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ORIGINAL ARTICLE
Mitochondrial disease genes COA6,COX6B and SCO2
have overlapping roles in COX2 biogenesis
Alok Ghosh, Anthony T. Pratt, Shivatheja Soma, Sarah G. Theriault,
Aaron T. Griffin, Prachi P. Trivedi and Vishal M. Gohil*
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA
*To whom correspondence should be addressedat: Texas A&M University, 301 Old Main Drive, ILSB 2146A, College Station, TX 77843, USA. Tel: +1 9798476138;
Fax: +1 9798459274; Email: vgohil@tamu.edu
Abstract
Biogenesis of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain, is a complex process
facilitated by several assembly factors. Pathogenic mutations were recently reported in one such assembly factor, COA6, and our
previous work linked Coa6 function to mitochondrial copper metabolism and expression of Cox2, a copper-containing subunit
of CcO. However, the precise role of Coa6 in Cox2 biogenesis remained unknown. Here we show that yeast Coa6 is an orthologue
of human COA6, and like Cox2, is regulated by copper availability, further implicating it in copper delivery to Cox2. In order to
place Coa6 in the Cox2 copper delivery pathway, we performed a comprehensive genetic epistasis analysis in the yeast
Saccharomyces cerevisiae and found that simultaneous deletion of Coa6 and Sco2, a mitochondrial copper metallochaperone, or
Coa6 and Cox12/COX6B, a structural subunit of CcO, completely abrogates Cox2 biogenesis. Unlike Coa6 deficient cells, copper
supplementation fails to rescue Cox2 levels of these double mutants. Overexpression of Cox12 or Sco proteins partially rescues
the coa6Δphenotype, suggesting their overlapping but non-redundant roles in copper delivery to Cox2. These genetic data are
strongly corroborated by biochemical studies demonstrating physical interactions between Coa6, Cox2, Cox12 and Sco proteins.
Furthermore, we show that patient mutations in Coa6 disrupt Coa6–Cox2 interaction, providing the biochemical basis for
disease pathogenesis. Taken together, these results place COA6 in the copper delivery pathway to CcO and, surprisingly, link it
to a previously unidentified function of CcO subunit Cox12 in Cox2 biogenesis.
Introduction
Defects in the function and formation of the mitochondrial re-
spiratory chain (MRC) manifest clinically in mitochondrial dis-
eases, one of the most common classes of inborn errors of
metabolism (1). A subset of MRC disorders can be attributed to
the deficiency of MRC complex IV, commonly known as cyto-
chrome c oxidase (CcO). CcO is the terminal enzyme of the MRC
that catalyzes the reduction of molecular oxygen to water and
generates an electrochemical gradient that drives mitochondrial
adenosine triphosphate (ATP) synthesis. CcO is an evolutionarily
conserved multi-subunit enzyme complex whose catalytic core
is composed of three subunits: Cox1, Cox2 and Cox3, which are
encodedbymitochondrialDNAinbothyeastandhumans(2).
The other structural subunits, which are encoded by nuclear
DNA, surround the catalytic core to form the CcO holoenzyme.
In addition to the protein subunits, CcO contains several cofac-
tors including two copper centers (Cu
A
and Cu
B
), two heme
groups (heme a and a
3
), a magnesium ion and a zinc ion (3).
The assembly of a fully mature, catalytically active CcO is an
extremely complex process that requires a number of assembly
factors to bring together the mitochondrial and nuclear DNA-
encoded subunits with their metal cofactors.
CcO biogenesis is a modular process that begins with the
independent maturation of the core subunits Cox1, Cox2 and
Cox3, followed by the addition of other nuclear-encoded subunits
(4,5). There are ∼40 assembly factors discovered to date that
facilitate different steps of CcO assembly (2). For example, 22
assembly factors are required for the expression and membrane
Received: August 21, 2015. Revised: November 6, 2015. Accepted: December 7, 2015
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
Human Molecular Genetics, 2016, 1–12
doi: 10.1093/hmg/ddv503
Advance Access Publication Date: 15 December 2015
Original Article
1
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insertion of the catalytic core subunits, 9 assembly factors are
required for copper delivery to copper A (Cu
A
) and copper B (Cu
B
)
site in Cox2 and Cox1 subunits, respectively, and 4 factors are
required for heme biosynthesis and insertion into the catalytic
core (2). Unlike assembly factors required for the expression and
insertion of mitochondrial DNA-encoded catalytic subunits, all
the factors required for copper delivery and heme insertion to
the Cox1and Cox2 subunitsare conservedin yeast and humans (2).
Patients suffering from CcO deficiency exhibit multi-systemic
and tissue-specific disorders, primarily affecting organs with
higher energy demands including the brain, skeletal muscle
and heart (6,7). CcO deficiency leads to early onset, autosomal re-
cessive disorders with fatal clinical outcomes (6,7). The combin-
ation of human genetics and knowledge of CcO assembly factors
from Saccharomyces cerevisiae has led to the discovery of multiple
CcO disease genes. While a few mutations can be attributed
to the structural subunits of CcO, including COX1, COX2, COX3,
COX4 and COX6B (7–10), the majority of patient mutations are
found in genes encoding assembly factors including LRPPRC,
TACO1, FASTKD2, PET100, COX10, COX14, COX15, COX20,
SURF1, SCO1, SCO2, COA3, COA5 and COA6 (11–28). Although
CcO deficient patients display heterogeneous clinical presenta-
tions, it has been noted that mutations in the assembly factors
involved in the same pathway exhibit similar clinical pheno-
types. For example, patients with pathogenic mutations in the
copper metallochaperones SCO1 and SCO2, which are involved
in copper delivery to the Cu
A
site of CcO subunit COX2, typically
develop neonatal encephalopathy and hypertrophic cardiomy-
opathy (18–21). Similarly, patient mutations in COA6 also result
in neonatal hypertrophic cardiomyopathy (23,24); however, the
precise role of COA6 in CcO assembly has remained unknown.
We first reported that COA6 is essential for CcO assembly in
yeast, zebrafish and human cells possibly by delivering copper
to COX2 subunit (29). While up to nine factors have been impli-
cated in copper delivery to CcO subunits COX1 and COX2, the pre-
cise role of many of them remains obscure (2). The components
of the copper delivery pathway that have been reconstituted
in vitro suggest that COX17 receives copper from a mitochondrial
matrix pool (30) and donates it to copper metallochaperones
COX11 and SCO1/SCO2 (31), which ultimately transfer copper to
the Cu
B
site in COX1 and Cu
A
site in COX2, respectively (32,33).
Multiple other proteins, including COA6, have been implicated
in this copper delivery pathway, but it is not clear where they
act in the pathway.
