(Received for publication, April 28, 1997, and in revised form, May 29, 1997)
From the Department of Biological Sciences, Columbia University, New York, New York 10027
The respiratory defect of Saccharomyces cerevisiae mutants assigned to complementation group G4 of a pet strain collection stems from their failure to synthesize cytochrome oxidase. The mutations do not affect expression of either the mitochondrially or nuclearly encoded subunits of the enzyme. The cytochrome oxidase deficiency also does not appear to be related to mitochondrial copper metabolism or heme a biosynthesis. These data suggest that the mutants are likely to be impaired in assembly of the enzyme. A gene designated COX15 has been cloned by transformation of mutants from complementation group G4. This gene is identical to reading frame YER141w on chromosome 5. To facilitate further studies, Cox15p has been expressed as a biotinylated protein. Biotinylated Cox15p fully restores cytochrome oxidase in cox15 mutants, indicating that the carboxyl-terminal sequence with biotin does not affect its function. Cox15p is a constituent of the mitochondrial inner membrane and, because of its resistance to proteolysis, probably is largely embedded in the phospholipid bilayer of the membrane. The present studies further emphasize the complexity of cytochrome oxidase assembly and report a new constituent of mitochondria involved in this process. The existence of COX15 homologs in Schizosaccharomyces pombe and Caenorhabditis elegans suggests that it may be widely distributed in eucaryotic organisms.
The completion of the Saccharomyces cerevisiae genome sequence (1) has spawned new large scale projects designed to unravel the functions of the numerous unknown reading frames. A substantial fraction of the total information in yeast chromosomal DNA is comprised of PET genes that are essential for the biogenesis of respiratory competent mitochondria. Mutations in these genes were shown in the early 1950s (2, 3) to affect the ability of yeast to respire. Renewed efforts to mutationally saturate for this class of genes (4, 5) have helped to expand our knowledge of the extent and nature of the contribution made by the nucleus toward maintenance of respiring mitochondria.
Biochemical studies of pet mutants1 have revealed that the assembly of respiratory chain enzymes is governed by an unexpectedly large number of genes. For example, some three dozen complementation groups have been reported to consist of mutants displaying a selective deficiency in cytochrome oxidase (4, 6). In addition to mutations in the structural genes, these strains are also affected in: 1) processing of the mitochondrial cytochrome oxidase pre-mRNAs (7-9), 2) translation of the resultant mRNAs (10, 11), 3) heme a biosynthesis (12), 4) copper import and transfer to the apoenzyme (13, 14), and 5) as yet poorly understood events in the pathway leading to the functional enzyme (15-17). To learn more about the assembly of this heteroligomeric membrane complex, we have continued to screen for, and to analyze pet mutants with lesions in cytochrome oxidase. In this article, we report the properties of mutants from complementation group G4 whose cytochrome oxidase deficiency results from mutations in COX15 (identical to reading frame YER141w on chromosome 5). We show that mutations in this gene have no effect on the expression of the mitochondrial or nuclear cytochrome oxidase genes. Since cox15 mutants also do not appear to be impaired in heme a synthesis and copper metabolism, the product of this gene (Cox15p) is most likely involved in some aspect of the assembly process itself. We also present evidence that Cox15p is imported into mitochondria and is a constituent of the inner membrane.
The genotypes and sources of the strains of S. cerevisiae used in this study are listed in Table I. The compositions of the media for growth of yeast have been described elsewhere (21).
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Unless otherwise indicated, wild-type and mutant yeast were grown to stationary phase in YPGal (2% galactose, 1% yeast extract, and 2% peptone) and mitochondria were prepared by the procedure of Faye et al. (22), except that Glusulase was replaced by Zymolyase 20,000 (ICN Biomedicals, Inc.) to convert cells to spheroplasts. Difference spectra of reduced versus oxidized cytochromes of mitochondrial extracts were taken at room temperature (20). Cytochrome oxidase activity was measured as described previously (20).
