From the Department of Medical Genetics, University
of Alberta, Edmonton, Alberta T6G 2H7, Canada and the ¶ Department
of Biological Sciences, Columbia University,
New York, New York 10027
Received for publication, September 26, 2002, and in revised form, November 7, 2002
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ABSTRACT |
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We have characterized Cox16p, a new cytochrome
oxidase (COX) assembly factor. This protein is encoded by
COX16, corresponding to the previously
uncharacterized open reading frame YJL003w of the yeast genome.
COX16 was identified in studies of COX-deficient mutants
previously assigned to complementation group G22 of a collection of
yeast pet mutants. To determine its location, Cox16p was
tagged with a Myc epitope at the C terminus. The fusion protein, when expressed from a low-copy plasmid, complements the mutant and is
detected solely in mitochondria. Cox16p-myc is an integral component of
the mitochondrial inner membrane, with its C terminus exposed to the
intermembrane space. Cox16 homologues are found in both the human and
murine genomes, although human COX16 does not
complement the yeast mutant. Cox16p does not appear to be involved in
maturation of subunit 2, copper recruitment, or heme A biosynthesis.
Cox16p is thus a new protein in the growing family of eukaryotic COX
assembly factors for which there are as yet no specific functions
known. Like other recently described nuclear gene products involved in
expression of cytochrome oxidase, COX16 is a candidate for
screening in inherited human COX deficiencies.
Cytochrome oxidase
(COX),1 the terminal enzyme
of the mitochondrial respiratory chain, catalyzes the transfer of
electrons derived from sugars, fats, and amino acids to molecular
oxygen. The mammalian enzyme consists of 13 subunits and has a well
characterized structure (1). The three largest subunits, which are
encoded in mitochondrial DNA, form the catalytic core of the enzyme,
with subunits 1 and 2 binding the prosthetic groups required for
electron transfer. There are two heme A molecules, both of which are
located in the hydrophobic interior of subunit 1 and, based on their
spectral properties, are denoted hemes a and
a3. In addition, there are two copper atoms that
form the binuclear CuA site in subunit 2 and a single
copper atom (designated CuB) located adjacent to the heme
a3 site in subunit 1 (2). The nuclear genome
contributes the remaining 10 subunits, which are thought to play a
primarily structural role.
In contrast to the wealth of information about the structure of
cytochrome oxidase, the mechanism by which the holoenzyme complex is
assembled remains unclear. Studies in the yeast Saccharomyces cerevisiae have provided much of the information regarding the proteins involved in the COX assembly pathway. Some of these proteins are involved in recruiting copper to mitochondria and COX (3-5). Others have been implicated in heme A biosynthesis (6, 7) and transport
and maturation of the mitochondrially encoded subunits (8-10). Despite
these advances, the precise functions of a number of COX assembly
factors remain unknown (11-13). It is also unclear whether all of the
proteins required for COX assembly have been identified. This
information is important for understanding the mechanism of COX
assembly and for elucidating the genetic basis of human COX
deficiencies (14, 15).
Here we report the identification and characterization of Cox16p, a new
COX assembly factor encoded by a previously uncharacterized yeast open
reading frame (YJL003w; GenBankTM accession number Z49278).
Mutations in COX16 result in a failure to complete assembly
of cytochrome oxidase, and cox16 mutants are
respiration-deficient. Cox16p does not appear to function in
mitochondrial copper homeostasis or in the synthesis of heme A. The
phenotype of cox16 mutants also argues against a role of Cox16p in processing of Cox2p or membrane insertion of the
mitochondrially encoded subunits of COX. Like many of the other COX
assembly factors, Cox16p is an integral component of the mitochondrial
inner membrane and appears to exist in a higher molecular weight
complex. Recent additions to the data base reveal that COX16
has homologues in Schizosaccharomyces pombe, as well as in
the murine and human genomes. Human COX16 is thus a
candidate gene for human COX deficiencies in which mutations in the
known COX assembly factors have not been identified.
Strains and Media--
The genotypes of the strains used in this
study are indicated in Table
I. The composition of media for growth
and analysis of yeast strains has been described elsewhere (16).