Our initial findings linkingCOA6 to COX2 biogenesis and mito-
chondrial copper metabolism have been corroborated by two re-
cent studies that show reduced stability of nascent COX2 in COA6
deficient cells and that COA6 binds to copper in vitro (34,35). While
these studies have also identified physical interactions between
COA6 and copper metallochaperones SCO1 and SCO2, the func-
tional significance of the interaction is not known. Here, we have
used genetic epistasis analysis in yeast to demonstrate that COA6
is essential for Cox2 expression in the absence of SCO2 and
COX12, a structural subunit of CcO. This synthetic interaction be-
tween Coa6, Sco2 and Cox12 proteins suggests their overlapping
functions in Cox2 biogenesis. Consistent with this observation,
we find that overexpression of Cox12 and Sco proteins partially
rescues the respiratory deficient growth phenotype of coa6Δcells.
We further substantiate these genetic data by demonstrating a
physical interaction between Coa6, Cox12, Cox2 and Sco proteins.
Taken together, our study not only places Coa6 in the copper
delivery pathway to Cox2, but also implicates COX12, a yeast
orthologue of the human mitochondrial disease gene COX6B,in
copper metabolism and Cox2 biogenesis.
Results
Yeast and human COA6 are orthologues
Previously, we showed that COA6 is an evolutionarily conserved
protein that is required for the expression of CcO subunits includ-
ing COX2 in yeast, zebrafish and human cells (29). Sequence
alignment of yeast and human COA6showed that these two pro-
teins are highly conserved except in the N-terminal region (29),
possibly because of differences in N-terminal mitochondrial tar-
geting sequences in these two species. The sequence conserva-
tion and their common role in CcO expression suggested that
yeast and human COA6 are orthologues. To experimentally test
the functional similarity of these two proteins, we performed a
complementation experiment by heterologous expression of
human COA6 in yeast coa6Δcells. We episomally expressed
yeast COA6 (yCOA6), human COA6 (hCOA6)oryeast–human hy-
brid COA6 (hyCOA6)incoa6Δcells and tested for their ability to
rescue the respiratory growth deficiency of coa6Δcells. The
hyCOA6 was constructed by fusing the gene segments corre-
sponding to the N-terminus of yeast Coa6 (amino acid residues
1–24) and the C-terminus of human COA6 (amino acids residues
57–125) containing the evolutionarily conserved Cx
9
Cx
n
Cx
10
C
motif (Fig. 1A). As expected, yCOA6 completely rescued the re-
spiratory growth defect of coa6Δcells, but hCOA6 was not able
to rescue this growth defect (Fig. 1B). However, hyCOA6 was
able to completely restore respiratory growth (Fig. 1B) suggesting
that yeast and human COA6 are orthologues.
Coa6 is regulated by mitochondrial biogenesis factors
Having established the orthologous relationship between the
human and yeast Coa6 protein, we decided to use yeast as a
model system to study the function and regulation of Coa6.To
study the regulation of the endogenous yeast Coa6 protein, we
generated polyclonal antibodies against native Coa6 protein.
We confirmed antibody specificity by detecting a Coa6 specific
band of ∼12 kDa in cellular extracts from wild type (WT) and
Coa6 overexpressing cells (Fig. 2A). We hypothesized that Coa6,
as a mitochondrial protein, would be regulated by mitochondrial
biogenesis factors including carbon source, growth phase and the
presence of mitochondrial DNA. Growth of yeast cells in respiro-
fermentable (YPGal) or non-fermentable (YPGE) media is known
to stimulate mitochondrial biogenesis, and accordingly, Coa6
levels were higher in cells grown in YPGal and YPGE medium
compared with fermentable medium (YPD) (Fig. 2B). In glucose
containing YPD medium, Coa6 expression increased late in the
growth phase, likely because of derepression of glucose-mediated
inhibition of mitochondrial biogenesis (Fig. 2B). Interestingly, we
found that an increase in Coa6 levels precedes an increase in
Cox2 levels (Fig. 2B), implying thatthe presence of Coa6 is essential
for Cox2 expression. Consistent with a previous report, which
showed a suppression of mitochondrial biogenesis in mitochon-
drial DNA deficient ρ
0
yeast cells (36), we observed a dramatic
decrease in Coa6 levels in ρ
0
cells (Fig. 2C), further confirming
that Coa6 levels are regulated by mitochondrial biogenesis.
It has been shown that steady-state levels of some of the CcO
assembly factors are reciprocally regulated. For example, levels of
Cmc1, a copper-binding mitochondrial intermembrane space
(IMS) protein, increase in response to deletion of Cmc2, another
CcO assembly factor (37). To tie Coa6 to CcO assembly factors
involved in copper metabolism, we explored the possibility that
Coa6 levels are regulated by other CcO assembly factors. There-
fore, we measured Coa6 levels in various yeast strains lacking
known copper metallochaperones including Cox11, Cox17, Sco1
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and Sco2 and twin Cx
9
C motif-containing proteins implicated
in CcO assembly, including Cox19, Cox23, Cmc1 and Cmc2.
We found that the levels of Coa6 did not change in the knockout
strains tested (Fig. 2D). Interestingly, we noticed that the levels
of Pgk1, a glycolytic enzyme used as a loading control, increased
in all CcO assembly factor mutants that are completely devoid of
Cox2 (Fig. 2D). Therefore, we used a non-specific band to confirm
equal loading (Fig. 2D). The increase in Pgk1 could be a homeo-
static mechanism to maintain cellular ATP production in Cox2
deficient cells that are unable to produce mitochondrial ATP.
In a reciprocal experiment, we measured the levels of copper
metallochaperones and CcO subunits, Cox17, Sco1, Sco2, Cmc1,
Cox12 and Cox2 in coa6Δcells and, except for a decrease in the
Cox2 and Cox12 levels, we did not find any significant change
in the levels of the other proteins measured (Supplementary
Material, Fig. S1). This result suggests that Coa6 abundance is
independent of the presence of these CcO assembly factors and
vice-versa.
Coa6 levels change in response to extracellular copper
abundance
A recent study has shown that levels of iron-containing proteins
in the MRC decrease in response to increasing amounts of the
iron chelator deferoxamine in mouse muscle cells (38). Since
Coa6 has been implicated in the copper delivery pathway to CcO
(29) and has recently been shown to bind copper (34,35), we
hypothesized that Coa6 levels would alter in response to copper
chelation and supplementation. In order to identify the optimal
concentration of copper-specific chelator bathocuproinedisulfonic
acid (BCS)and copper, WT yeast were grown in respiro-fermentable
YPGal medium supplemented with increasing amounts of BCS
or copper chloride (CuCl
2
). We found that 25 μor more of BCS re-
duced yeast growth in YPGal medium by limiting bioavailable cop-
per required for respiratory growth, whereas up to 100 μcopper
supplementation did not alter yeast growth (Fig. 3A and B). We ob-
served that Coa6 levels decreased with increasing concentrations
Figure 1. Heterologous expression of yeast–human hybrid COA6 rescues respiratory growth defect of yeast coa6Δcells. (A) Schematic representationof yeast Coa6, human
COA6 and hybrid COA6proteins. The sequence from yeast Coa6 is shown in white, and the sequence from human COA6 is shown in gray.(B) Ten-fold serial dilutions of WT
and coa6Δcells transformed with either empty vector (pRS 416) or with pRS416 expressing yeast COA6 (yCOA6), human COA6 (hCOA6) or hybrid COA6 (hyCOA6) were
spotted on fermentable (YPD) and non-fermentabl e (YPGE) growth media. Plates were incubated at 30°C and 37 °C and images were taken after 2–4 days of growth.
