Am J Physiol Cell Physiol 291: C1129 –C1147, 2006.
First published June 7, 2006; doi:10.1152/ajpcell.00233.2006.
Invited Review
Assembly of mitochondrial cytochrome c-oxidase, a complicated and highly
regulated cellular process
Flavia Fontanesi,1 Ileana C. Soto,2 Darryl Horn,2 and Antoni Barrientos1,2
Departments of 1Neurology and 2Biochemistry and Molecular Biology, The John T. Macdonald Foundation
Center for Medical Genetics, University of Miami Miller School of Medicine, Miami, Florida
systems biology; proteomics; genomics; transcription; translation
EUKARYOTIC CELLS are able to produce energy, in the form of
ATP molecules, by two different pathways. Energy can be
generated via glycolysis or by oxidation of glucose to ethanol
or lactic acid. In aerobic conditions, the complete oxidation of
carbohydrates, proteins, and fats to CO2 and H2O, via the
efficient mitochondrial respiratory chain (MRC) and oxidative
phosphorylation (OXPHOS) system, generates an ATP yield
⬃15 times higher than glycolysis. The MRC and OXPHOS
system are composed of five enzymatic complexes (I to V)
embedded in the inner mitochondrial membrane and two mobile electron carriers (ubiquinone and cytochrome c). Electrons
are donated from reducing equivalents, NADH and FADH2, to
complex I and complex II, respectively, and flow down an
electrochemical gradient in the MRC until complex IV, which
catalyzes the reduction of molecular oxygen, the final acceptor
of electrons, to water. Complexes I, III, and IV use the energy
liberated by the electron flux to pump protons from the mitochondrial matrix to the intermembrane space, generating a
proton gradient across the mitochondrial inner membrane that
is used by complex V to drive the synthesis of ATP from ADP
Address for reprint requests and other correspondence: A. Barrientos, Depts.
of Neurology and Biochemistry and Molecular Biology, The John T. Macdonald Foundation Center for Medical Genetics, Univ. of Miami Miller School
of Medicine, Miami, FL 33136 (e-mail: [email protected]).
http://www.ajpcell.org
and inorganic phosphate. To adjust the production of energy to
the variable energetic requirements of the cell, mitochondrial
respiration and OXPHOS are tightly regulated depending on
environmental stimuli. In the case of the unicellular yeast
Saccharomyces cerevisiae, the metabolism of the cell has to
respond to the availability of oxygen and carbon sources
(reviewed in Ref. 59), whereas in mammalian cells the carbon
source has a very limited effect on respiration, which is mostly
regulated by oxygen concentration (104), muscle contraction
(91), and hormone levels (reviewed in Refs. 155, 218). Which
mechanisms regulate the MRC activity? According to the
chemiosmotic hypothesis proposed by Mitchell in 1961, the
activity of the MRC is regulated exclusively by the proton
gradient across the mitochondrial inner membrane (133, 134).
ADP uptake into mitochondria stimulates ATP synthase activity, resulting in a decrease of the proton gradient, which will
finally lead to the stimulation of mitochondrial respiration
(133, 134). However, an additional regulatory mechanism of
mitochondrial respiration known as physiological control has
been proposed (reviewed in Refs. 102, 124). It is based on the
finding that complex IV of the respiratory chain, cytochrome
c-oxidase (COX), is inhibited at a high intramitochondrial
ATP-to-ADP ratio (66), through ATP binding to subunit IV of
the mammalian enzyme, subunit Va of the yeast enzyme (8,
97). This mechanism of respiratory control is switched on by
0363-6143/06 $8.00 Copyright © 2006 the American Physiological Society
C1129
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Fontanesi, Flavia, Ileana C. Soto, Darryl Horn, and Antoni Barrientos.
Assembly of mitochondrial cytochrome c-oxidase, a complicated and highly
regulated cellular process. Am J Physiol Cell Physiol 291: C1129 –C1147, 2006.