Cloning and Disruption of COX16--
The yeast genomic library
used to clone COX16 was constructed from partial
Sau3A fragments of nuclear DNA of the respiratory-competent haploid strain S. cerevisiae D273-10B/A1. C25/U1, a mutant
from complementation group G22 (8), was transformed with the yeast genomic library as described previously (11), and the COX16 gene identified by isolating subclones capable of conferring
respiratory competence on C25/U1. The COX16 gene was
disrupted by insertion of a 1.1-kb URA3 fragment at the
internal HindIII site and transformation of the
respiratory-competent haploid strain, W303-1A, with the linear
fragment containing the disrupted gene and flanking sequences. The
transformation yielded the cox16 mutant
aW303 Construction of pMGL5 and the COX16-myc Fusion--
To
overexpress a Cox16p-myc fusion protein, we first constructed pMGL5, a
yeast/Escherichia coli shuttle plasmid containing a Myc
epitope tag in the backbone of the multicopy vector YEp351 (17).
Briefly, a 3.3-kb Nar1-AatII fragment of YEp351
containing the LEU2 marker and the 2-µm origin of
replication, was used to replace a 3.8-kb
Nar1-AatII fragment in YCpmyc111 (a gift from Dr.
Troy Harkness, modified from Gietz and Sugino (18)). This converted the
CEN plasmid into an episomal plasmid. To make the Myc fusion,
COX16 was amplified by PCR, using primers that introduced a
PstI site 216 nucleotides upstream of the start codon
(5'-gttattagactgcagatacacttcc-3') and a SmaI site two
nucleotides upstream of the termination codon (5'-cgttttgaatgttcccgggcattc-3'). The 416-bp PCR product was ligated to
pMGL5, resulting in an in-frame fusion of Cox16p to the Myc epitope at
the C terminus. The same COX16 fragment was also fused to
the Myc sequence in the CEN plasmid YCpmyc111. Both constructs were
verified to be in-frame by automated sequencing (LiCor, Lincoln, NE).
Miscellaneous Methods--
Transformation of yeast was carried
out by the method of Schiestl and Gietz (19). Yeast strains were grown
to stationary phase in YPGal, and mitochondria were isolated as
described previously (20). For analysis of in vivo labeled
mitochondrial translation products, strains were grown in YPGal and
labeled with [35S]methionine in the presence of
cycloheximide (21). The labeling reaction was terminated after 15 min
by addition of an excess of 80 mM cold methionine and 80 µg/ml puromycin (0 time). Samples of the cultures were incubated at
30 °C and collected at 15 and 30 min, pelleted, and resuspended in
75 µl of a mixture containing 1.8 M NaOH, 1 M
Mitochondrial preparations used for the inner membrane
localization were isolated as described by Glick (23). Density gradient centrifugation was carried out using mitochondria (3 mg) from aW303 cox16 Mutants Are Defective in Cytochrome Oxidase
Assembly--
S. cerevisiae strains C25 and E699 of
complementation group G22 of a pet mutant collection (8) are
respiration-deficient due to a specific loss of cytochrome oxidase.
Spectral analysis of mitochondria isolated from C25 and E699 show a
partial or complete loss of the 605-nm cytochrome
aa3 peak, respectively (Fig.
1A). Since the respiratory
defect is complemented by
As in other COX assembly-defective mutants (20), the steady state
concentrations of subunits 1, 2, and 3 are very low in the
cox16 mutant (Fig. 1B). With the exception of
subunit 5, which is slightly reduced, the other nuclear-encoded
subunits are present at normal concentrations. In vivo
labeling of C25 in the presence of cycloheximide to block cytoplasmic
protein synthesis, indicated that the three mitochondrially encoded
subunits of cytochrome oxidase are correctly expressed (Fig.
1C), although labeling of Cox2p appeared to be slightly less
than in wild type. To assess if Cox16p participates in export of the
Cox2p precursor, the size and stability of this mitochondrial gene
product was compared in different COX null mutants sharing the same
nuclear background. The results of the in vivo pulse-chase
labeling experiment confirmed that only processed Cox2p is detected in
the cox16 null mutant aW303
Analysis of mitochondrial hemes in both the C25/U1 and cox16
null mutant revealed the presence of heme A and heme O (data not shown)
in amounts seen with most other COX assembly mutants (7), suggesting
that Cox16p is not involved in heme A biosynthesis. Finally, the
respiratory deficiency of C25/U1 and the cox16 null mutant
were not rescued by supplementation of the growth medium with copper,
calcium, iron, magnesium, manganese, or zinc (data not shown). This
result does not, however, exclude Cox16p from having a function in
transport or insertion of one of the heavy metals known to be
associated with COX.