Extraction and Separation of Mitochondrial HemesMitochondria were suspended in 0.5 M sorbitol, 20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA at a protein concentration of 15-20 mg/ml. Extraction of mitochondrial hemes was achieved by addition of mitochondria to acidified acetone such that the final concentration of protein was 3 mg/ml in 83% acetone (acetone:HCl = 97.5:2.5). The mixture was vortexed for 30 s and centrifuged for 5 min in a Beckman Microfuge. The clarified extract was collected and mixed with an equal volume of 50% acetonitrile. Insoluble material formed was removed by a second centrifugation. The extract was then neutralized with 1.65 M ammonium hydroxide. The neutralized extract equivalent of 1.5 mg of mitochondrial protein was applied to a Phenomenex (Torrance, CA) 3.9 × 300-mm C18 Bondclone column. The column was developed by a modification of the method of Sone and Fujiwara (23). Hemes were eluted at a flow rate of 1 ml/min using a 30-50% acetonitrile gradient over the first 5 ml, followed by a 50-75% linear acetonitrile gradient over the subsequent 35 ml. The acetonitrile solutions contained 0.05% trifluoroacetic acid. The elution times of protoheme, heme a, and heme o were determined prior to each set of runs with standards. The elution of heme compounds was monitored at 400 nm.
Cloning of the COX15The wild-type COX15 gene was cloned by transformation of E412/L1 and N7-211/L1 with a yeast genomic library by the method of Beggs (24). The library used for the transformation was constructed from partial Sau3A fragments of nuclear DNA (averaging 7-10 kb) from the respiratory competent haploid strain S. cerevisiae D273-10B/A1. The purified DNA fragments were ligated to the BamHI site of YEp13 (25). E412/L1 or N7-211/L1 were grown in YPGal to early stationary phase and approximately 108 cells were transformed with 50 µg of plasmid DNA. Part of the transformation mixture (10%) was plated on minimal glucose to estimate the total number of plasmid-bearing transformants, and the rest of the cells were plated on rich glycerol medium (2% peptone, 1% yeast extract, 3% glycerol, 2% ethanol, 1.2 M sorbitol) to select respiratory competent clones. Sixteen transformants whose respiratory competency and leucine prototrophy co-segregated were used to isolate and characterize the complementing plasmids.
Construction of COX15-BIO Fusion GeneA gene expressing
biotinylated Cox15p was constructed by introducing a XbaI
site 12 nucleotides before the termination codon. The polymerase chain
reaction primer complementary to the sense strand was
5-ttataatggtctagaggctaactt. The second polymerase chain reaction
primer was identical to the sense strand except for two nucleotides.
This primer (5
-tcgtttattaagcttcctattgta) introduced a
HindIII site 72 nucleotides upstream of the COX15 initiation codon. The 1.6-kb fragment amplified from pG4/ST3 was transferred to YEp352-Bio7 (17). The resultant plasmid (pG4/ST10) contained an in-frame fusion of COX15 to a 270-nucleotide
long sequence coding for the biotinylation site of bacterial
transcarboxylase (26). The fusion gene was transferred from the
multicopy plasmid to the integrative plasmid YIp352 (27). After
linearization at the NcoI site in the plasmid-borne
URA3 gene, the COX15 fusion gene sequence was
integrated at the ura3 locus of the cox15 mutant aWL
COX15.
COX15 was disrupted at the
internal BglII site, either with a 1.8-kb BamHI
fragment containing HIS3, or alternatively, with a 3-kb
BglII fragment containing LEU2. In both cases,
pG4/ST4 was digested with BglII and the linearized plasmid
ligated with the respective disruptors. The mutant alleles were
recovered as linear SacI fragments and used to transform
either W303-1A (with cox15::HIS3) or LL20 (with
cox15::LEU2) by the one-step gene replacement method (28). Respiratory defective transformants prototrophic for
either of the markers were crossed to a cox15 mutant and to a o tester strain. Integration of the disrupted alleles in
chromosomal DNA was verified genetically by the crosses to the
cox15 mutants, and in the case of the
cox15::LEU2 allele, also by Southern blot analysis
of genomic DNA.
Standard procedures were used for the preparation and ligation of DNA fragments, and for transformation and recovery of plasmid DNA from Escherichia coli (29). The preparation of yeast nuclear DNA and the conditions for the Southern hybridizations, were as described by Myers et al. (30). DNA was sequenced by the method of Maxam and Gilbert (31). Proteins were separated by PAGE either in the buffer system of Laemmli (32) or by the method of Schagger and von Jagow (33). Immunodetection of proteins on Western blot was carried out with 125I-protein A (34). Protein concentrations were determined by the method of Lowry et al. (35).
Approximately 70 independent mutants have been assigned to complementation group G4 of a pet collection (4). G4 mutants are respiratory deficient as a result of recessive mutations in a nuclear gene, causing a specific deficiency in cytochrome oxidase. The enzymatic properties of N7-211, a representative of this complementation group have already been reported in an earlier mutant screen (20). Based on assays of respiratory chain enzymes and spectral analysis of mitochondrial cytochromes, this mutant was shown to have a specific cytochrome oxidase deficiency (20).