First published June 7, 2006; doi:10.1152/ajpcell.00233.2006.—Cytochrome coxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, plays
a key role in the regulation of aerobic production of energy. Biogenesis of
eukaryotic COX involves the coordinated action of two genomes. Three mitochondrial DNA-encoded subunits form the catalytic core of the enzyme, which contains
metal prosthetic groups. Another 10 subunits encoded in the nuclear DNA act as a
protective shield surrounding the core. COX biogenesis requires the assistance of
⬎20 additional nuclear-encoded factors acting at all levels of the process. Expression of the mitochondrial-encoded subunits, expression and import of the nuclearencoded subunits, insertion of the structural subunits into the mitochondrial inner
membrane, addition of prosthetic groups, assembly of the holoenzyme, further
maturation to form a dimer, and additional assembly into supercomplexes are all
tightly regulated processes in a nuclear-mitochondrial-coordinated fashion. Such
regulation ensures the building of a highly efficient machine able to catalyze the
safe transfer of electrons from cytochrome c to molecular oxygen and ultimately
facilitate the aerobic production of ATP. In this review, we will focus on describing
and analyzing the present knowledge about the different regulatory checkpoints in
COX assembly and the dynamic relationships between the different factors involved in the process. We have used information mostly obtained from the suitable
yeast model, but also from bacterial and animal systems, by means of large-scale
genetic, molecular biology, and physiological approaches and by integrating
information concerning individual elements into a cellular system network.
Invited Review
C1130
REGULATION OF COX ASSEMBLY
COX STRUCTURE AND ASSEMBLY
In the yeast Saccharomyces cerevisiae, COX is formed by
11 different subunits of dual genetic origin. The three largest
subunits, Cox1p, Cox2p, and Cox3p, are encoded in the mitochondrial DNA (mtDNA) and form the catalytic core of the
enzyme. These subunits are transmembrane proteins, embedded in the mitochondrial inner membrane with 12, 2, and 7
transmembrane ␣-helices, respectively, and coordinate heme A
and copper cofactors, which are responsible for the electron
transfer activity. Cox1p contains two redox centers, one
formed by a low-spin heme A and another by a high-spin heme
a3 and CuB. A third redox center of the enzyme is formed by
the two copper ions present in the CuA site of Cox2p. The CuA
active site accepts electrons from cytochrome c and rapidly
reduces heme A. From heme A, the electrons are transferred
intramolecularly to the heme a3-CuB binuclear center, where
molecular oxygen binds to be reduced (11). For each electron
transferred, two protons are pumped across the mitochondrial
inner membrane. Cox3p does not contain prosthetic groups,
and, although its function is unknown, it could be involved in
the assembly and/or stability of subunits 1 and 2 or in the
modulation of the access of oxygen to the binuclear center (30,
161). Interestingly, studies performed on bacterial COX,
formed exclusively of the three core subunits, have shown that
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Cox3p could play a role in modulating proton transfer through
subunits 1 and 2 (95).
In addition to the mtDNA-encoded subunits that are synthesized by the transcriptional/translational machinery present in
the mitochondrial matrix, the enzyme is composed of several
small nuclear-encoded subunits, synthesized in the cytoplasm
and imported into mitochondria. The nuclear-encoded subunits
surround the catalytic core of the enzyme and are not essential
to catalyze oxygen reduction and proton vectorial transfer. The
specific functions of these subunits are not yet completely
understood, but it is known that they are involved in the
assembly/stability and dimerization of the enzyme. They also
play a significant role in protecting the catalytic core from
reactive oxygen species (ROS) and in modulating the catalytic
activity of the enzyme. The importance of these subunits was
demonstrated by the complete loss of COX activity and, in
consequence, cellular respiration of yeast strains carrying null
mutations in the nuclear genes encoding for subunits Cox4p,
two isoforms of Cox5p (Cox5ap and Cox5bp), Cox6p, Cox7p,
and Cox9p, suggesting an essential role for these subunits in
assembly/stability of the enzyme (3, 36, 62, 195, 215). Subunit
Cox5ap is also involved in catalysis modulation. As mentioned
above, ATP binding to the matrix domain of Cox5ap, when the
intramitochondrial ATP-to-ADP ratio is high, causes a decrease in the efficiency of energy transduction or decreased
H⫹/e⫺ stoichiometry of proton pumping (188). Moreover, the
COOH-terminal domain of Cox5p, conserved between both
isoforms, interacts with subunit 2, and it was proposed to have
a regulatory function preventing the backflow of water or
protons from the cleft formed between Cox1p and Cox2p (32,
98, 198). COX can exist in the mitochondrial inner membrane
as either a monomer or a dimer, the latter being considered the
active form of the enzyme, in which two monomers are
connected by a cardiolipin molecule (103, 197).