These data are representative of three independent experiments.
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of BCS, whereas copper supplementation modestly increased
Coa6 levels (Fig. 3C). Similarly, Cox2 levels decreased drastically
under copper limiting conditions, while copper supplementation
slightly increased Cox2 (Fig. 3C). The decrease in Coa6 with BCS
supplementation is more pronounced in mitochondrial samples
isolated from chromosomal hemagglutinin (HA) tagged-Coa6
cells (Fig. 3D). These results suggest that, like the copper-contain-
ing protein Cox2, Coa6 expression is dependent on bioavailable
copper, further implicating it in mitochondrial copper
metabolism.
Coa6 acts in parallel with Cox12 and Sco2 to maintain
Cox2 levels
Our previous results showed that copper supplementation res-
cues the coa6Δrespiratory-deficient phenotype (29) and our cur-
rent study shows that Coa6 levels are regulated by copper
availability. Together, these studies strongly suggest that Coa6
plays a role in copper delivery to CcO. In order to place Coa6 in
the CcO copper delivery pathway, a genetic epistasis analysis
was performed where coa6Δcells were crossed with the deletion
strains of the known copper metallochaperones and Cx
9
C
proteins implicated in CcO assembly. The resulting double
knockouts were extensively phenotyped in different growth con-
ditions (Supplementary Material, Table S1). Analysis of the
growth phenotypes of both the parental single knockouts and
the double knockouts identified strong synthetic lethal interac-
tions of COA6 with SCO2 and COX12 (Fig. 4A and B). To probe for
the mechanism that leads to synthetic lethality of double knock-
outs in respiratory media, we measured levels of the copper-
containing CcO subunit Cox2. Cox2 levels are decreased in
coa6Δ,sco2Δand cox12Δto different degrees, but completely
absent in both coa6Δsco2Δand coa6Δcox12Δcells (Fig. 4C and D).
Exogenous supplementation of copper fails to rescue the growth
defect and Cox2 deficiency of either double knockout (Fig. 4A–D).
These data suggest that Coa6, Sco2 and Cox12 play overlapping
roles in the copper delivery to Cox2.
Coa6 physically interacts with Cox2, Cox12
and Sco proteins
Genetic interactors tend to participate in a common pathway and
are more likely to physically interact (39). To test whether Coa6
physically interacts with Cox2 and copper metallochaperones,
we performed co-immunoprecipitation experiments using anti-
HA and anti-Cox2 antibodies. Reciprocal co-immunoprecipita-
tion experiments showed that Coa6 specifically interacts with
Cox2 protein (Fig. 5A) and not with Cox1 or other proteins of
the MRC (data not shown). In order to rule out the possibility
that the interaction was merely due to Coa6 overexpression, we
Figure 2. Coa6 expression is regulated by mitochondrial biogenesis factors and is independent of CcO assembly factors. (A) Total cellular protein was extracted from
BY4741 WT, coa6Δand coa6Δtransformed with pRS426-COA6 and subjected to SDS PAGE/western blot. Yeast Coa6 protein was detected using purified polyclonal Coa6
antibody; NS designates a non-specific protein band detected by poly clonal Coa6 antibody. Porin (Por1) was used as a loading control. (B) Western blot ana lysis of
Coa6 and Cox2 protein levels in total cellular extracts from BY4741 WT yeast cells grown in YPD, YPGal or YPGE to early logarithmic, mid-logarithmic (ML) and ear ly
stationary (ES) growth phase. Por1 was used as a loading control. (C) Western blot analysis of Coa6 and Cox2 levels in BY4741 WT and ρ
0
cells grown in YPGal medium
to ML growth phase. Pgk1 was used as a loading control. As indicated, 25 or 50 μg of protein was loaded in each lane. (D) Western blot analysis of Coa6, Cox2 and Pgk1
protein levels in the indicated knoc kout strains grown to ML growth ph ase in YPGal mediu m. Since Pgk1 levels increased in cells completely lacking Cox2, a non-
specific (NS) band was used as a loading control. The data in panels B-D are representative of three independent experiments.
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constructed chromosomal HA-tagged Coa6 cells, where Coa6HA
is expressed from its endogenous promoter. In this case, we
found a similar interaction between Coa6HA and Cox2 (Fig. 5B).
Furthermore, to confirm that the interaction we observed was
not because of an interaction between Cox2 andthe HA tag itself,
we performed an immunoprecipitation experiment using poly-
clonal anti-Coa6 antibodies on WT and coa6Δmitochondria.
We again found Coa6–Cox2 interaction and additionally, upon
probing with other antibodies, we identified physical interactions
between Coa6 and Sco1, Sco2 and Cox12, but not between Coa6
and Cu
B
center-containing Cox1 or copper metallochaperone
Cox17 (Fig. 5C). These results suggest that Coa6 exists as a part
of a multimeric protein complex in mitochondria. To detect
Coa6-containing complex(es), we performed a blue native poly-
acrylamide gel electrophoresis (BN PAGE)/western analysis
on mitochondrial extracts and found that Coa6 is part of three
high molecular weight complexes of ∼60, 140 and 200 kDa
(Fig. 5D). We thus conclude that Coa6 and its interacting partners
form complexes that may participate in Cox2 metallation.
Coa6, Cox12 and Sco proteins have overlapping functions
Our genetic interaction study suggested that Coa6 acts in parallel
with Cox12 and Sco2 in Cox2 biogenesis; these proteins are thus
likely to have overlapping functions. To test this hypothesis, we
overexpressed COX12,SCO2 and SCO1,incoa6Δcells and scored
for rescue of respiratory growth on YPGE plates at 37°C. As a con-
trol, we expressed other proteins involved in Cox2 biogenesis
including COX17, COX19, COX20 and COX23 in coa6Δcells. We
also expressed the yeastmim ic(Sco2 E161K) of the most common
human SCO2 patient mutation (E140K) (20)incoa6Δcells to test
for its ability to suppress coa6Δgrowth defect (Fig. 6A). As seen
in Figure 6B, the coa6Δrespiratory growth defect was partially
suppressed by overexpression of SCO1,SCO2, COX12 and COX20
but not by COX17, COX19 and COX23. Interestingly, the Sco2
E161K mutant also partially rescued coa6Δgrowth phenotype
suggesting that E161 is not essential for yeast Sco2 function
(Fig. 6B). To further interrogate yeast Sco2 function, we expressed
yeast mimics (E161K and S246F) of two human SCO2 patient
mutations (E140K and S225F) (20)incoa6Δsco2Δcells. We found
that while E161 is not essential, S246 is critical for coa6Δsco2Δ
growth and yeast Sco2 function (Fig. 6C). While the rescue of
the respiratory growth defect of coa6Δcells by overexpression of
Sco proteins and Cox20 is not surprising since these proteins
are known to cooperate in the late stages of Cox2 biogenesis
(40), the ability of COX12 to suppress the coa6Δgrowth defect
is surprising and links Cox12 to the copper delivery pathway
to Cox2.
