs are indicated.

 
The function of Cox24p in splicing of the COX1 precursor was assessed by hybridization of total mitochondrial RNAs with probes from introns aI1, aI2, aI3, aI4, and aI5{gamma}. The results of these Northern analyses indicated that the cox24 mutant is able to splice group II introns aI1 and aI5{gamma}. Both introns are stable and accumulate in the wild-type and mutant (Fig. 4). The concentration of aI2, the third stable group II intron of COX1, is significantly lower in the mutant and is present at nearly wild-type levels in the revertant W303{Delta}COX24/R2. The results with probes against group I introns aI3 and aI4 are more difficult to interpret because the excised products are unstable and normally are degraded. The aI4 probe detected high molecular weight pre-cursors that are much less abundant in wild type and are substantially reduced in the revertant. In the case of the aI3 probe, several of the partially processed intermediates seen in wild type are absent in the mutant but are partially restored in the revertant (Fig. 4). These results point to multiple roles of Cox24p in COX1 mRNA processing, including excision of both group I and group II introns.

Suppression of the cox24 Null Mutant by mtDNA with Different Intron Compositions—The COX1 processing defect was also tested by examining the suppressor activities of mitochondrial genomes differing in their intron compositions. These were introduced into a {rho}0 derivative of the cox24 null mutant by cytoduction (49). As might be predicted, the absence of COB introns did not relieve the respiratory deficiency of the mutant (CK5112 in Fig. 5). This was also true of the cox24 mutant with a COX1 gene lacking the aI4 intron (WIO4). The cox24 mutant, however, was partially rescued by mtDNA lacking introns aI1, aI2, aI3, and aI5{gamma} (GF134-6D). These results are consistent with Northern hybridizations with intron-specific probes, indicating impaired processing of aI2 and aI3 in the mutant (Fig. 4).

Cox1p Expression Is Inhibited in a cox14-cox24 Double Mutant—The incomplete suppression of the cox24 mutant by a mitochondrial genome devoid of introns suggested that in addition to transcript processing Cox24p may have another function, either in translation or assembly of cytochrome oxidase. Mutations in genes required for maturation of the COX1 precursor transcript or translation of the mRNA display the absence of Cox1p among the normal complement of mitochondrial translation products. Translation of Cox1p is also compromised by mutations that prevent assembly of the subunits into the functional complex (36). Synthesis of Cox1p, in all such assembly defective mutants can be rescued when the original mutation is combined with a cox14 null mutation (36). This circumstance permits mutations that prevent formation or translation of the Cox1p mRNA to be discriminated from mutations affecting a post-translational assembly step. A regulatory model of Cox1p translation has been invoked to explain the epistatic effect of the cox14 mutation over other mutations affecting post-translational events in COX assembly (36).


Figure 5
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  		FIGURE 5.
Suppression of the cox24 null mutant by mtDNA with different introns in the COX1 and COB genes. Serial dilutions of wild-type and mutant strains were spotted on rich glucose (YPD) and rich ethanol/glycerol (YPEG) plates. The photograph was taken after incubation at 30 °C for 2 days. The intron compositions of the strains are indicated in parentheses for each strain. W303/I0 is a respiratory competent strain devoid of all introns. All the other strains have a cox24 null allele in a W303 nuclear background. {Delta}COX24/A21 has mtDNA of D273-10B/A21 (34), which lacks the first three introns of COB (bI1, bI2, and bI3) and introns aI5{alpha} and aI5beta of COX1. The notation bI+ indicates the presence of all the COB introns. For further details of how the strains were constructed refer to Table 1.

 
This experimental paradigm was used to examine expression of Cox1p in a cox24-cox14 double mutant with intronless mtDNA. The results of in vivo translation assays indicated that not only did inclusion of the cox14 mutation fail to enhance Cox1p synthesis over and above that seen in the single cox24 null mutant, but instead exerted an inhibitory effect (Fig. 6). Because COX24 is adjacent to MSS51 (32), which is also required for Cox1p synthesis (46, 50, 51), the possibility existed that the cox24 null allele interferes with normal expression of Mss51p. This seems unlikely in view of a similar inhibition of Cox1p synthesis when the cox14 mutation was combined with the cox24 point mutation of C149 (not shown).