The majority of the information regarding the dimeric structure of COX was obtained by crystallographic resolution of the
structure of the bovine heart enzyme (198), showing that the
cytochrome c binding site (two molecules of cytochrome c
bind to the dimer in a cooperative way) is located in a cleft at
the interface between the two monomers (24, 57), and both the
mitochondrial-encoded subunit Cox1p and some of the nuclearencoded subunits participate to form the four subunit-subunit
contacts that stabilize the dimeric structure (116). Interestingly, a
strain carrying a null mutation of cox8 was found to retain a
residual COX activity of 80% of the wild-type strain, resulting
from the inability of the enzyme to dimerize, which reduces the
efficiency of electron transfer between cytochrome c and COX
(151). Finally, the deletion of COX13, coding for subunit 10, does
not affect COX activity, suggesting that the enzyme can be
assembled in the absence of this subunit (188).
At present, the process of COX assembly is only partially
known, probably because there is a fast turnover of the hydrophobic core subunits in COX assembly-defective mutants,
which is particularly exacerbated in yeast as explained below.
Already in the early 1980s, studies that used rat liver mitochondria suggested that COX biogenesis occurs in a sequential
process, with the different subunits being added in an ordered
manner (208). Analyses of the human enzyme by Blue-Native
electrophoresis have allowed a proposed assembly pathway
characterized by at least three discrete assembly intermediates
that probably represent rate-limiting steps in the process (143).
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cAMP-dependent phosphorylation, and it is switched off by
calcium-induced dephosphorylation of COX (21). The phosphorylation event converts COX into the rate-limiting step of
respiration (11, 205), thus highlighting the key role of COX in
regulating the rate of respiration and ATP synthesis. This
allosteric regulation of COX is short term because it results in
a change of enzyme kinetics, it is immediate, and does not
require protein synthesis and assembly. A long-term regulation
of COX also exists and consists of controlling the number of
the enzyme molecules present in the mitochondrial inner membrane and/or controlling the subunit isoform composition that
confers the different catalytic properties of enzymes. This
regulation requires protein synthesis and assembly.
COX is a multimeric enzyme, with its assembly involving
the interplay of two different genomes and requiring the
assistance of a large number of assembly factors acting at
several levels of the process. The purpose of this review is to
summarize the actual knowledge about the process of COX
assembly and the multiple levels of its regulation. Although
some information about the bacterial and animal enzymes will
also be reviewed, we will mostly focus on the S. cerevisiae
system. Although still fragmentary, a large body of information
about the COX assembly process and its regulation in yeast is
available because of the amenability of this model organism for
large-scale mutagenesis and subsequent selection of respiratory
and COX mutants (130, 199) and for genomic and proteomic
studies (12, 87, 158). There is no doubt that to decipher the
intricacies of a biological mechanism complex fundamentally
critical for cellular bioenergetics, such as the COX assembly
process, the multidisciplinary approach should integrate data
resulting from biochemical and genetic analyses of genome,
transcriptome, proteome, and metabolome. In this light, systems biology can indeed reveal new insights and relevance on
COX assembly and its regulation.
Invited Review
REGULATION OF COX ASSEMBLY
C1131
In accordance with the model proposed by Nijtmans et al.
(143), biogenesis of Cox1p is the first step of the process and
corresponds to the first assembly intermediate. The second
intermediate is possibly formed by CoxIp, CoxIVp (the human
homologue of the yeast subunit Cox5ap), and perhaps CoxVap
(yeast subunit 6) (212). The third intermediate contains all of
the subunits except subunits VIa and VIIa or VIIb (corresponding to the yeast subunits 10 and 7, respectively), the addition of
which concludes the formation of the holoenzyme (143, 212).
Studies performed with yeast mutants have shown that the
COX assembly process is conserved between yeast and human.
The fact that, in yeast COX assembly mutants, the amount of
newly synthesized Cox1p is translationally downregulated (15)
(explained below) probably prevents the formation of detectable amounts of assembly intermediates in most COX mutants.
However, it is well known that Cox1p is necessary for the
assembly of Cox2p, Cox3p, and Cox4p, and recent analyses of
yeast cox2 mutants have revealed the presence of subassemblies in yeast similar to the ones described in human cells (92).