Figure 3. Coa6 levels are regulated by copper availability. WT yeast cells were grown in YPGal medium with increasing concentrations of (A) the copper chelator, BCS or
(B) CuCl
2
. Growth was analyzed by measuring absorbance at600 nm. (C) Western blot analysis of Coa6 and Cox2 levels in whole cell protein lysate prepared from WT yeast
cells grown to ML growth phase in YPGal medium with increasing amounts of BCS or CuCl
2
.(D) Westernblot analysis of Coa6HA and Cox2 levels in mitochondria isolated
from chromosomally HA-tagged COA6 cells grown in YPD to ES growth phase with i ncreasing amounts of BCS. Por1 was used as a loading control. The blots are
representative of at least two independent experiments.
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Sequence alignment and structural analysis of both yeast and
mammalian Coa6 and Cox12 proteins shows presence of a con-
served Cx
9
Cx
n
Cx
10
C motif (Supplementary Material, Fig. S2A)
with a similar predicted tertiary structure (Supplementary Mater-
ial, Fig. S2B), strongly supporting their overlapping function. To
further dissect the interaction between Coa6 and Cox12, we per-
formed a reciprocal experiment, whereby we tried to rescue the
respiratory growthdeficient phenotype of cox12Δwith COA6 over-
expression. Interestingly, we found that instead of rescuing,
COA6 overexpression enhanced the cox12Δgrowth defect (Sup-
plementary Material, Fig. S2C). Taken together, our results sug-
gest overlapping but non-redundant roles of Coa6, Sco1 Sco2,
and Cox12 in Cox2 maturation.
Pathogenic mutations within the conserved Cx
9
Cx
n
Cx
10
C
motif of Coa6 disrupt its interaction with Cox2
Previously, we showedthat patient mutations in the Cx
9
Cx
n
Cx
10
C
motif of Coa6 are pathogenic and that the conserved cysteine re-
sidues of the motif are essential for Coa6 function (29). Patient
mutations reduced the stability of Coa6, partly explaining the
mechanism of pathogenesis (29). However, it was not clear if
reduced levels of Coa6 were sufficient to cause pathogenesis. In
addition to Coa6 stability, it is possible that patient mutations
may disrupt Coa6 function by preventing its interactions with
its binding partners. Therefore, we tested the effect of patient
mutations ( p.W26C and p.C68A) as well as a mutation in a con-
served cysteine residue (p.C25A) on Coa6 interactions (Fig. 7A).
Co-immunoprecipitation with anti-HA antibodies confirmed
that the patient mutations severely disrupted Coa6–Cox2 inter-
action while only mildly affecting the Coa6–Sco1 interaction
(Fig. 7B). These results demonstrate that the Coa6 residues mu-
tated in the mitochondrial disease patient are essential for its
interaction with Cox2, providing the biochemical basis for the
disease pathogenicity.
Discussion
Recent advances in genomic technologies have identified patho-
genic mutations in a plethora of uncharacterized genes; however,
understanding the function of these disease genes has remained
a major bottleneck in elucidating disease pathogenesis. Recently,
we uncovered an evolutionarily conserved role of a previously
uncharacterized mitochondrial disease gene, COA6,inCcO
assembly and mitochondrial copper metabolism (29); however,
its precise role in these processes remained unknown. In this
study, we place Coa6 in the mitochondrial copper delivery
pathway to CcO subunit Cox2. We also show that Coa6 has an
overlapping function with a mitochondrial copper metallocha-
perone, Sco2 and a CcO subunit, Cox12, in Cox2 biogenesis. Like
Coa6, mutations in the human orthologues of Sco2 and Cox12
have been shown to result in hypertrophic cardiomyopathy
(10,20,21,23,24). Thus, our study not only links three different
mitochondrial disease genes known to cause hypertrophic car-
diomyopathy to the mitochondrial copper delivery pathway,
but also provides mechanistic insights into Cox2 biogenesis
and uncovers genetic redundancies in the copper delivery
pathway.
We present several lines of evidence demonstrating that
Coa6 is a novel member of the mitochondrial copper delivery
pathway to Cox2. First, simultaneous deletion of Coa6 and Sco2,
a well-known mitochondrial copper metallochaperone, leads to a
Figure 4. Coa6, Sco2 and Cox12 have an overlapping but non-redundant role in Cox2 expression. (A) Ten-fold serially diluted WT, coa6Δ,sco2Δand coa6Δsco2Δcells were
spotted on YPGE plates with and without 5 μCuCl
2
.(B) Ten-fold serially diluted WT,coa6Δ,cox12Δand coa6Δcox12Δcells were spotted on synthetic complete (SC) galactose
medium with and without 5 μm CuCl
2
. Plates were incubatedat 30°C and images were taken after 3 days of growth. (Cand D)WT, single knockouts of Coa6, Sco2, Cox12 and
double knockouts coa6Δsco2Δand coa6Δcox12Δwere grownto ML phase in YPGal liquidmedium with and without 5 μm CuCl
2
. Cox2 levels in total cellular protein extractsof
WT and mutant cells were analyzed by western blot. Pgk1 was used as a loading control.
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synthetic growth defect and complete absence of Cox2 in yeast
cells (Fig. 4A and C). Second, overexpression of copper metallo-
chaperones Sco1 and Sco2 partially rescues the coa6Δrespiratory
deficient phenotype (Fig. 6). Third, Coa6 physically interacts with
Sco1, Sco2 and Cox2, providing biochemical evidence suggesting
these proteins operate in the same pathway (Fig. 5C). These re-
sults obtained using the yeast model system are consistent
with two recent studies in human cell lines showing physical
interactions of COA6 with SCO2 and COX2 (34), and COA6 with
SCO1 and COX2 (35). Together, these findings establish an evolu-
tionarily conserved role of Coa6 in copper delivery to Cox2.
Furthermore, overexpression experiments with known Cox2 bio-
genesis factors helped place Coa6 in the copper delivery pathway
to Cox2. Inability of Cox17, Cox19 and Cox23, well-known IMS
proteins involved in copper delivery to mitochondria, to rescue
the coa6Δgrowth defect suggests that these proteins act up-
stream of Coa6. Conversely, rescue of the coa6Δphenotype
by Sco1, Sco2, Cox12 and Cox20 suggests that these proteins
either act downstream of Coa6 or in a parallel pathway of copper
delivery and Cox2 maturation.