To better understand the biochemical basis for this phenotype, we have
carried out a more complete analysis of cytochrome oxidase proteins.
The failure of G4 mutants to express cytochrome oxidase is not due to a
block of transcription or processing of the mitochondrially encoded
(36) pre-mRNAs for subunits 1-3, or translation of the resultant
mRNA. This is borne out by in vivo pulse labeling of
mitochondrially synthesized proteins in whole cells poisoned with
cycloheximide to inhibit cytoplasmic protein synthesis. Under these
conditions, incorporation of [35S]methionine into
subunits 1, 2, and 3 is as efficient in the G4 mutant as in wild-type
(Fig. 1). Even though synthesis of these endogenously
produced proteins appears to be normal, their steady-state concentrations are dramatically lower in the mutants. This is evident
from the immunoblot analysis of subunits 1 and 2 in mitochondria of
aW303COX15 (see below for construction of this mutant) shown in Fig.
2. Subunit 3 could not be tested because of a lack of a
suitable antibody. Proteolytic degradation of these hydrophobic membrane proteins is commonly observed in mutants unable to complete assembly of the enzyme (13, 14, 17).
Since the synthesis of cytochrome oxidase depends on the expression of
six nuclearly encoded subunits, the steady-state concentrations of
these constituents were also compared in mitochondria from aW303COX15 and the parental strain W303-1A. These imported
components of the enzyme were examined in intact mitochondria, in
submitochondrial particles, and the soluble protein fraction released
from sonically disrupted mitochondria. The antibodies used for these
analyses detect subunits 4, 5, 6, and the smaller polypeptides
(subunits 7, 8, and 8a). The latter, however, are not resolved in the
gel and migrate as a single composite band. The results of the
immunoblots shown in Fig. 2 indicate that the mitochondrial content of
the imported subunits in the mutant is not significantly different from
the wild-type (Fig. 2).
In other studies we have noted that a sizeable fraction of subunits 4 and 6 are recovered in the soluble protein fraction following sonic disruption of mitochondria.2 The soluble forms of subunits 4 and 6 probably represent a mitochondrial pool of unassembled subunits. The relative distribution of subunits 4, 5, and 6 in the soluble and membrane fractions revealed an increase in the amounts of unassembled subunits 4 and 6, but not of subunit 5 which co-fractionates exclusively with the submitochondrial vesicles. The increase of soluble subunit 4 in the mutant is explained by the depletion of subunit 1, with which it is associated in the enzyme complex (38). X-ray crystallographic data indicate that in the bovine enzyme, subunit 6 is complexed to subunit 5, the latter being almost completely embedded in the phospholipid bilayer (38). The increase in soluble subunit 6 is surprising in view of the normal concentration of subunit 5 in the mutant, all of which is in the membrane. The recovery of a larger fraction of subunit 6 in the soluble fraction suggests a more labile association of subunits 5 and 6 when the normal complex is not formed. The presence in the G4 mutant of subunits 4, 5, and 6 (this probably also applies for the low-molecular weight subunits that are not resolved by PAGE) as mature proteins, and at concentrations comparable to those seen in wild-type mitochondria, argues against an effect of the mutation on expression, transport, or processing of these polypeptides.
Cytochrome oxidase utilizes heme a and copper as obligatory electron carriers (39). In addition to their functional roles, these prosthetic groups are required for the assembly of a stable enzyme. Mutations interfering with heme a synthesis (40) or mitochondrial copper metabolism (13, 14) lead to a deficiency of cytochrome oxidase activity, concomitant with proteolysis of subunits 1 and 2 of the enzyme. The enhanced turnover of these proteins under copper or heme a limiting conditions is probably a consequence of a more open and less stable quaternary structure.
The cytochrome oxidase deficiency of some mutants impaired in copper delivery to mitochondria can be suppressed by high copper concentrations in the medium (13). The failure of copper to rescue G4 mutants tends to argue against, but does not exclude, a defect in mitochondrial copper transport or transfer to the apoenzyme. Similarly, the presence of heme a, albeit at low concentrations in the mutant mitochondria (data not shown) made it unlikely that the lesion is related to the synthesis of this prosthetic group.