Assembling the structural subunits forming COX involves
an elaborated set of pathways (Fig. 1). How the expression of
subunits encoded in two physically separated genomes is
coordinated, how the proteins find each other in the mitochondrial inner membrane, and at what extent COX assembly is a
protein-assisted process are three examples of essential questions from which a complete answer is not yet available despite
the efforts of a large number of laboratories. Several considerations allow us to understand the complexity of the COX
assembly process and the complexity of its regulation. 1)
Because of the dual-genetic origin of COX, the expression of
COX subunits has to be coordinated and regulated to form a
fully assembled enzyme in which the 1:1 stoichiometry of the
structural subunits is always respected. 2) The dual-genetic
origin also leads to the necessity of two different pathways of
transcription, translation, maturation, and mitochondrial import. In addition to the structural subunits, the assembly of
COX requires the function of a large number of protein factors
necessary for all steps in the process. The analysis of assembly
intermediates described above has allowed insights into the
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overview of the assembly process, but information about the
players involved and their specific roles has been very limited.
To disclose the factors involved in COX assembly, a profitable
strategy has been to systematically analyze yeast mutants
defective in COX assembly, with the goal of identifying the
functions of the gene products responsible for this phenotype
and, by this means, reconstruct the different steps of the
assembly pathway. Screens of nuclear respiratory-deficient
mutants have revealed the existence of ⬎20 nuclear genes
coding for factors other than structural subunits, which selectively affect expression of this respiratory complex in yeast
(130, 199). They are involved in all the stages of the COX
assembly process (reviewed in Refs. 12, 87, 106), including
mitochondrial transcription (166), processing of mitochondrial
transcripts (177, 178), mitochondrial translation (15, 52, 139,
152), membrane insertion of the structural subunits (83, 85),
copper homeostasis and insertion into the apoenzyme (41, 70,
71, 93), and heme biosynthesis and incorporation to subunit 1
(18, 19, 146). 3) The availability of the two cofactors, responsible for the catalytic activity of the enzyme, is essential for
COX assembly. Copper is an essential cofactor of key enzymes
in the cell, but its reactivity with dioxygen may lead to toxicity
(202). For this reason, free aquo-copper ions are virtually
absent in the cytoplasm and they are always bound to specific
proteins necessary for the trafficking of copper within cells
(202). Nevertheless, recent data have suggested the presence of
a pool of nonproteinaceous copper present in the mitochondrial
matrix (50). Heme a is a unique heme compound present
exclusively in COX, and it is synthesized through a catalytic
pathway that takes place inside mitochondria. Copper and
heme A are deeply embedded in the core of the enzyme,
suggesting a coordinated and simultaneous mechanism of
translation, maturation, and membrane insertion of the subunits
carrying the prosthetic groups. In addition to copper and heme,
COX also contains magnesium, sodium, and zinc ions, the
function of which remains to be clarified. 4) The mitochondrialencoded subunits are hydrophobic proteins, in particular
Cox1p and Cox3p, and they tend to aggregate in an aqueous
environment. For this reason, the expression and maturation of
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Fig. 1. Dual-genetic involvement in the cytochrome c-oxidase
(COX) assembly process. COX biogenesis involves the coordinated action of 2 genomes. Three proteins, encoded in the
mitochondrial DNA (mtDNA), form the catalytic core of the
enzyme, which contains heme A and copper prosthetic groups.
Ten other proteins are encoded in the nuclear DNA. In addition
to the structural subunits, COX biogenesis requires the assistance
of ⬎20 additional nuclear-encoded factors acting at all levels of
the process (some indicated here). The process is regulated in
both nuclear-mitochondrial directions in a highly unique
crosstalk network. IMS, intermembrane space; MIM, mitochondrial inner membrane; MOM, mitochondrial outer membrane.
Invited Review
C1132
REGULATION OF COX ASSEMBLY
REGULATION OF COX ASSEMBLY
Regulation of COX biogenesis and activity in response to
changing environmental or physiological conditions plays a
central role in the physiology and metabolism of the adapting
cells. The study of these regulatory processes has been approached from the perspective of multiple individual disciplines together with the methodology used by systems biology,
which focuses on the iterative and integrative study of biological systems in response to perturbations, as defined by Auffray
et al. (10).
Multiple levels of regulation characterize the COX assembly
process, including availability of subunits and assembly factors
regulated at the transcriptional and translational levels, availability of cofactors, protein import into mitochondria and
membrane insertion, and coordination of sequential or simultaneous steps of the process (Fig. 1). In this section, we will
summarize the present knowledge about several pathways
involved in regulation of COX assembly in the yeast S. cerevisiae.