Previously, we showed that copper supplementation was able
to rescue coa6Δcells, but how copper supplementation is able to
bypass the complete absence of Coa6 remained an open question
(29). In light of our current findings that Coa6, Sco2 and Cox12
have an overlapping function, it is conceivable that either Sco2
or Cox12 can substitute for Coa6 when excess copper is available.
We suggest Sco2 as the more likely candidate to perform this
function, because we are not able to detect any Cox2 in coa6Δs-
co2Δcells even after copper supplementation (Fig. 4C). We do de-
tect a faint band corresponding to Cox2 in copper supplemented
coa6Δcox12Δcells (Fig. 4D), presumably because Sco2 is able to
function in these cells to form Cox2, albeit at a much lower
level. This model can also explain the mild respiratory deficient
phenotypes of individual deletions of COA6 and SCO2 cells,
since the absence of one is partially compensated by the other
and vice-versa. The function of Sco2 in yeast cells has remained
enigmatic for many years because of the absence of an overt
respiratory deficient phenotype of sco2Δcells (41), but our results
now firmly tie Sco2 function to Cox2 biogenesis and provide a
system to uncover the role of Sco2 in vivo (Fig. 6C).
The interaction between Coa6 and Cox12 is more intriguing,
considering that Cox12, a structural subunit of CcO, has never
been linked to the copper delivery pathway (42). Interestingly,
COX6B, the mammalian homolog of Cox12, and Coa6 have highly
Figure 5. Coa6 physically interactswith Cox2, Cox12 and Sco proteins. (A) Western blot detection of Coa6-HA and Cox2 proteins in mitochondrial extracts from coa6Δcells
transformed with either empty vector ( pAG423) or pAG423-COA6HA before (input) or after immunoprecipitation with anti-HA and anti-Cox2 antibodies. (B) Western blot
detection of Coa6-HA and Cox2 proteins in mitochondrial extracts from WT and chromosomally HA-tagged COA6 cells before (input) or after immunoprecipitation with
anti-HA antibody. (C) Western blot detection of nativeCoa6, Cox1, Cox2, Cox12, Sco1 and Sco2 proteins in mitochondrial extracts fromWT and coa6Δcells before (input) or
after immunoprecipit ation with anti-Coa6 antibody. (D) BN PAGE/western blot detection of Coa6-containing complexes from 1% digito nin solubilized mitoc hondria
isolated from WT and coa6Δcells grown in YPGE with 5 μCuCl
2
. NS designates a non-specific protein band.
Human Molecular Genetics |7
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homologous sequences, including a conserved Cx
9
Cx
n
Cx
10
C
motif, and are predicted to have similar structures (Supplemen-
tary Material, Fig. S2A and B), which combined with their
synthetic lethal interaction (Fig. 4B) strongly supports their over-
lapping function in Cox2 maturation. Consistent with this possi-
bility, we observed that overexpression of Cox12 is able to
partially rescue respiratory growth deficiency of coa6Δcells. Add-
itional evidence in support of overlapping functions of Coa6 and
Cox12 comes from a human genetics study showing that a muta-
tion in COX12/COX6B results in hypertrophic cardiomyopathy
(10), a signature clinical presentation in patients with mutations
in COA6 and SCO2 (20,21,23,24). Interestingly, Coa6 overexpres-
sion in cox12Δfails to rescue the growth defect (Supplementary
Material, Fig. S2C), which suggests that Cox12 has an additional
function that is not compensated by Coa6.
A recent paper showed that Coa6 is part of a single oligomeric
complex in human cells (34). Similarly, we identified Coa6 in mul-
timeric complexes in yeast mitochondria (Fig. 5D). We predict
that these complexes are likely to be Cox2 assembly intermedi-
ates composed of assembly factors required for copper delivery
to Cox2. Our prediction is supported by co-immunoprecipitation
experiments showing physical interactions of Coa6 with Cox2,
Cox12, Sco1 and Sco2 proteins (Fig. 5A–C). The differences in
the size and number of Coa6-containing complexes in yeast
and humans could be due to differences in the Cox2 assembly
processes in these two organisms. In yeast, modular assembly
of mitochondrial encoded Cox1 and Cox3 subunits have been
delineated (5,43), however Cox2 assembly intermediates have
not yet been characterized. Our findings together with the
reagents developed in this study provide the necessary tools to
delineate the Cox2 assembly process in detail.
Towards understanding the molecular basis of pathogenesis,
it was recently shown that the human COA6 (p.W59C) mutant is
mislocalized to the mitochondrial matrix and thus would not re-
tain its function (34). In contrast, a subsequent study suggested
that the COA6 ( p.W59C) mutant retains some functionality and
that pathogenesis results from an increased aggregation state
of the mutant protein rather than mislocalization (35). Here, we
find that yeast Coa6 with the patient mutation (p.W26C) no long-
er physically interacts with Cox2 (Fig. 7B). However, since we are
able to still detect interaction of the mutant Coa6 with Sco1, an
integral membrane protein that has most of its soluble part
facing the IMS, it is unlikely that the mutant Coa6 is mislocalized
to mitochondrial matrix. Therefore, we argue that the cause of
pathogenesis in the patient with these Coa6 mutations is loss
of interaction between mutant Coa6 and Cox2.
Based on our genetic and biochemical interaction studies, we
propose a model placing the Coa6 and Cox12 in the copper deliv-
ery to the Cu
A
site for Cox2 maturation (Fig. 8). Since human COA6
has been shown to bind copper with high affinity, it is possible
that Coa6 acts as a metallochaperone. However, an in vitro dem-
onstration of Cu
A
site formation by Coa6 and its interacting
partners will be required to dissect its precise molecular function
in copper delivery.
Figure 6. Overexpressionof Cox12, Cox20 and Sco proteins partially rescues coa6Δrespiratory defect. (A) Sequence alignment of conserved regions of human SCO2 and its
yeast homolog. Arrows indicateamino acid residues ( glutamic acid E140and serine S225) shown to be mutated in human mitochondrial disease patients. Twomutations
in yeast Sco2, E161K and S246F, that mimic the patient mutations are also indicated with arrows. (B) Ten-fold serially diluted WT cells transformed with pRS416 empty
vector and coa6Δcells transformed with pRS416 empty vector or pRS416 vector expressing either COA6 ,SCO1,SCO2,COX12, COX20, COX17, COX19, COX23 and sco2 E161K
were spotted on YPGE plates at 30°C and 37°C. Images weretaken after 3 days for cells grown at 30°C and 6 days forcells grown at 37°C. (C) Ten-fold serially diluted WTand
coa6Δsco2Δcells transformed with pRS416 empty vector or pRS416 vector expressing either SCO2,or sco2 with patient mutation E161K or S225F were spotted on YPD or YPGE
plates and incubated at 30°C. Images were taken after 2 days for cells grown on YPD and 4 days for cells grown on YPGE.