Cox24p Homologues Are Confined to Species of Saccharomyces—A search of the current data bases disclosed the presence of COX24 homologues within but not outside of the Saccharomyces genus. The alignment of Cox24p from S. cerevisiae, Saccharomyces paradoxus, Saccharomyces bayanus, and Saccharomyces mikatae is shown in Fig. 7A. The carboxyl-terminal 39 residues, which include the premature termination codon of C149, comprise a very hydrophilic and basic domain that is identical in the four species.

Although the amino-terminal half is also conserved among the different species, there are fewer identities in this region. The protein has a centrally located putative hydrophobic membrane-spanning domain (Fig. 7B). This feature is consistent with the solubility properties of Cox24p (see below).

Cox24 Localization and Extraction Properties—To localize Cox24p, COX24 was modified so as to express the protein with a carboxyl-terminal hemagglutinin tag. This hybrid gene was introduced into a cox24 null mutant either on a multicopy plasmid (W303{Delta}COX24/ST16) or by integration at the chromosomal ura3 locus (W303{Delta}COX24/ST17). The respiratory defect of the mutant was partially rescued in both transformants. The tagged protein (Cox24p-HA) was readily detected in mitochondria of W303{Delta}COX24/ST16 with a monoclonal antibody against the hemagglutinin tag (Fig. 8A). The signal in mitochondria from the transformant with the integrated gene, however, was difficult to detect. This suggests that Cox24p is a very low abundance protein. Cox24-HA remained in the membrane fraction (submitochondrial particles) following sonic disruption of mitochondria. Surprisingly it was not extracted by alkaline treatment of submitochondrial membranes with sodium carbonate but was solubilized when the membranes were treated with dodecyl maltoside (Fig. 8A).


Figure 6
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  		FIGURE 6.
Mitochondrial protein synthesis in cox14-cox24 double mutants. Mitochondrial translation products were labeled in vivo with [35S]methionine and total cellular proteins were separated as in Fig. 1C. The strains labeled were: W303/I0 (wild-type), aW303{Delta}COX24/I0 (a cox24 null mutant), W303{Delta}COX14/I0 (a cox14 null mutant), and aW303{Delta}COX14,{Delta}COX24/I0 (a cox14-cox24 null double mutant).

 
Cox24p-HA was localized in the inner membrane in an orientation facing the matrix side. This is evident from its lack of sensitivity to proteinase K in mitochondria and mitoplasts, the latter lacking an intact outer membrane (Fig. 8A). The presence of the soluble matrix marker {alpha}-ketoglutarate dehydrogenase in the mitoplasts indicated that the inner membrane remained intact following hypotonic shock. Cytochrome b2, a soluble intermembrane marker was depleted in the mitoplasts fraction confirming lysis of the outer membrane. This is also supported by the susceptibility to proteinase K of Sco1p, an inner membrane protein extending into the intermembrane space (7). The above localization of Cox24p-HA was confirmed for the wild-type protein with an antibody raised against a 12-residue long peptide in the carboxyl-terminal conserved domain (Fig. 8B).


Figure 7
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  		FIGURE 7.
Alignment of Cox24p in different species of Saccharomyces. A, the sequences of Cox24p in S. cerevisiae, S. bayanus, S. mikatae, and S. paradoxus were aligned with the ClustalW program (52). The black and gray boxes highlight identical and conserved residues, respectively. The position of the deletion in the C149 allele, which creates a stop codon immediately after the mutation, is identified by the arrow. B, hydropathy profile of Cox24p according to Kyte and Doolittle (53).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our studies show that Cox24p, encoded by reading frame YLR204W on chromosome XII, is a mitochondrial protein facing the matrix side of the inner membrane. Null mutations in this gene, for which we propose the name COX24, elicit a growth defect on non-fermentable carbon sources as a result of a deficiency in cytochrome oxidase. Assays of mitochondrial protein synthesis in whole cells and Western analysis of COX subunit polypeptides indicated that cox24 mutants are severely impaired in the synthesis of Cox1p, the heme-bearing subunit of this respiratory complex. Unlike most cytochrome oxidase mutants, which lack the {alpha} absorbance bands of cytochromes a and a3 at 605 nm, the cox24 mutant shows evidence of a weak absorption band with a maximum at 595 nm indicative of a non-native environment of heme A. At present we do not know if this absorption stems from heme A that may be associated with a small residual amount of the Cox1p present in the mutant or with some other mitochondrial protein.