The yeast S. cerevisiae is an aerobic-anaerobic facultative
organism. When grown in medium containing glucose, it
produces energy and ethanol through glycolysis and fermentation, while the genes encoding proteins involved in the respiratory metabolism are repressed. When glucose is exhausted
and if oxygen is present, yeast cells are able to respire by use
of ethanol, previously produced as a carbon source. This
requires a dramatic change of the metabolism from fermentation to respiration, called diauxic shift. During the diauxic
shift, the respiratory genes are derepressed and induced by the
action of several transcription factors (reviewed in Ref. 175).
COX activity is modulated according to the growth conditions
and cell metabolism. It is low in cells grown in the presence of
glucose, and, thus with fermenting, it is induced fivefold after
the diauxic shift (31, 153). COX activity is also low in cells
grown in copper-depleted media (213), and it is absent in cells
grown in anaerobiosis and in heme-deficient mutants (119,
164, 214). Regulation of COX biogenesis allows for the modulation of COX activity in response to substrate availability
AJP-Cell Physiol • VOL
and oxygen concentration. The level of fully assembled and
functional enzyme is principally controlled by the availability
of the structural subunits and assembly factors, depending
mostly on transcriptional regulation of nuclear and mitochondrial COX genes and by translational regulation of mitochondrial COX subunits.
TRANSCRIPTIONAL REGULATION OF NUCLEAR COX
ASSEMBLY GENES BY OXYGEN
All of the nuclear genes encoding for COX subunits (COX4,
COX5a, COX6, COX7, COX8, and COX9), with the exception
of COX5b, are aerobic genes (31, 157). They are optimally
expressed at oxygen pressure of 200 ␮M, corresponding to the
atmospheric oxygen concentration, but they are also expressed
at lower levels in anaerobic conditions (33). COX5b, however,
is a hypoxic gene exclusively expressed at oxygen concentrations below 0.5 ␮M (33). The oxygen regulation of these genes
was exhaustively studied in cultures grown in medium containing galactose, a nonrepressing sugar, to minimize the effects of carbon sources on the expression of COX genes (33).
Analyses of the data obtained revealed that the expression of
the aerobic genes is dependent on the oxygen concentration,
not merely by its presence or absence. With the exception of
COX5a, which is transcribed at stationary levels for oxygen
concentrations between 200 ␮M and 10 ␮M, the other aerobic
genes showed a marked decline (40 –70%) of expression between 200 and 100 ␮M oxygen, whereas their expression
remained nearly constant between 100 and 10 ␮M. Under 10
␮M oxygen, all of the aerobic genes present a gradual decline
in expression with a drastic decline at 1 ␮M oxygen for COX4,
COX6, COX7, COX8, and COX9 and 0.5 ␮M oxygen for
COX5a (33). Given the sharp decline of expression of the
dose-response curves, 1 and 0.5 ␮M oxygen are considered the
thresholds for the expression of these aerobic genes, although
they present a residual expression variable from 7% of COX4
to 39% of COX9, in complete anaerobiosis (2.5% CO2 in
oxygen-free nitrogen) (33). The levels of COX5b mRNA are
undetectable for oxygen concentrations ⬎0.5 ␮M, indicating
that the hypoxic gene is more tightly regulated than the aerobic
genes (33). Kinetic analysis of oxygen-regulated induction/
repression (exemplified in Fig. 2) indicated that, although only
COX8 is fully expressed 90 min after a shift from anaerobiosis
to air, the other aerobic genes reach expression between 25%
(COX9) and 80% of their atmospheric steady-state levels after
2 h (33). Even more complex are the kinetics of gene expression after shift from air to anaerobiosis. In this case, the aerobic
genes present a small increase for the first 10 –20 min after the
shift, followed by a rapid decrease in expression during 20 min
and finally a slow continued decrease thereafter. However, the
overall decrease in speed could not be totally explained by the
downregulation of transcription, and it was proposed that a
second mechanism involving mRNA-specific degradation exists (33, 56). The induction of the hypoxic gene COX5b, after
a shift from aerobic to anaerobic growth conditions, is markedly slower than the induction of aerobic genes during a shift
of opposite direction. The full expression of COX5b requires in
fact 12 h (157), whereas the analysis of the repression kinetics
showed that only 20 min after a shift from anaerobiosis to air
COX5b mRNAs are undetectable (33). In summary, these data
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these proteins during the assembly process require the activity
of specific chaperones. 5) The protective role of chaperones is
also emphasized by the fact that COX assembly takes place in
the proximity of the sites of ROS production in the mitochondrial membrane, making both the protein subunits and the
metal cofactors possible targets of oxidation. In addition,
alterations in the assembly process could produce inefficient
enzymes that could lead to an increase in the production of
ROS. For this reason, COX biogenesis has to be precisely
regulated, especially the synthesis and assembly of subunits
Cox1p and Cox2p, containing active metal centers. 6) Different
isoforms of nuclear-encoded subunits confer to the enzymes’
different kinetic properties, and their expression is highly
regulated in response to a plethora of stimuli. In the case of the
yeast S. cerevisiae, only one nuclear-encoded subunit exists in
two isoforms, the above-mentioned Cox5a and Cox5b proteins,
which are differently regulated by oxygen concentration (206).