Cloning and Disruption of COX16--
To identify the gene
responsible for the cytochrome oxidase deficiency of G22 mutants,
C25/U1 was transformed with a yeast genomic library, and uracil
prototrophic clones were checked for growth on non-fermentable carbon
sources. A transformant, C25/U1/T1, rescued both the uracil auxotrophy
and respiratory deficiency and was used to obtain the recombinant
plasmid pG22/T1, which contained an insert of genomic DNA ~7 kb in
length. This plasmid restored respiration when back transformed into
the mutant. Subcloning generated the construct pG22/ST2, which
conferred respiratory competence to C25/U1, and sequencing of this
construct identified the gene responsible for the complementation.
Comparison of the putative COX16 open reading frame to the
current databases revealed that the COX16 gene is identical
to open reading frame YJL003w on chromosome X (GenBank accession number
Z49278) in the yeast genome.
COX16 was disrupted to ascertain that the respiratory
deficiency of G22 mutants is caused by mutations in this gene. The
mutant allele was obtained by inserting a 1-kb HindIII
fragment containing the URA3 gene at an internal
HindIII site of COX16 in pG22/ST2, yielding
pG22/ST4. The respiratory-competent haploid strain W303-1A was
transformed with a linear BglII-SphI fragment of
pG22/ST4. An uracil-independent and respiration-deficient transformant
(aW303
Cox16p consists of 118 amino acid residues with a predicted mass of
14.1 kDa. The sequence includes one potential transmembrane domain
(Fig. 3). The presence of a mitochondrial
targeting sequence at the N terminus (Fig. 3B) is predicted
by the P-Sort program (//psort.nibb.ac.jp/helpwww2.html). The
amino acid composition of Cox16p is biased toward acidic residues, and
analysis of the putative mature protein reveals a pI of 5.0 (//ca.expasy.org/tools/pi_tool.html). Cox16p does not reveal any
homology to proteins of known function in the most recent databases,
nor does it appear to have any identifiable functional domains that
might provide clues about its function.
Expression and Mitochondrial Location of a cox16-myc Fusion
Protein--
The location of Cox16p was studied with a Myc-tagged
protein. Because of the presence of a potential cleavable N-terminal signal sequence, the Myc epitope was added at the C terminus. Growth of
transformants, expressing the tagged protein from either a high copy
episomal (aW303
A low molecular weight protein in mitochondria from aW303
Almost all of the COX assembly factors identified to date are
constituents of the mitochondrial inner membrane, as would be expected
from their involvement in assembly of a hydrophobic multimeric complex
of this membrane. The prediction of a membrane-spanning domain in
Cox16p suggested that it might be an integral membrane protein. This
is confirmed by the solubility properties of the protein. Titration of
aW303
Western analysis of intact mitochondria and mitoplasts from
aW303
Some of the COX assembly factors characterized to date appear to be
part of higher molecular weight homo- or heteroligomeric complexes (11,
21, 33, 34). The native molecular mass of Cox16p-myc was
estimated, by sedimentation of a 1% deoxycholate extract of
mitochondria in a 7 to 20% linear sucrose gradient, to be ~84 kDa
for both aW303 Cox16p Has Mammalian Homologs--
Searches of current protein and
expressed sequence tag databases indicate that Cox16p appears to
have a human (HSCP203; accession number NP_057552) and a murine
(accession number NP_079737) homologue. The human cDNA was
originally identified in a screen for novel proteins expressed in CD34+
hematopoietic stem/progenitor cells (35). Human COX16 is
located on the long arm of chromosome 14, in the interval
14q22.1-14q24.3 (LOC51241). Fig. 5
presents an alignment of the Cox16 proteins from yeast, humans, and
mice. This analysis reveals that the highest sequence conservation is in the region of the transmembrane domain and the C-terminal half. The
four Cox16 proteins shown in Fig. 5 share 24% identity and 40%
conserved residues.
To test if the human gene can complement the yeast cox16
mutant the cDNA for human COX16 was amplified by PCR
from a HeLa cell cDNA library. The cDNA was cloned into a yeast
expression vector containing the ADH1 promoter and
terminator in a YEp351 backbone,3 allowing
constitutive expression of proteins from cDNAs with their own start
codons. This construct (pCOX16H/ST1) did not complement the respiratory
deficiency of the aW303 The yeast COX16 gene product reported in this study is
a mitochondrial protein with an essential function in COX assembly. The
absence of cytochrome oxidase in cox16 mutants is
accompanied by a loss of the cytochrome aa3
spectral signal and a marked reduction in the steady state
concentrations of the mitochondrial-encoded but not nuclear-encoded
subunits of COX. The exception is Cox5p, which is also reduced in the
cox16 mutant but not in other previously studied COX
mutants. It is unclear whether this observation is relevant to the
function of Cox16p.