Cloning and Sequencing of COX15A gene capable of conferring
wild-type growth to G4 mutants on non-fermentable carbon sources was
cloned by transformation of N7-211/L1 and E412/L1 with a genomic
library of yeast nuclear DNA constructed in the shuttle vector YEp13
(25). The plasmids recovered from the different respiratory competent
yeast transformants had either identical or overlapping fragments of
genomic DNA. The recombinant plasmid pG4/T2 (Fig. 3) was
used to subclone and localize the gene. The insert of this plasmid was
dissected at an internal BamHI site into two fragments. Of
the two, only the shorter 2.3-kb fragment starting at the internal
BamHI site, and extending to the distal end of the insert,
complemented the mutants (Fig. 3). Further subcloning of this region
indicated that the internal BglII and PstI sites
were likely to be in the gene (see pG4/ST3-ST7 in Fig. 3).
The 2.3-kb insert of pG4/ST3 was sequenced by the method of Maxam and Gilbert (31). The nucleotide sequence revealed two open reading frames, one of which included the BglII and PstI sites inferred from the subcloning results to be in the gene. This open reading frame is located on chromosome 5 next to, and in an orientation opposite to, MAG (3-methyladenine DNA glycosylase) (41). In keeping with the nomenclature adopted to designate genes required for expression of cytochrome oxidase, this gene has been designated COX15 (GenBank accession L38643).
COX15 codes for a protein of Mr = 54,667. The amino-terminal 76 residues constitute a hydrophilic sequence with a preponderance of basic amino acids. This sequence, which is not conserved in two Cox15p homologs (see below), probably serves as a mitochondrial import signal. The amino acid composition and the primary sequence of the protein suggests a hydrophobic protein.
Two homologs of Cox15p were found in current protein data bases. One is
an unidentified protein of the fission yeast Schizosaccharomyces pombe (GenBank accession Z70043); the other homolog is a protein of Caenorhabditis elegans (GenBank accession Z49130). An
alignment of the three proteins indicates 28% identity between the
three proteins in an 375-amino acid span (Fig. 4). The
S. pombe protein contains a carboxyl-terminal extension of
150 residues that is absent in the other proteins. The homology of the
different Cox15p's is also evident from their hydropathy profiles. The
yeast and nematode proteins have 7-8 analogously spaced hydrophobic
domains of sufficient lengths to span a phospholipid bilayer (shown for the S. cerevisiae Cox15p in Fig. 4).
In Situ Disruption of COX15
COX15 was disrupted at
the internal BamHI site, either with a 3-kb BglII
fragment containing LEU2 or with a 1.8-kb BamHI
fragment containing HIS3. The LEU2 and
HIS3 disrupted alleles were substituted for the normal
chromosomal copies of the gene in the respiratory competent haploid
strains LL20 (20) and W303-1A, respectively. Respiratory deficient
transformants were verified to be complemented by a o tester
strain but not by G4 mutants, confirming the presence of the disruption
alleles. In the case of the disruption in LL20, this was also shown
directly by Southern analysis of genomic DNA.
The spectral properties (Fig. 5) and enzyme activities
of mitochondria indicated the mutant constructs to lack cytochrome oxidase but not other components of the respiratory chain. The cytochrome oxidase deficiency of the cox15 mutant constructs
and their failure to be complemented by G4 mutants argues that the latter have mutations in COX15.
Localization of Cox15p
The subcellular location of Cox15p was
studied by expressing it as a biotinylated protein from constructs in
which the coding sequence of COX15 was fused at the 3 end
to a bacterial biotinylation signal sequence (see "Materials and
Methods"). Transformation of the cox15 mutant WL
COX15
with the fusion gene either on a multicopy plasmid (pG4/ST10) or in an
integrative vector (pG4/ST11) yielded respiratory competent clones
(WL
COX15/ST10 and WL
COX15/Int, respectively) that grew on
non-fermentable carbon sources with rates comparable to wild-type
yeast. The COX15-BIO gene construct on multicopy and in
single copy also restored a full complement of cytochrome oxidase (Fig.
5).
Wild-type yeast as well as the transformants harboring the fusion gene
were fractionated to obtain mitochondria and total post-mitochondrial
supernatant proteins. The biotinylated proteins in the two fractions
were visualized on Western blots with a peroxidase-conjugated avidin
detection system. Mitochondria from both transformants, but not from
the wild-type strain, exhibit a unique biotin-containing protein with
an apparent size of 48 kDa (Fig. 6). This protein is
present in higher abundance in the mutant transformed with the fusion
gene on a multicopy plasmid. The biotinylated protein is not detected
in the post-mitochondrial supernatant fraction of the wild-type strain
or the two transformants. These results indicate that the hybrid
protein expressed from the fusion gene is biotinylated in
vivo and is localized in mitochondria. The ability of yeast to
biotinylate both cytoplasmic and mitochondrial proteins has been
demonstrated previously (44-46). Whether biotinylation of
mitochondrial proteins occurs in the cytoplasm or subsequent to import
has not been investigated and is not currently known. The presence in
mitochondria of biotin-dependent enzymes, however, tends to
argue that there may be a separate organellar biotinylation system.