8|Human Molecular Genetics
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Materials and Methods
Yeast strains and culture conditions
All strains used in this study are listed in Supplementary Mater-
ial, Table S2 and were confirmed by polymerase chain reaction as
well as by replica plating on dropout plates. The WT yeast BY4741
cells expressing the COA6 gene with a 3′chromosomal HA tag
was constructed as previously described (44) using primers listed
in Supplementary Material, Table S3. Yeast cells were grown in
YP (1% yeast extract, 2% bactopeptone) medium with 2% dextrose
(YPD), 2% galactose (YPGal) or 3% glycerol + 1% ethanol (YPGE) as
a carbon source. Synthetic media was prepared with 0.17% yeast
nitrogen base, 0.5% ammonium sulfate, 0.2% dropout amino acid
mix and contained either 2% dextrose, 2% galactose or 3% gly-
cerol as a carbon source. Solid media additionally contained 2%
agar. Growth medium was supplemented with BCS or CuCl
2
wherever indicated. Growth was measured spectrophotometric-
ally at 600 nm in liquid medium or by spotting on solid plates.
Transformed yeast cells were grown in selection media to pre-
vent loss of the plasmid. Double knockout yeast strains (Supple-
mentary Material, Table S2) were constructed by sporulation of
the diploids followed by tetrad dissection on YPD medium. The
identities of all double mutant strains were confirmed by their
genotypes. BY4741 ρ
0
cells were obtained by culturing WT cells
in the presence of ethidium bromide (25 μg/ml) for 2 days.
BY4741 cox17Δcells were constructed by one-step gene disrup-
tion using a hygromycin cassette (44).
Plasmids
Yeast COA6 gene was cloned into three different plasmids: (1)
a single copy plasmid ( pRS416) under control of the native
promoter; (2) a multi-copy plasmid (pRS426) under control of the
native promoter and (3) a Gateway cloning plasmid ( pAG423-
GPD-ccdB-HA) as described previously (29). The hCOA6 (PubMed
Gene ID: 388753) and the hyCOA6 gene constructs were codon
optimized for yeast, synthesized using GeneArt
®
Gene Synthesis
(Life Technologies) and clonedinto pRS416 plasmid underthe con-
trol of the yeast Coa6 promoter. Yeast SCO1, SCO2, COX12, COX17,
COX19, COX20 and COX23 were cloned into pRS416 vector under
native yeast promoter. Point mutations in SCO2 were introduced
by site-directed-mutagenesis using QuickChange Lightning kit
from the Agilent Technologies. All the primers used in this study
are listed in Supplementary Material, Table S3. All constructs were
confirmed by DNA sequencing.
Coa6 purification and antibody generation
Yeast COA6 was first cloned into a pET28a-His
6
-GFP-TEV plasmid
using EcoRI and XhoI restriction sites. This construct was then
transformed into Rosetta DE3 Escherichia coli cells to express re-
combinant Coa6 protein. Protein purification was performed
using a HisTrap™HP column (GE Healthcare Life Sciences)
and a gel filtration Superdex 200 column (GE Healthcare Life
Sciences). Purified Coa6 (1.5 mg) was used to generate rabbit poly-
clonal antisera (Rockland Immunochemicals, Inc.). In order to
obtain purified Coa6 antibody, rabbit antisera was incubated
with Coa6 protein coupled to Affi-Gel 10 beads (Biorad) for 2–4h
at room temperature in a 5 ml Qiagen column. The column was
then washed with 50–100 ml phosphate buffered saline (PBS)
and antibodies were eluted using 0.2 glycine, 500 mNaCl,
(pH 2.0) buffer into a tube containing 1.5 Tris (pH 8.8) to neu-
tralize the pH. Aliquots of purified antibody were stored at −20°C.
Figure 7. Patient mutations disrupt Coa6 int eraction with Cox2. (A) Schematic
representation of human and yeast Coa 6 proteins highl ighting the co nserved
Cx
9
Cx
n
Cx
10
C motif with conserved residue s that are mutated in human
mitochondrial disease patient. (B) Western blot detection of Coa6-HA, Cox2 and
Sco1 protein s from mitochondrial extracts of coa6Δcells transformed with
either empty vec tor pAG423 or pA G423 expressing COA6 HA,orCOA6 mutants
before (input) or after immunoprecipitation with anti-HA antibodies.
Figure 8. A proposed role of Coa6 in the mitochondrial copper deliverypathway to
Cox2. Coa6 associates with Cox2,Cox12 and Sco proteins to form a copper delivery
complex for Cu
A
center biogenesis. Previ ously, it has bee n shown that Cox17
transfers copper to Sco protein s, but the mecha nism by which Cox1 7 receives
copper from a li gand bound (-L) copper pool is not known. Although Cmc1,
Cox19 and Cox23 are implicated in the copper deliver y pathway, we did not
detect Coa6 interaction with any of th ese proteins. OMM, outer mitochondrial
membrane; IMM, inner mitochondrial membrane; IMS, intermembrane space.
Human Molecular Genetics |9
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Whole cell yeast protein extraction and isolation
of mitochondrial fractions
Yeast cells (60–80 mg wet weight) were suspended in 350 μl
SUMEB buffer (1.0% sodium dodecyl sulfate (SDS), 8 urea,
10 mMOPS,pH6.8,10mEDTA) containing 1 mPMSF and
protease inhibitor cocktail (Roche Diagnostics). Cells were trans-
ferred to a fresh tube containing 350 mg of acid-washed glass
beads (Sigma-Aldrich) and were vortexed three times for 1 min
each, with 30 s incubation on ice between every vortex step.
Lysed cells were kept on ice for 10 min to reduce bubbles and
then heated at 70°C for 10 min. Cell debris and glass beads were
spun down at 14 000 g for 10 min at 4°C. The supernatant was
transferred to a separate tube and protein was quantified by the
Pierce BCA protein assay kit (Thermo Scientific). Mitochondria
were isolated from yeast cells as described previously (45)and
mitochondrial protein concentration was quantified using a
BCA assay.
SDS-polyacrylamide gel electrophoresis, BN PAGE
and western blotting
SDS-polyacrylamide gel electrophoresis (SDS PAGE) and BN PAGE
were performed to separate denatured and native protein com-
plexes. For SDS PAGE, whole cell yeast protein lysate (50 μg) or
mitochondria (20 μg) were separated and blotted onto a polyviny-
lidene difluoride membrane. For BN PAGE sample preparation,
yeast mitochondria (40 μg) were solubilized in 1% digitonin and
soluble lysate was resolved on a 3–12% gradient native PAGE
Bis-Tris gel (Life Technologies). Membranes were blocked for 1 h
in 5% nonfat milk dissolved in Tris-buffered saline with 0.1%
Tween 20 (TBST-milk), followed by overnight incubation with pri-
mary antibody in TBST-milk at 4°C. Primary antibodies were used
at the following dilutions: coa6, 1:1,000; Cox1, 1:1000 (Abcam
110270), Cox2, 1:10 000 (Abcam 110271); HA, 1:10 000 (Sigma
H9658); Sco1, 1:500; Sco2, 1:600 and Cox17, 1:250 (from Dr Alexan-
der Tzagoloff); Cox12, 1:1000 (from Dr Chris Meisinger); Cmc1,
1:500 (from Dr Antoni Barrientos); Porin, 1:50 000 (Abcam
110326); Pgk1, 1:50 000 (Life Technologies 459250).