7) Finally, multiple points of quality control of the assembly
process and degradation of unassembled subunits and/or incomplete enzymes play an important role in COX biogenesis
(7, 147, 160).
Invited Review
REGULATION OF COX ASSEMBLY
suggest that aerobic and hypoxic genes are regulated through
different signal transduction pathways and that cells adapt
faster to environmental changes in which the oxygen concentration increases rather than a switch to anaerobic conditions. It
appears also surprising that, in anaerobic growth conditions,
there is some expression of genes encoding for subunits of
COX, an enzyme of the respiratory metabolism that requires
oxygen as substrate. To try to explain this observation it is
necessary to distinguish between two conditions: growth at low
oxygen concentration (below 0.5 ␮M) and growth in complete
anaerobiosis. In the first case, COX expression can be explained considering the different kinetic properties that the two
different isoforms of subunit 5 confer to the enzyme. The
transmembrane ␣-helix of Cox5p interacts with Cox1p and,
depending on the isoform, is able to alter the protein environment around the binuclear center in a way that does not alter
the Km of the enzyme but does alter the turnover number (4).
The Cox5bp isoenzyme has a higher maximal turnover number, and, although in the presence of Cox5ap the access of
electrons to heme a3 is limited, electron transfer from heme a
to heme a3 is three- to four times faster in the presence of
Cox5bp (4). In conditions of low-oxygen tension, the synthesis
of subunit Cox5bp, instead of subunit Cox5ap, confers an
advantage to the cells that can respire better because they
assemble a COX enzyme catalytically more efficient for the
mitochondrial production of energy (89).
The amount of fully assembled COX decreases with decreasing oxygen concentrations, which results from the explained transcriptional repression of nuclear COX genes together with a posttranscriptional regulation of the mitochondrial genes COX1 and COX2 (158). At oxygen concentrations
below 0.1 ␮M, COX activity is undetectable (164). However,
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even in complete anaerobiosis, COX genes are transcribed and,
although with no direct correspondence to the mRNAs levels,
both mtDNA-encoded and most nuclear-encoded COX subunits are present in protomitochondria, indicating that COX
subunits are translated and imported in the complete absence of
oxygen (33, 56). However, subunit 4 is detected only in very
reduced amounts (0.2%), and subunit 8, involved in COX
dimerization, is undetectable in protomitochondria by Western
analysis (56).
Measurement of respiration by providing oxygen to anoxic
cells indicated that, even in the absence of oxygen, a functional
respiratory chain is maintained (58). This finding led to the
hypothesis that the respiratory chain can use a final electron
acceptor other than oxygen (58). This hypothesis was supported by the fact that COX is involved, probably acting as an
oxygen sensor, in the induction of hypoxic genes (111) and the
alternative acceptor could play a role in this induction. In
oxygen-limiting growth conditions, the importance of the expression of hypoxic genes encoding for mitochondrial resident
proteins can be understood considering that even during anaerobic growth the cells need to maintain many metabolic
pathways that take place inside mitochondria. In addition, we
can easily think that the presence of a minimum amount of
fully assembled COX in the mitochondria of anoxic cells will
allow for an immediate response of the cell to sudden increases
in oxygen concentration, being already able to respire while
increasing the extent of COX biogenesis.
The oxygen regulation of nuclear COX genes is mediated
by heme that acts as an intracellular effector of the environmental signal (oxygen concentration) (165, 194, 196).
Experimentally, in heme-deficient mutants, the expression
of COX genes is independent of oxygen levels. In these
mutants, hypoxic genes are derepressed and aerobic genes
are repressed, whereas the supplementation of hemin to the
medium complements the mutant phenotype (89, 140).
Heme was considered the ideal effector of aerobic regulation because heme biosynthesis requires oxygen. Two enzymes of the biosynthetic pathway use oxygen as substrate
(112), one of them being the product of HEM13, which
catalyzes the rate-limiting step of the heme synthesis (112).