Like most COX assembly factors characterized to date, Cox16p is an
integral component of the mitochondrial inner membrane. The protease
protection experiments indicate that the C terminus of Cox16p-myc is
exposed to the cytoplasmic side of the inner membrane. This orientation
implies that the hydrophobic sequence, proximal to the N terminus, acts
as a stop-transfer signal (36). The location of the N-terminal
transmembrane domain combined with the orientation of Cox16p in the
inner membrane implies that the active region is in the hydrophilic
C-terminal half and is located in the intermembrane space. At present,
all the COX assembly factors with this orientation have Cox2p as their
target. Sco1p is inferred to function in maturation of the
CuA site (28), the Imp protease is responsible for removal
of the Cox2p N-terminal presequence (37), and Cox20p is a chaperone of
the Cox2p precursor that is required for the proteolytic processing
step (10). The presence of stable and mature Cox2p in the mutant
excludes the involvement of Cox16p in export or proteolytic processing
of the N-terminal extension. Attempts to relate Cox16p to maturation of
a metal center in the enzyme have also been negative. Copper and
other heavy metals, known to be present in COX, do not rescue
cox16 mutants when added to the growth medium. This result
does not, however, rule out an indirect role for Cox16p in the
acquisition of any one of these metals by the enzyme.
Studies of the yeast Saccharomyces cerevisiae have shed
considerable light on the widely differing roles that the dozen or more
currently known nuclear-encoded factors play in COX assembly (15).
Homologous proteins for most of these assembly factors have been
identified in many other genomes, including human (38-41). The yeast
model has also facilitated the attribution of some autosomal recessively inherited human COX deficiencies to mutations in genes coding for proteins involved in heme A biosynthesis (42), copper homeostasis (41, 43), and still other poorly understood aspects of COX
expression (44). The human COX16 homologue does not
complement a yeast cox16 mutant, which may indicate a
different function for human COX16 or may be due to the
heterologous context. The inability of a mammalian gene to exert its
function in a yeast background is not without precedent. Only about
one-third of the human COX assembly factors identified to date
complement corresponding yeast mutants (38, 40). We tend to think,
therefore, that human COX16 has the same function as yeast
Cox16p. The discovery of a new COX assembly factor provides another
candidate gene for sequencing in patients with autosomal recessively
inherited COX deficiencies without identified mutations.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Strains used in this study
COX16.
-mercaptoethanol, and 0.01 M phenylmethylsulfonyl fluoride. Proteins were precipitated by addition of an equal volume of
50% trichloroacetic acid. The mixture was centrifuged, and the pellet
washed once with 0.5 M Tris, twice with water, and resuspended in 50 µl of sample buffer (22). Labeled mitochondrial proteins were separated on a 12.5% polyacrylamide gel containing 6 M urea and 6% glycerol, and the gel was dried prior to autoradiography.
COX16/myc1, which were extracted with 1% deoxycholate and 0.5 M NaCl and loaded onto 2.4 ml of 7-20% sucrose gradients
(11). Lactate dehydrogenase (0.3 mg) was added to the extract as an internal standard, along with 2.5 mg hemoglobin. Gradients were chilled
at 4 °C for 1 h before addition of the extract and, following loading of the samples, centrifuged in a Beckman Optima TLX tabletop ultracentrifuge (Beckman Coulter, Fullerton, CA) for 12 h at
54,000 rpm. Protein concentrations were determined by the method of
Lowry et al. (24). Cytochrome oxidase activity and
mitochondrial cytochrome spectral analyses were carried out as
described by Tzagoloff et al. (25). Mitochondrial proteins
were separated by SDS-PAGE with 12% gels (22), 15% gels with glycerol
(26), or 16.5% gels containing 6 M urea (27). Western
analysis was carried out as described previously using antibodies
against subunits of cytochrome oxidase or Sco1p (28) or commercial
antibodies to the Myc epitope tag (Sigma). Immunoblots were
visualized using enhanced chemiluminescence (Amersham Biosciences).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
0 mutants, the cytochrome
oxidase lesion stems from recessive mutations in a nuclear gene, which
has been designated as COX16.
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Fig. 1.
Spectral analysis and steady state levels of
COX subunits in cox16 mutants. A, spectra
of mitochondrial cytochromes. Mitochondria were prepared from the two
wild type strains D273-10B/A1 (D273) and W303-1A
(W303) and from the cox16 mutants C25, E699, and
aW303 COX16 (
COX16) and extracted at a protein
concentration of 5 mg/ml with potassium deoxycholate (11). Difference
spectra of the oxidized (potassium ferricyanide) versus
reduced (sodium dithionite) extracts were recorded at room temperature.