Immunoprecipitation
Immunoprecipitation with anti-HA was performed using mito-
chondrial extracts of coa6Δcells transformed with empty vector
(pAG423), pAG423 expressing COA6HA or COA6 mutants (W26C,
C25A, C68A). Mitochondrial protein (3 mg) were solubilized with
RIPA buffer (Thermo Scientific) with protease inhibitor cocktail
(Roche Diagnostics) for 1 h at 4°C and then centrifuged at
14 000 g for 10 min. After removal of insoluble materials, mito-
chondrial lysates (input) were incubated overnight with anti-
HA antibody at 4°C. Protein A-agarose beads were then mixed
for 2 h at 4°C to bind antibodies. After washing the beads four
times with buffer containing 50 mTris, pH 7.4, 0.25% deoxycho-
late, 1% NP-40, 150 mNaCl, 1 mEDTA and once with PBS,
beads were suspended in NuPAGE LDS sample buffer and boiled
for 5 min prior to performing SDS PAGE. For immunoprecipita-
tion with anti-Coa6 antibodies, mitochondria were isolated
from WT and coa6Δcells grown in YPGE with 5 μCuCl
2
and
then solubilized with buffer containing 20 m4-(2-hyrdox-
yethyl)-1-piperazineethanesulfonic acid, pH 7.4, 100 mNaCl,
1mCaCl
2
, 1.5% digitonin and 10% glycerol with protease inhibi-
tor cocktail (Roche Diagnostics) for 30 min in a rotator at 4°C.
Insoluble mitochondrial fraction was pelleted at 20 000 g and the
remaining soluble supernatant wasused for immunoprecipitation.
Immunoprecipitation was performed using Dynabeads
®
Protein
G Immunoprecipitation Kit (Life Technologies) as per manufac-
turer’sprotocol.Briefly, anti-Coa6 was coupled to Dynabeads
®
Protein G and incubated with mitochondrial extract in a rotator
for 2 h at room temperature. After three washes, proteins were
eluted and boiled with NuPAGE LDS sample buffer prior to SDS
PAGE and western blotting.
Supplementary Material
Supplementary Material is available at HMG online.
Acknowledgements
We thank Dr Miriam L. Greenberg for the BY4741 and BY4742 wild
type yeast cells; Dr Alexander Tzagoloff, Dr Chris Meisinger and
Dr Antoni Barrientos for their generous gift of antibodies and
Dr David Barondeau and Dr Jared Rutter for pET28aGFP-TEV
and pRS plasmids, respectively. We thank Charli Baker and
Shrishiv Timbalia for their technical support.
Conflict of Interest statement. None declared.
Funding
This work was supported by the National Institutes of Health
award (R01GM111672) and the Welch Foundation grant (A-1810)
to V.M.G.
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12 |Human Molecular Genetics
at Texas A&M College Station on January 8, 2016http://hmg.oxfordjournals.org/Downloaded from
Strain
YPD
YPGal
YPGE
SCD
SCGal
SCG
BY4741 WT
++++
++++
++++
++++
++++
++++
BY4742 coa6Δ
++++
++++
++++
++++
++++
++++
BY4741 cox11Δ
++++
++++
-
++++
++
-
STY2 coa6Δcox11Δ
++++
++++
-
++++
++
-
BY4741 cox12Δ
++++
++++
-
++++
+++
-
STY3 coa6Δcox12Δ
++++
++++
-
++++
++
-
BY4741 cox17Δ
++++
++++
-
++++
-
-
STY1 coa6Δcox17Δ
++++
++++
-
++++
-
-
BY4741 cox19Δ
++++
+++
-
++++
++
-
STY4 coa6Δcox19Δ
++++
+++
-
++++
++
-
BY4741 cox23Δ
++++
+++
-
++++
++
-
STY5 coa6Δcox23Δ
++++
+++
-
++++
++
-
BY4741 cmc1Δ
++++
++++
++++
++++
++++
++
STY6 coa6Δcmc1Δ
++++
++++
++++
++++
++++
++++
BY4741 sco1Δ
++++
+++
-
++++
++
-
STY9 coa6Δsco1Δ
++++
+++
-
++++
++
-
BY4741 sco2Δ
++++
++++
++++
++++
++++
++++
STY10 coa6Δsco2Δ
++++
++++
-
++++
++++
++
BY4741 pic2Δ
++++
++++
++++
++++
++++
++++
STY11 coa6Δpic2Δ
++++
++++
+++
++++
++++
+++
! !
Supplementary Table 1. Genetic interaction study of Coa6 with known CcO assembly factors
involved in copper metabolism. Serial dilutions of wild type (WT), single knockouts, and double
knockouts were spotted on rich (YP) or synthetic complete (SC) media with different carbon
sources as indicated and incubated at 30ᴼC for 2-5 days. The plates were scored according to
the number of dilutions that grew. Maximal growth of all four dilutions is indicated using “++++,”
while no growth is indicated by “-”.
!
!
!
!
!
!