The partial suppression of the cox24 null mutant by an intronless mitochondrial genome points to a role of Cox24p in intron processing. This is confirmed by Northern analysis of the mitochondrial COB and COX1 RNAs, both of which are transcribed from genes with multiple introns. Whereas processing of the COB mRNA was only marginally retarded in the mutant, the COX1 exon probe detected mainly large precursor transcripts with an almost complete absence of the mature COX1 mRNA. Hybridization of mitochondrial RNAs to specific COX1 intronic probes revealed that the mutant is blocked in processing of introns aI2 and aI3. The requirement of Cox24p for aI2 and aI3 splicing is also supported by the suppressor activity of mtDNA containing a COX1 gene with only the aI4 intron.

The accumulation of high-molecular weight intermediates with the fourth (aI4) intron indicated that the cox24 mutation affects either excision or stability of this group I intron. Splicing of aI4 depends on the expression of a maturase encoded in the COB bI4 intron, which also functions in excision of bI4 itself (56). The presence in the mutant of a translatable COB mRNA excludes a deficiency of the bI4 maturase as a plausible explanation for the accumulation of splicing intermediates with aI4. The partial rescue of the mutant by a COX1 gene lacking all introns except the aI4 intron suggests that a failure to process the upstream aI2 or aI3 introns may exert a polar effect on excision of aI4.

Because the cox24 mutant is blocked in splicing of both group II (aI2) and group I (aI3) introns, it is unlikely that Cox24p is involved in catalyzing some specific step of the mechanisms by which these two different types of introns are spliced. A more plausible explanation is that Cox24p affects processing of the COX1 precursor in a more general way, perhaps by stabilizing a splicing competent conformation of the COX1 RNA similar to that proposed for Cbp2p in splicing of bI5 (57).

Mutants with lesions in genes that code for proteins dedicated exclusively to splicing of introns acquire wild-type growth properties on respiratory substrates when their target introns are absent in mtDNA (58). The finding that suppression of the growth defect and restoration of Cox1p synthesis in the cox24 mutant harboring an intronless mitochondrial genome is only partial, indicates that in addition to splicing of COX1 introns, Cox24p has yet another function. The phenotype of the cox14-cox24 double mutant with an intronless COX1 gene points to a possible function of Cox24p in translation of Cox1p. In previous studies the cox14 null allele was shown to restore Cox1p synthesis in mutants arrested at post-translational stages of COX assembly (36). This was not true of mutants with genetic lesions in genes such as MSS51 and PET309 that are required for translation of Cox1p (36). The negative effect of the cox14 mutation on Cox1p translation in the double mutant argues against a post-translational role of Cox24p in assembly and is more consistent with a requirement of this protein for Cox1p translation.

Maturation of the COX1 transcript (16-20, 26, 28-31) and translation of the resultant mRNA (4, 36, 46, 50, 51) are complex events requiring the intervention of a large number of gene products. Some of these accessory proteins are organized in large complexes that are associated the inner membranes facing the matrix where they influence different aspects of COX1 and COB expression (27, 59). A dual role of Cox24p in mitochondrial RNA processing and translation would suggest that this protein may also be a constituent of such a multifunctional complex.