The position of cytochrome aa3 is marked.
B, Western analysis of COX subunits. Mitochondria were
isolated from the wild type W303-1A (W303), the mutant
aW303
COX16 (
COX16), aW303
COX16/myc1
(myc1) and aW303
COX16/myc2 (myc2), which are
the cox16 mutant transformed with the
COX16-myc fusion in a high-copy and a CEN
plasmid, respectively. Subunits 1, 2, and 3 of cytochrome oxidase
(Cox1, Cox2, Cox3) were analyzed by separating 10 µg of mitochondrial
protein on 12% polyacrylamide gels. The nuclear-encoded subunits
(Cox4; Cox5; Cox6; Cox7, 7a, 8) were detected by separating 20 µg of
mitochondrial protein on a 16.5% polyacrylamide/6 M urea
gel. Following transfer to nitrocellulose, the blots were probed with
subunit-specific antibodies and visualized using enhanced
chemiluminescence. C, the wild type strains D273-10B/A1 and
the mutant C25 were grown in YPGal and labeled in vivo with
[35S]methionine in the presence of cycloheximide (46).
Mitochondria were isolated and separated by SDS-PAGE on a 7.5-15%
linear polyacrylamide gel. The identities of the labeled proteins are
marked in the margin: ribosomal protein (Var1); subunit 1 (Cox1), subunit 2 (Cox2), and subunit 3 (Cox3) of cytochrome oxidase; cytochrome b
(Cytb); subunit 6 (Atp6), subunit 8 (Atp8) and subunit 9 (Atp9) of ATPase.
COX16 after the 15-min pulse
(Fig. 2). Since proteolytic cleavage of
the presequence requires export of the N-terminal transmembrane domain
to the intermembrane space (29), these results exclude Cox16p from
having a function in membrane insertion of the N-terminal domain of the
precursor. The pulse-chase results also make it unlikely that Cox16p is
involved in membrane insertion of the C-terminal domain of Cox2p. By
comparison, the turnover of Cox2p in a cox18 mutant was
greatly increased compared with the wild type or other COX mutants
(e.g. cox15). Cox18p is required for the export
of the C-terminal domain of Cox2p (30), and the rapid degradation of
the protein probably occurs as a consequence of exposure of the C
terminus to proteolytic enzymes of the inner membrane and/or matrix. In
contrast, Cox2p turnover was similar in the cox15 and
cox16 mutants during the 30-min chase (Fig. 2). Since Cox15p
functions in heme A synthesis (31), the turnover rate of Cox2p in
cox15 mutants is unlikely to be due to a problem with
membrane insertion, and the decreased amount of Cox2p in cox16 and cox15 mutants is likely related to the
general block in COX assembly. The reduction of newly synthesized Cox1p
observed in the three mutants is a hallmark of most COX assembly
mutants examined to
date.2
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Fig. 2.
Turnover of in vivo labeled
mitochondrial translation products in wild type and cox16
mutants. The parental wild type (W303-1A), a
cox18 null mutant ( COX18), a cox16
null mutant (
COX16), and a cox15 null mutant
(COX15) were grown in YPGal and labeled with
[35S]methionine in the presence of cycloheximide for 15 min as described under "Materials and Methods." Excess methionine
was added, and samples were taken after the indicated times of chase at
30 °C. Mitochondrial translation products were analyzed on a 12.5%
polyacrylamide gel containing 6 M urea and 6% glycerol and
are identified in the margin as described in Fig. 1.
COX16) was determined by Southern blot analysis of genomic DNA
to have the URA3 insertion in COX16 (not shown).
The biochemical phenotype of aW303
COX16 was ascertained to be
similar to that of G22 mutants. It displays a selective absence of
cytochromes aa3 and a loss of cytochrome oxidase
activity. The cytochrome oxidase deficiency, combined with the lack of
complementation of aW303
COX16 by C25 and E699, constitutes strong
evidence that the mutations in these strains are allelic with the
cox16 disruption.
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Fig. 3.
The COX16 gene encodes a
small, acidic protein. A, a hydropathy plot of Cox16p
predicts a single membrane-spanning segment. B, the primary
amino acid sequence of Cox16p is shown with the potential mitochondrial
targeting sequence (as determined by the P-SORT program) denoted by the
bar above the sequence. The predicted transmembrane domain
is indicated by the bar underneath the sequence.