Nevertheless, two interesting observations must be discussed further. First, COX genes are expressed in the
complete absence of oxygen. At this respect, it is necessary
to consider that Hem13p can also function in the absence of
oxygen, probably by using another electron carrier as substrate (23, 111) and that a small amount of heme is also
present in cells grown anaerobically (58, 90). Second, the
mRNA levels of COX aerobic genes decrease with decreasing oxygen tension for concentrations between 200 and 1
␮M, whereas in the same range of oxygen concentrations
the intracellular level of heme remains quite constant (112).
It has been suggested that heme could regulate the expression of COX genes by changes in heme concentration or by
changes in heme redox states. The two models are not
exclusive, and it was proposed that heme could act as the
ligand of a transcription factor, modulating COX gene
expression in a concentration-dependent manner for low
oxygen concentrations (0 –1 ␮M), and that heme acts as or
affects the function of a transcription factor/oxygen-sensor
protein depending on its redox state for higher oxygen
concentrations (1–200 ␮M) (33). In consequence, in the
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Fig. 2. Kinetics of regulation of nuclear COX genes expression by oxygen.
Variation in the amount of relative mRNA levels of the nuclear aerobic COX
gene COX5a (solid lines) and of the nuclear hypoxic COX gene COX5b
(broken lines) after a shift from anaerobiosis to aerobiosis (A) and from
aerobiosis to anaerobiosis (B). The x-axis represents a range of time of ⬃2 h
after the shift, and the ordinate represents the relative amount of transcript from
zero to their steady-state level in the 2 growing condition. Modified from Refs.
33 and 110.
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Invited Review
C1134
REGULATION OF COX ASSEMBLY
oxygen-dependent regulation of COX nuclear genes, heme
acts as an intermediary between the environmental condition
(oxygen concentration) and specific transcription factors.
Two heme-dependent transcriptional activators have been
identified so far: the Hap1 protein and the Hap complex
(Hap2/3/4/5) (reviewed in Refs. 111, 225). Although Hap1p
mediates only the oxygen regulation of aerobic genes and its
activity is modulated by heme binding (204, 221), the Hap
complex mediates the regulation in response to two environmental stimuli, oxygen concentration and carbon source
availability (67, 78, 131). The genes regulated by the Hap
complex are repressed when the cells grow in medium
containing glucose even in the presence of oxygen/heme
(67, 75). How heme modulates the activity of Hap complex
is still unclear, but it has been proposed that heme can
regulate the catalytic subunit of the complex (Hap4p) posttranslationally (67). The oxygen regulation of the COX
aerobic genes COX4, COX5a, and COX6 is mediated by the
Hap complex (34, 193, 194, 196).
The inverse regulation by oxygen and heme of the genes
encoding the two isoforms of subunit 5 was extensively
studied (Fig. 3). Although the gene encoding the aerobic
isoform COX5a is induced by the Hap complex, the hypoxic
gene COX5b is repressed by the heme-dependent transcriptional repressor Rox1/Reo1 (196). In consequence, in aerobic growth conditions and in the presence of heme, Hap2/
3/4/5 induces the expression of COX5a and Rox1/Reo1
represses the expression of COX5b, with the result that only
the aerobic isoform of subunit 5 is synthesized in the cell.
Instead, when the cells grow in anaerobiosis or low oxygen
tension, i.e., in conditions of absence or low heme concentration, neither Hap complex nor Rox1/Reo1 can respectively activate or repress the target COX genes, and the
hypoxic isoform of subunit 5 is expressed (196). The oxygen-dependent action of Ord1/Ixr1, a second transcriptional
repressor on COX5b gene that binds in the same target
sequence as Rox1/Reo1 (113), was also reported. Ord1/Ixr1
AJP-Cell Physiol • VOL
represses specifically COX5b transcription, although it does
not affect either the expression of other hypoxic genes or
that of aerobic genes such as COX5a. The heme-dependent
repression operated by Rox1/Reo1 is not mediated through
a direct effect of heme on Rox1/Reo1 protein, but it is due
to a heme-dependent transcriptional activation of the ROX1
gene, operated by Hap1p (123, 225). It is interesting to note
that, although the repression of COX5b is mediated indirectly by Hap1p, and therefore in a way depends exclusively
on the environmental oxygen concentration, the oxygendependent transcriptional regulation of the COX aerobic
genes is mediated by the Hap complex. Moreover, studies
on the regulation of COX6 have demonstrated that, for this
gene, the transcriptional regulation is independent of Hap1p
(194). In consequence, the oxygen regulation is secondary
to the regulation by the carbon source. Although small-scale
experimental data to define the mechanism of oxygen regulation of the other COX nuclear-encoded subunits are not
available, systematic analysis of yeast gene expression has
indicated that the other COX genes are also regulated by the
Hap complex (34, 76).