Figure 8
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  		FIGURE 8.
Solubilization and localization of Cox24p. A, mitochondria were prepared from W303{Delta}COX24/ST16 expressing Cox24p with an HA tag at its carboxyl terminus. The mitochondria were sonically irradiated for 5 s with a Branson sonifier and subjected to centrifugation at 300,000 x gav for 15 min. The supernatant consisting of soluble proteins from the matrix and intermembrane space were collected, and the pellet consisting of submitochondrial particles was suspended in the starting volume of buffer. Submitochondrial particles were mixed with an equal volume of 0.2 M sodium carbonate and incubated on ice for 30 min. The submitochondrial particles were also adjusted to a final concentration of 1% dodecyl maltoside. Following centrifugation of the sodium carbonate and detergent-treated samples at 300,000 x gav for 15 min, the supernatants were collected, and the pellets were suspended in the starting volumes of buffer. Equivalent volumes of each fraction were separated by SDS-PAGE on a 12% polyacrylamide gel, transferred to nitrocellulose, and the Western blot was reacted with a mouse monoclonal antibody against the HA tag. Proteins were visualized as in Fig. 1D. The fractions loaded on the gel were: mitochondria (Mit), supernatant after sonic disruption of mitochondria (SS), submitochondrial particles (SMP), carbonate extract (CS), carbonate pellet (CP), dodecyl maltoside extract (LS), and dodecyl maltoside pellet (LP). Cox24p-HA is identified in the margin. B, mitochondria were prepared by the method of Glick and Pon (54) from the W303{Delta}COX24/ST16 expressing Cox24p-HA and from wild-type strain W303-1A. The mitochondria (Mt) were converted to mitoplasts (Mplast) by the method of Glick (55). Both mitochondria and mitoplasts were incubated on ice for 1 h in the absence or presence of 0.1 mg/ml proteinase K (Prot. K). Following addition of phenyl-methylsulfonyl fluoride to inhibit proteinase K, mitochondria were isolated by centrifugation at 14,000 x g for 10 min. Equivalent amounts (40 µg) of mitochondria and mitoplasts were separated by SDS-PAGE, transferred to nitrocellulose, and the Western blots were first treated either with a monoclonal antibody against the HA tag (Ab HA), or with a rabbit polyclonal antibody against Cox24p (Ab Cox24p). The Western blots were also reacted with rabbit polyclonal antibodies against Sco1p, cytochrome b2 (Cyt. b2), and {alpha}-ketoglutarate dehydrogenase (KGD). Proteins were visualized as in A after a secondary reaction with peroxidase-conjugated anti-mouse or anti-rabbit IgG.

 
The cox24 point mutant C149 is part of a collection of respiratory deficient pet strains that was previously used to identify 18 complementation groups defective in splicing of the COX1 precursor (60). These mutants were found to exhibit a range of different phenotypes based on their patterns of COX1 transcripts. One of the mutants (N266) reported in that study had a COX1 RNA phenotype very similar to that of cox24 mutants. N266 had normal COB mRNA but was deficient in COX1 mRNA. It accumulated splicing intermediates with aI4 and showed no evidence of aI2 processing. The similarity of the two mutants suggests that the defective protein in N266 may be functionally related to Cox24p. Further studies of this possibility will require identification the gene, which until now has proven difficult to clone by complementation.

COX24 is transcribed from an open reading adjacent to, but in the opposite strand from, MSS51. Based on the presence of an ABF1 binding site between the two genes, it was speculated that their products may have related functions (32). Our results, particularly the implication of Cox24p in translation of Cox1p, lend further credence to this possibility.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Research Grant GM45952. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a grant from the Conselho Nacional Desenvolvimento Cientifico e Tecnologico (CNPq) Fundação Amparo a Pesquisa São Paulo (FAPESP). Current address: Dept. of Genetics, IBB-UNESP, Botucatu/SP 18607-741, Brazil. Back

2 Current address: Dept. of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011. Back

3 To whom correspondence should be addressed. Tel.: 212-854-2920; Fax: 212-856-8246; E-mail: spud{at}cubpet2.bio.columbia.edu.