COX16/myc1) or low copy CEN plasmid (aW303
COX16/myc2) on non-fermentable carbon sources
(ethanol/glycerol), was indistinguishable from that of transformants
expressing an untagged version of Cox16p. Restoration of growth on
these substrates correlated with the recovery of cytochrome oxidase.
This was evident from the spectra (not shown) and Western analysis of
COX subunits (Fig. 1B). The concentrations of the three
mitochondrially encoded subunits and subunit 5 in the transformants are
similar to the wild type levels, regardless of the vector used to
express the Myc-tagged Cox16p (Fig. 1B).
COX16/myc1
and aW303
COX16/myc2 is detected with the Myc antibody (Fig.
4A). This band is absent in
the post-mitochondrial supernatant fraction from these strains and
is also undetectable in wild type and mutant mitochondria.
Based on its migration, this protein has an apparent mass of 21 kDa,
which is 6 kDa larger than the combined size of native Cox16p and the
additional 1 kDa contributed by the Myc epitope. The reason for the
discrepancy, which may be even larger if Cox16p has a cleavable signal,
is not clear but is probably related to anomalous binding of SDS by
this acidic protein.
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Fig. 4.
Cox16p is a mitochondrial inner membrane
protein. A, Western analysis of mitochondria
(Mit) and postmitochondrial supernatant (PMS)
fractions from the wild type strain W303-1A (W303), the
cox16 mutant aW303 COX16 (
COX16), and the
transformants aW303
COX16/myc1 (myc1) and
aW303
COX16/myc2 (myc2), expressing a Myc-tagged Cox16p
from a high copy plasmid and from a CEN plasmid, respectively.
Mitochondrial (20 µg) or postmitochondrial supernatant fractions (40 µg) were separated in 12% polyacrylamide gels and transferred to
nitrocellulose, and the blot probed with a monoclonal antibody specific
to the Myc epitope tag. B, mitochondria from
aW303
COX16/myc1 were extracted in the absence (
) or presence (+)
of 0.5 M NaCl and increasing concentrations of deoxycholate
(DOC). Following centrifugation at 100,000 × g, the pellet (containing mitochondrial membrane with
non-extracted protein) and supernatant (containing proteins extracted
from the mitochondrial membrane) fractions were normalized back to the
starting volume of mitochondria and separated on a 15% gel and
analyzed as described in A. C, mitochondria with
intact outer membranes were isolated and converted to mitoplasts by the
method of Glick and Pon (47). The equivalent of 1 mg of mitochondrial
protein starting material was incubated in the presence or absence of
0.016 mg of proteinase K for 30 min, and each fraction analyzed for the
presence of the Myc-tagged Cox16p or Sco1p (mitochondrial copper
transport). MT = mitochondria; MP = mitoplasts; p = pellet; S = supernatant; PK = proteinase K; + or
= presence
or absence of proteinase K.
COX16/myc1 mitochondria with deoxycholate in the presence of
0.5 M NaCl showed that extraction of Cox16p-myc requires a
minimum of 0.25% detergent (Fig. 4B).
COX16/myc1 revealed Cox16p to be a constituent of the
mitochondrial inner membrane as it is only found in the pelleted,
mitoplast fraction (Fig. 4C). Treatment with proteinase K
caused Cox16p to be degraded in mitoplasts but not mitochondria. The
decreased signal seen in the proteinase K-treated mitochondria is
probably due to a subpopulation of mitochondria with damaged outer
membranes. Similar results were obtained when mitochondria and
mitoplasts were probed with antibody against Sco1p, an inner membrane
protein protruding into the intermembrane space (32). Under these
conditions, subunit 5 is protected from digestion by proteinase K in
mitoplasts (data not shown).
COX16/myc1 and aW303
COX16/myc2 (data not shown).
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Fig. 5.
Cox16p has murine and human homologs. An
alignment of S. cerevisiae (Sc) and S. pombe (Sp) Cox16ps with the murine (Mm) and
human (Hs) Cox16 homologs. The alignment was generated by
the ClustalW program (//www.ebi.ac.uk/clustalw/) and shaded with the
Boxshade Program (//www.ch.embnet.org/software/BOX_form.html).
Identical residues are shaded in black, and conservative
replacements are shaded in gray.
COX16 parent strain even after prolonged
incubation on a non-fermentable carbon source (7 days), whereas the
yeast COX16 gene restores growth on ethanol/glycerol after
one night. These results indicate that the human gene is homologous,
but not orthologous, to yeast COX16.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (to D. M. G.) and by research Grant GM50187 from the National Institutes of Health (to A. T.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Natural Sciences and Engineering Research Council PGS'A' Studentship.