The hydrophobic nature of the Cox15p sequence suggested it was likely
to be a membrane component. This was ascertained by subfractionation of
mitochondria from WLCOX15/Int, expressing chromosomally integrated
COX15-BIO. Mitochondria were disrupted by sonic irradiation
and separated into submitochondrial vesicles and soluble intermembrane
and matrix proteins. Western analysis of the two fractions disclosed an
exclusive association of biotinylated Cox15p with the membranes. Cox15p
behaves as an integral membrane protein based on its solubility
properties. As demonstrated in Fig. 6, extraction of the protein
requires at least 0.25% deoxycholate and 0.5 M NaCl,
conditions indicative of an integral membrane component.
To ascertain whether Cox15p is located in the outer or inner membrane,
mitochondria were lysed under hypotonic conditions and sonically
irradiated to fragment the outer and inner membranes (47). The mixture
was separated on an isopycnic sucrose gradient and the distribution of
biotinylated Cox15p was compared with inner (subunit 5 of cytochrome
oxidase) and outer membrane (porin) markers. Even though the method
achieves a separation of outer membranes relatively free of inner
membrane contamination in the upper part of the gradient, the denser
inner membranes contain substantial amounts of outer membrane. This is
seen in the bimodal distribution of porin (Fig. 7).
Cox15p-Bio fractionates as a single peak with a distribution similar to
cytochrome oxidase subunit 5, indicating that it is an inner membrane
protein.
Treatment of mitochondria and mitoplasts with proteinase K did not elicit loss of the protein. Under the same conditions an inner membrane protein containing an extramembrane domain facing the intermembrane space underwent proteolysis in mitoplasts but not in mitochondria (data not shown). This suggests that most of the Cox15p sequence must be embedded in the phospholipid bilayer and whatever domains exist outside the membrane are protected from attack by proteinase K. A largely membrane localization of Cox15p is supported by the 7-8 putative transmembrane domains making up most of the protein. Assuming Cox15p has 8 transmembrane segments and insertion into the membrane is coupled to transport, the biotinylated carboxyl terminus would be expected to face the matrix side of the membrane. By the same token, 7 transmembrane segments would dictate a localization of the carboxyl terminus in the intermembrane space. As indicated above, biotinylated Cox15p is resistant to proteinase K in mitoplasts. The protein was also resistant to proteolysis when mitochondria were sonically disrupted in the presence of proteinase K. These observations indicate that, independent of its location with respect to the two faces of the inner membrane, the carboxyl-terminal bacterial sequence with covalently bound biotin is not available to the protease, perhaps because it is masked by some other protein. Treatment of mitochondria with 0.1% deoxycholate prior to addition of proteinase K, however, led to a quantitative loss of Cox15p, thereby excluding the possibility that the protein is inherently resistant to proteolysis.
An estimate of the molecular weight of biotinylatd Cox15p was obtained
by sedimentation analysis of a detergent-solubilized extract of
WLCOX15/Int mitochondria in a sucrose gradient. The sedimentation of
Cox15p-Bio relative to known size markers suggests a native molecular
weight approximately two times that of the monomer (Fig.
8). Several other proteins involved in cytochrome oxidase assembly (17)3 have also been found
to be larger than indicated by their monomer molecular weight, possibly
because they may be part of larger heteroligomeric complexes. Attempts
to obtain evidence for the existence of a complex of Cox15p with other
proteins by biochemical or genetic means, however, were
unsuccessful.
The production of respiratory chain enzymes, particularly cytochrome oxidase, has been shown to depend on numerous nuclear genes, not all of whose functions are understood at present (4-6). The products of these genes act at all stages of assembly, including maturation of mitochondrial cytochrome oxidase transcripts and steps unique to cytochrome oxidase, such as addition of the heme a and copper prosthetic groups. COX15 is representative of a large group of genes that catalyze events occurring late in the assembly pathway. Even though the precise function of this protein is not clear at present, our results show that it acts subsequent to the synthesis of the endogenous and imported subunits. Of the more extensively studied pet genes known to affect cytochrome oxidase, all except COX17 (13) appear to code for mitochondrial membrane proteins (15-17); and as demonstrated in this study for Cox15p, are components of the inner membrane. The co-localization of Cox15p and cytochrome oxidase in the same compartment of mitochondria is consistent with the notion that it may be directly involved in the assembly process.