4 The abbreviations used are: COX, cytochrome c oxidase; pet mutant, respiratory deficient mutant of yeast with a mutation in a nuclear gene; {rho}o/- mutant, respiratory deficient mutant with either large deletions in or lacking mitochondrial DNA; mtDNA, mitochondrial DNA; HA, hemagglutinin. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225[Abstract/Free Full Text]
  2. McEwen, J. E., Ko, C., Kloeckner-Gruissem, B., and Poyton, R. O. (1986) J. Biol. Chem. 261, 11872-11879[Abstract/Free Full Text]
  3. Seraphin, B., Simon, M., and Faye, G. (1988) EMBO J. 7, 1455-1464[Medline] [Order article via Infotrieve]
  4. Manthey, G. M., and McEwen, J. E. (1995) EMBO J. 14, 4031-4043[Medline] [Order article via Infotrieve]
  5. Mulero, J. J., and Fox, T. D. (1993) Mol. Biol. Cell 4, 327-1335
  6. Costanzo, M. C., and Fox, T. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2677-2681[Abstract/Free Full Text]
  7. Glerum, D. M., Shtanko, A., and Tzagoloff, A. (1996) J. Biol. Chem. 271, 20531-20535[Abstract/Free Full Text]
  8. Hiser, L., Di Valentin, M., Hamer, A. G., and Hosler, J. P. (2000) J. Biol. Chem. 275, 619-623[Abstract/Free Full Text]
  9. Barros, M. H., Nobrega, F. G., and Tzagoloff, A. (2002) J. Biol. Chem. 277, 9997-10002[Abstract/Free Full Text]
  10. Manon, S., and Guerin, M. (1989) Biochim. Biophys. Acta 985, 127-132[Medline] [Order article via Infotrieve]
  11. Bonitz, S. G., Coruzzi, G., Thalenfeld, B. E., Tzagoloff, A., and Macino, G. (1980) J. Biol. Chem. 255, 11927-11941[Abstract/Free Full Text]
  12. Dujon, B. (1989) Gene (Amst.) 82, 91-114[CrossRef][Medline] [Order article via Infotrieve]
  13. Lazowska, J., Meunier, B., and Macadre, C. (1994) EMBO J. 13, 4963-4972[Medline] [Order article via Infotrieve]
  14. Guo, W., W., Moran, J. V., Hoffman, P. W., Henke, R. M., Butow, R. A., and Perlman, P. S. (1995) J. Biol. Chem. 270, 15563-15570[Abstract/Free Full Text]
  15. Szczepanek, T., Macadre, C., Meunier, B., and Lazowska, J. (1994) Gene (Amst.) 139, 1-7[CrossRef][Medline] [Order article via Infotrieve]
  16. Lazowska, J., Jacq, C., and Slonimski, P. P. (1980) Cell 2, 333-348
  17. Kennell, J. C., Moran, J. V., Perlman, P. S., Butow, R. A., and Lambowitz, A. M. (1993) Cell 73, 133-146[CrossRef][Medline] [Order article via Infotrieve]
  18. Wenzlau, J. M., Saldanha, R. J., Butow, R. A., and Perlman, P. S. (1989) Cell 56, 421-430[CrossRef][Medline] [Order article via Infotrieve]
  19. Seraphin, B., Faye, G., Hatat, D., and Jacq, C. (1992) Gene (Amst.) 113, 1-8[CrossRef][Medline] [Order article via Infotrieve]
  20. Lazowska, J., Claisse, M., Gargouri, A., Kotylak, Z., Spyridakis, A., and Slonimski, P. P. (1989) J. Mol. Biol. 205, 275-289[CrossRef][Medline] [Order article via Infotrieve]
  21. McGraw, P., and Tzagoloff, A. (1983) J. Biol. Chem. 258, 9459-9468[Abstract/Free Full Text]
  22. Stepien, P. P., Margossian, S. P., Landsman, D., and Butow, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6813-6817[Abstract/Free Full Text]
  23. Gampel, A., and Tzagoloff, A. (1989) Mol. Cell Biol. 9, 5424-5433[Abstract/Free Full Text]
  24. Golik, P., Szczepanek, T., Bartnik, E., Stepien, P. P., and Lazowska, J. (2004) FEMS Yeast Res. 4, 477-485[Medline] [Order article via Infotrieve]
  25. Margossian, S. P., Li, H., Zassenhaus, H. P., and Butow, R. A. (1996) Cell 84, 199-209[CrossRef][Medline] [Order article via Infotrieve]
  26. Huang, H. R., Rowe, C. E., Mohr, S., Jiang, Y., Lambowitz, A. M., and Perlman, P. S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 163-168[Abstract/Free Full Text]
  27. Krause, K., Lopes de Souza, R., Roberts, D. G., and Dieckmann, C. L. (2004) Mol. Biol. Cell 15, 2674-2683[Abstract/Free Full Text]
  28. Herbert, C. J., Labouesse, M., Dujardin, G., and Slonimski, P. P. (1988) EMBO J. 7, 473-483[Medline] [Order article via Infotrieve]