Supported by Grant MDACU01991001 from the Muscular Dystrophy Association.
** CIHR New Investigator and an Alberta Heritage Foundation for Medical Research Scholar. To whom correspondence should be addressed: Dept. of Medical Genetics, University of Alberta, 8-33 Medical Sciences Bldg., Edmonton, AB T6G 2H7, Canada. Tel.: 780-492-4563; Fax: 780-492-1998; E-mail: moira.glerum@ualberta.ca.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M209893200
2 A. Barrientos and A. Tzagoloff, unpublished studies.
3 D. M. Glerum and D. L. Adams, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviation used is: COX, cytochrome oxidase.
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1. | Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., and Yoshikawa, S. (1996) Science 272, 1136-1144[Abstract] |
2. | Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi, H., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., and Yoshikawa, S. (1995) Science 269, 1069-1074[Medline] [Order article via Infotrieve] |
3. |
Glerum, D. M.,
Shtanko, A.,
and Tzagoloff, A.
(1996)
J. Biol. Chem.
271,
20531-20535 |
4. |
Glerum, D. M.,
Shtanko, A.,
and Tzagoloff, A.
(1996)
J. Biol. Chem.
271,
14504-14509 |
5. |
Carr, H. S.,
George, G. N.,
and Winge, D. R.
(2002)
J. Biol. Chem.
277,
31237-31242 |
6. |
Nobrega, M. P.,
Nobrega, F. G.,
and Tzagoloff, A.
(1990)
J. Biol. Chem.
265,
14220-14226 |
7. | Barros, M. H., Carlson, C. G., Glerum, D. M., and Tzagoloff, A. (2001) FEBS Lett. 492, 133-138[CrossRef][Medline] [Order article via Infotrieve] |
8. | Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225 |
9. |
Hell, K.,
Neupert, W.,
and Stuart, R. A.
(2001)
EMBO J.
20,
1281-1288 |
10. |
Hell, K.,
Tzagoloff, A.,
Neupert, W.,
and Stuart, R. A.
(2000)
J. Biol. Chem.
275,
4571-4578 |
11. |
Glerum, D. M.,
Koerner, T. J.,
and Tzagoloff, A.
(1995)
J. Biol. Chem.
270,
15585-15590 |
12. |
Mashkevich, G.,
Repetto, B.,
Glerum, D. M.,
Jin, C.,
and Tzagoloff, A.
(1997)
J. Biol. Chem.
272,
14356-14364 |
13. | Forsha, D., Church, C., Wazny, P., and Poyton, R. O. (2001) Biochem. Soc. Trans. 29, 436-441[Medline] [Order article via Infotrieve] |
14. | Shoubridge, E. A. (2001) Am. J. Med. Genet. 106, 46-52[CrossRef][Medline] [Order article via Infotrieve] |
15. | Barrientos, A., Barros, M. H., Valnot, I., Rotig, A., Rustin, P., and Tzagoloff, A. (2002) Gene 286, 53-63[CrossRef][Medline] [Order article via Infotrieve] |
16. | Myers, A. M., Pape, L. K., and Tzagoloff, A. (1985) EMBO J. 4, 2087-2092[Abstract] |
17. | Hill, J. E., Myers, A. M., Koerner, T. J., and Tzagoloff, A. (1986) Yeast 2, 163-167[Medline] [Order article via Infotrieve] |
18. | Gietz, R. D., and Sugino, A. (1988) Gene 74, 527-534[CrossRef][Medline] [Order article via Infotrieve] |
19. | Schiestl, R. H., and Gietz, R. D. (1989) Curr. Genet. 16, 339-346[Medline] [Order article via Infotrieve] |
20. |
Glerum, D. M.,
Muroff, I.,
Jin, C.,
and Tzagoloff, A.
(1997)
J. Biol. Chem.
272,
19088-19094 |
21. |
Barrientos, A.,
Korr, D.,
and Tzagoloff, A.
(2002)
EMBO J.
21,
43-52 |
22. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
23. | Glick, B. S. (1995) Methods Enzymol. 260, 224-231[Medline] [Order article via Infotrieve] |
24. |
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275 |
25. | Tzagoloff, A., Akai, A., and Needleman, R. B. (1975) J. Biol. Chem. 250, 8228-8235[Abstract] |
26. | Glerum, D. M., and Tzagoloff, A. (1998) Anal. Biochem. 260, 38-43[CrossRef][Medline] [Order article via Infotrieve] |
27. | Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368-379[Medline] [Order article via Infotrieve] |
28. |
Dickinson, E. K.,
Adams, D. L.,
Schon, E. A.,
and Glerum, D. M.