Strain
Genotype
Source
BY4741 WT
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0
Greenberg lab
BY4742 WT
MAT α, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0
Greenberg lab
BY4742 coa6Δ
MAT α, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0, coa6Δ::NatMX4
This study
BY4741 COA6HA
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0
This study
BY4741 ρ0
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0
This study
BY4741 coa6Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, coa6Δ:: KanMX4
Open Biosystems
BY4741 sco1Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, sco1Δ::KanMX4
Open Biosystems
BY4741 sco2Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, sco2Δ::KanMX4
Open Biosystems
BY4741 cox11Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, cox11Δ::KanMX4
Open Biosystems
BY4741 cox12Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, cox12Δ::KanMX4
Open Biosystems
BY4741 cox17Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, cox17Δ::HphMX4
This study
BY4741 cox19Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, cox19Δ::KanMX4
Open Biosystems
BY4741 cox23Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, cox23Δ::KanMX4
Open Biosystems
BY4741 cmc1Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, cmc1Δ::KanMX4
Open Biosystems
BY4741 cmc2Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, cmc2Δ::KanMX4
Barrientos Lab
BY4741 pic2Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, pic2Δ ::KanMX4
This study
STY9 sco1Δcoa6Δ
MAT a, his3Δ1, leu2Δ0, ura3Δ0, met15Δ0, sco1Δ::KanMX4,
coa6Δ::clonNAT
This study
STY10 sco2Δcoa6Δ
MAT a, his3Δ1, leu2Δ0, ura3Δ0, lys2Δ0, sco2Δ::KanMX4,
coa6Δ::NatMX4
This study
STY2 cox11Δcoa6Δ
MAT a, his3Δ1, leu2Δ0, ura3Δ0, cox11Δ::KanMX4,
coa6Δ::NatMX4
This study
STY3 cox12Δcoa6Δ
MAT a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0, lys2Δ0,
cox12Δ::KanMX4, coa6Δ::NatMX4
This study
STY1 cox17Δcoa6Δ
MAT a, his3Δ1, leu2Δ0, ura3Δ0, lys2Δ0, met15Δ0,
cox17Δ::HphMX4, coa6Δ::NatMX4
This study
STY4 cox19Δcoa6Δ
MAT a, his3Δ1, leu2Δ0, ura3Δ0, lys2Δ0, cox19Δ::KanMX4,
coa6Δ::NatMX4
This study
STY5 cox23Δcoa6Δ
MAT a, his3Δ1, leu2Δ0, ura3Δ0, cox23Δ::KanMX4,
coa6Δ::NatMX4
This study
STY6 cmc1Δcoa6Δ
MAT a, his3Δ1, leu2Δ0, ura3Δ0, lys2Δ0, cmc1Δ::KanMX4,
coa6Δ::NatMX4
This study
STY11 pic2Δcoa6Δ
MAT a, his3Δ1, leu2Δ0, ura3Δ0, met15Δ0, pic2Δ::KanMX4,
coa6Δ::NatMX4
This study
!
Supplementary Table 2. Yeast strains used in this study.
!
Supplementary Table 3. Primers used in this study.
Name
Sequence (5’ ! 3ˊ)
Cloning in pRS416
SacI yCOA6+500bp upstream
Forward
ccctggGAGCTCgcaaagacgcgcagccaaaaagccgaacagttta
BamHI yCOA6 Reverse
gggactGGATCCtcactgatttcgttccctctgtttagcttcctgctcgat
XhoI 500bp upstream Reverse
ccctaaCTCGAGcgctatattactactattcctttc
XhoI hCOA6 Forward
cccggcCTCGAGatgggtccaggtggtccattattgtc
XhoI HyCOA6 Forward
cccggcCTCGAGatgggtttgttctctttcgacgg
BamHI h/HyCOA6 Reverse
cccattGGATCCtcaggacttagcagtagtttcag
Cloning in pAG423GPD
AttB1 yCOA6 Forward
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCatgggcttattttcatttgatggtggc
AttB2 yCOA6 Reverse
GGGGACCACTTTGTACAAGAAAGCTGGGTCctgatttcgttccctctgtttagc
Cloning in pYM14 chromosomal
HA tag
S3 yCOA6 Forward
aagagaatcgagcaggaagctaaacagagggaacgaaatcagCGTACGCTGCAGGTCGAC
S2 yCOA6 Reverse
atatatatgttaatatgagccaataactcactaaaaactcaATCGATGAATTCGAGCTCG
Site Directed mutagenesis
primers
yCOA6 W26C Forward
agaaagttgtgctgcgagtccagggacgc
yCOA6 W26C Reverse
gcgtccctggactcgcagcacaactttct
yCOA6 C68A Forward
gtggaaaatgaaaaatttgaggagaatgccgcccatagctgga
yCOA6 C68A Reverse
tccagctatgggcggcattctcctcaaatttttcattttccac
yCOA6 C25A Forward
gttcacagagaaagttggcctgggagtccagggacg
yCOA6 C25A Reverse
cgtccctggactcccaggccaactttctctgtgaac
ySCO2 E161K Forward
taatctgtcaagcttttctggacaaatgtcggggc
ySCO2 E161K Reverse
gccccgacatttgtccagaaaagcttgacagatta
ySCO2 S246F Forward
gggtcgatcaaatagaaaaatatgaaatggtccactaagtaatcctgg
ySCO2 S246F Reverse
ccaggattacttagtggaccatttcatatttttctatttgatcgaccc
Supplementary Figure 1. Levels of CcO assembly factors do not change in coa6Δ cells.
Western blot analysis of the indicated CcO subunits and assembly factors in total cellular
protein extract from WT and coa6Δ cells grown to mid-logarithmic growth phase in YPGal
medium. The blot was probed with the indicated antibodies. Pgk1 was used as a loading control.
25 2550 50
WT coa6
Pgk1
g
Sco1
Cox12
Cox2
Cmc1
Sco2
Cox17
Supplementary Figure 2. Coa6 and Cox12 exhibit sequence and structural similarity but
Coa6 overexpression retards cox12Δ growth.
(A) Sequence alignment of human COX6B, yeast Cox12, human COA6, and yeast Coa6
proteins performed using ClustalW. (B) The yeast Coa6 structure predicted on the basis of the
bovine COX6B structure (Protein Data Bank ID 1OCC) using Phyre2 software and was
visualized using PyMol. (C) WT and cox12Δ cells were transformed with empty vector, pRS416-
COX12, or pRS416-COA6 and spotted on SC Galactose plates. Cells were grown at 30°C and
images were taken after 3 days.
WT+pRS416
+pRS416
+COX12
+COA6
SC Glucose 30°C SC Galactose 30°C
A
COX6B (Cox12 ortholog)
crystal structure (PDB 1OCC)
Coa6 predicted structure
(Phyre2)
B
C
hCOX6B --------------------------MAED-METKIKNYK-TAPFDSRFPNQN-QTRNCW 31
yCOX12 --------------------------MADQ-ENSPLH----TVGFDARFPQQN-QTKHCW 28
hCOA6 MGPGGPLLSPSRGFLLCKTGWHSNRLLGDCGPHTPVSTALSFIAVGMAAPSMK-ERQVCW 59
yCOA6 --------------------------MGLFSFDGGKK--------ESQPPNTRSQRKLCW 26
hCOX6B QNYLDFHRCQKAM-------TAKGGDIS-VCEWYQRVYQSLCPTSWVTDWDEQR------ 77
yCOX12 QSYVDYHKCVN----------MKGEDFA-PCKVFWKTYNALCPLDWIEKWDDQR------ 71
hCOA6 GARDEYWKCLD----------ENLEDAS-QCKKLRSSFESSCPQQWIKYFDKRRDYLKFK 108
yCOA6 ESRDAFFQCLDKADILDAMDPKNSKSIKSHCKVENEKFEENCAHSWIKYFKEKR-VIDFK 85
hCOX6B ---AEGTF-PGKI------ 86
yCOX12 ---EKGIF-AGDINSD--- 83
hCOA6 EKFEAGQFEPSETTAKS-- 125
yCOA6 REQTIKRIEQEAKQRERNQ 104