  29. Labouesse, M. (1990) Mol. Gen. Genet. 224, 209-221[CrossRef][Medline] [Order article via Infotrieve]
  30. Valencik, M. L., Kloeckener-Gruissem, B., Poyton, R. O., and McEwen, J. E. (1989) EMBO J. 8, 3899-3904[Medline] [Order article via Infotrieve]
  31. Bousquet, I., Dujardin, G., Poyton, R. O., and Slonimski, P. P. (1990) Curr. Genet. 18, 117-124[CrossRef][Medline] [Order article via Infotrieve]
  32. Simon, M., Della Seta, F., Sor, F., and Faye, G. (1992) Yeast 8, 559-567[Medline] [Order article via Infotrieve]
  33. ten Berge, A. M., Zoutewelle, G., and Needleman, R. B. (1974) Mol. Gen. Genet. 131, 113-121[Medline] [Order article via Infotrieve]
  34. Tzagoloff, A., Akai, A., and Foury, F. (1976) FEBS Lett. 65, 391-396[CrossRef][Medline] [Order article via Infotrieve]
  35. Seraphin, B., Boulet, A., Simon, M., and Faye, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6810-6814[Abstract/Free Full Text]
  36. Barrientos, A., Zambrano, A., and Tzagoloff, A. (2004) EMBO J. 23, 3472-3482[CrossRef][Medline] [Order article via Infotrieve]
  37. Broach, J. R., Strathern, J. N., and Hicks, J. B. (1979) Gene (Amst.) 8, 121-133[CrossRef][Medline] [Order article via Infotrieve]
  38. Beggs, J. D. (1978) Nature 275, 104-109[CrossRef][Medline] [Order article via Infotrieve]
  39. Rothstein, R. J. (1983) Methods Enzymol. 101, 202-211[Medline] [Order article via Infotrieve]
  40. Hill, J. E., Myers, A. M., Koerner, T. J., and Tzagoloff, A. (1986) Yeast 2, 163-167[CrossRef][Medline] [Order article via Infotrieve]
  41. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  42. Maxam, A. M., and Gilbert, W. (1980) Methods Enzymol. 65, 49-56
  43. Faye, G., Kujawa, C., and Fukuhara, H. (1974) J. Mol. Biol. 88, 185-203[CrossRef][Medline] [Order article via Infotrieve]
  44. Tzagoloff, A., Akai, A., and Needleman, R. B. (1975) J. Biol. Chem. 250, 8228-8235[Abstract/Free Full Text]
  45. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  46. Barrientos, A., Barros, M. H., Valnot, I., Rotig, A., Rustin, P., and Tzagoloff, A. (2002) Gene (Amst.) 286, 53-63[CrossRef][Medline] [Order article via Infotrieve]
  47. Muroff, I., and Tzagoloff, A. (1990) EMBO J. 9, 2765-2773[Medline] [Order article via Infotrieve]
  48. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13
  49. Conde, J., and Fink, G. R. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3651-3655[Abstract/Free Full Text]
  50. Decoster, E., Vassal, A., and Faye, G. (1993) J. Mol. Biol. 232, 79-88[CrossRef][Medline] [Order article via Infotrieve]
  51. Perez-Martinez, X., Broadley, S. A., and Fox, T. D. (2003) EMBO J. 22, 5951-5961[CrossRef][Medline] [Order article via Infotrieve]
  52. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G., and Thompson, J. D. (2003) Nucleic Acids Res. 31, 3497-3500[Abstract/Free Full Text]
  53. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 147, 105-132
  54. Glick, B. S., and Pon, L. A. (1995) Methods Enzymol. 260, 213-223[Medline] [Order article via Infotrieve]
  55. Glick, B. S. (1995) Methods Enzymol. 260, 224-231[Medline] [Order article via Infotrieve]
  56. Labouesse, M., and Slonimski, P. P. (1983) EMBO J. 2, 269-276[Medline] [Order article via Infotrieve]
  57. Gampel, A., and Cech, T. R. (1991) Genes Dev. 5, 1870-1880[Abstract/Free Full Text]
  58. Hill, J., McGraw, P., and Tzagoloff, A. (1985) J. Biol. Chem. 260, 3235-3238[Abstract/Free Full Text]
  59. Naithani, S., Saracco, S. A., Butler, C. A., and Fox, T. D. (2003) Mol. Biol. Cell 14, 324-333[Abstract/Free Full Text]
  60. Pel, H. J., Tzagoloff, A., and Grivell, L. A. (1992) Curr. Genet. 21, 139-146[CrossRef][Medline] [Order article via Infotrieve]

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