(2000)
J. Biol. Chem.
275,
26780-26785 |
29. | Hell, K., Herrmann, J., Pratje, E., Neupert, W., and Stuart, R. A. (1997) FEBS Lett. 418, 367-370[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Saracco, S. A.,
and Fox, T. D.
(2002)
Mol. Biol. Cell
13,
1122-1131 |
31. |
Barros, M. H.,
Nobrega, F. G.,
and Tzagoloff, A.
(2002)
J. Biol. Chem.
277,
9997-10002 |
32. |
Beers, J.,
Glerum, D. M.,
and Tzagoloff, A.
(1997)
J. Biol. Chem.
272,
33191-33196 |
33. | Heaton, D. N., George, G. N., Garrison, G., and Winge, D. R. (2001) Biochemistry 40, 743-751[CrossRef][Medline] [Order article via Infotrieve] |
34. | Lode, A., Kuschel, M., Paret, C., and Rodel, G. (2000) FEBS Lett. 485, 19-24[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Zhang, Q. H., Ye, M., Wu, X. Y.,
Ren, S. X.,
Zhao, M.,
Zhao, C. J., Fu, G.,
Shen, Y.,
Fan, H. Y., Lu, G.,
Zhong, M., Xu, X. R.,
Han, Z. G.,
Zhang, J. W.,
Tao, J.,
Huang, Q. H.,
Zhou, J., Hu, G. X., Gu, J.,
Chen, S. J.,
and Chen, Z.
(2000)
Genome Res.
10,
1546-1560 |
36. | Neupert, W. (1997) Annu. Rev. Biochem. 66, 863-917[CrossRef][Medline] [Order article via Infotrieve] |
37. | Nunnari, J., Fox, T. D., and Walter, P. (1993) Science 262, 1997-2004[Medline] [Order article via Infotrieve] |
38. | Amaravadi, R., Glerum, D. M., and Tzagoloff, A. (1997) Hum. Genet. 99, 329-333[CrossRef][Medline] [Order article via Infotrieve] |
39. | Petruzzella, V., Tiranti, V., Fernandez, P., Ianna, P., Carrozzo, R., and Zeviani, M. (1998) Genomics 54, 494-504[CrossRef][Medline] [Order article via Infotrieve] |
40. | Glerum, D. M., and Tzagoloff, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8452-8456[Abstract] |
41. | Papadopoulou, L. C., Sue, C. M., Davidson, M. M., Tanji, K., Nishino, I., Sadlock, J. E., Krishna, S., Walker, W., Selby, J., Glerum, D. M., Coster, R. V., Lyon, G., Scalais, E., Lebel, R., Kaplan, P., Shanske, S., De, Vivo, D. C., Bonilla, E., Hirano, M., DiMauro, S., and Schon, E. A. (1999) Nat. Genet. 23, 333-337[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Valnot, I.,
von Kleist-Retzow, J. C.,
Barrientos, A.,
Gorbatyuk, M.,
Taanman, J. W.,
Mehaye, B.,
Rustin, P.,
Tzagoloff, A.,
Munnich, A.,
and Rotig, A.
(2000)
Hum. Mol. Genet.
9,
1245-1249 |
43. | Valnot, I., Osmond, S., Gigarel, N., Mehaye, B., Amiel, J., Cormier-Daire, V., Munnich, A., Bonnefont, J. P., Rustin, P., and Rotig, A. (2000) Am. J. Hum. Genet. 67, 1104-1109[Medline] [Order article via Infotrieve] |
44. | Zhu, Z., Yao, J., Johns, T., Fu, K., De, Bie, I., Macmillan, C., Cuthbert, A. P., Newbold, R. F., Wang, J., Chevrette, M., Brown, G. K., Brown, R. M., and Shoubridge, E. A. (1998) Nat. Genet. 20, 337-343[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Souza, R. L.,
Green-Willms, N. S.,
Fox, T. D.,
Tzagoloff, A.,
and Nobrega, F. G.
(2000)
J. Biol. Chem.
275,
14898-14902 |
46. |
Tzagoloff, A.,
and Meagher, P.
(1972)
J. Biol. Chem.
247,
594-603 |
47. | Glick, B. S., and Pon, L. A. (1995) Methods Enzymol. 260, 213-223[Medline] [Order article via Infotrieve] |