To understand whether the regulation of COX genes by
oxygen was dependent on the same COX activity, mitochondrial respiration or OXPHOS, Dagsgaard et al. (56) measured
the level of expression of COX aerobic genes in nuclear and
mitochondrial mutants in which COX activity and consequently respiration or OXPHOS were completely abolished.
The results indicated that neither COX activity, nor respiration,
nor OXPHOS affects the expression of these genes. However,
a marked reduction of the expression of COX genes, in both
aerobic and hypoxic cells, was observed in cells devoid of
mtDNA (␳o strains) or containing mtDNA carrying large deletions (␳⫺ strains), indicating that the mtDNA affects the
transcription of nuclear-encoded aerobic COX genes, in both
normoxic and anoxic growth conditions, through a signaling
pathway to the nucleus (56). In addition, the effects of oxygen
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Fig. 3. Transcriptional regulation of nuclear COX structural genes by oxygen concentration and by carbon source availability. In the presence of oxygen
concentrations equal to or higher than 0.5 ␮M, COX5a is induced in a heme-dependent way by the transcriptional activator Hap2/3/4/5. Heme also induces the
expression of the ROX1 gene though the transcriptional factor Hap1p. The product of ROX1 is a translational repressor that is able to bind the promoter of COX5b
and repress its expression. COX5a is also subjected to glucose repression (196). COX6 is a COX nuclear aerobic gene induced in the presence of oxygen
concentrations equal or higher than 1 ␮M in a heme-dependent way, through the action of the transcriptional activator Hap2/3/4/5 (193). COX6 is also repressed
by glucose, and the derepression pathway during diauxic shift is characterized by a series of phosphorylations-dephosphorylations involving the Snf complex
Ssn6p and the transcriptional activator Abf1p (179). Arrows represent a positive regulation, and bars represent a negative regulation.
Invited Review
REGULATION OF COX ASSEMBLY
and mtDNA on the expression of COX nuclear genes are
independent of each other (56).
TRANSCRIPTIONAL REGULATION OF NUCLEAR COX
ASSEMBLY GENES BY CARBON SOURCE
AJP-Cell Physiol • VOL
Data regarding carbon source regulation of the other nuclear
COX structural genes in S. cerevisiae derive essentially from
systematic analyses of yeast gene expression. The results
obtained from several microarray experiments have been reported over the past few years and have been analyzed for this
review to extract data concerning regulation of COX subunits
and COX assembly factors. DeRisi et al. (61) carried out a
comprehensive investigation of the temporal program of gene
expression (transcriptome) accompanying the metabolic shift
from fermentation to respiration by using DNA microarrays
containing virtually every gene of S. cerevisiae (61). Two other
investigations based on the genome-wide exploration of gene
expression patterns by microarray analysis were focused on the
analyses of yeast gene expression in response to a large
spectrum of environmental stresses ranging from osmotic and
heat shock stress to oxidative stress, nutrient starvation, and
acid and alkali stresses (45, 68). Gunji et al. (76) also analyzed
the role of specific transcriptional factors on the reprogramming of gene expression after several environmental stress
conditions. The same RNA microarrays were also used to
identify genes whose expression was affected by deletion of
the transcriptional activator HAP2 and HAP3 and expression
patterns of genes encoding for known transcriptional factors in
different growth conditions (76). By performing a similar kind
of experiment, Buschlen et al. (34) measured changes in the
expression of the yeast transcriptome in cells grown in medium
containing the fermentable, nonrepressing carbon source, galactose, induced by the deletion of HAP2 and HAP4. (34).
Instead, Lascaris et al. (115) used a different approach to
analyze the effect of Hap complex on the expression of yeast
genes to understand their former observation that overexpression of HAP4 is able to override the signals that normally result
in glucose repression of mitochondrial function. To compare
the expression profile in repressing growth conditions (media
supplement with glucose 3%) of wild-type cells and cells
overexpressing Hap4p more than fivefold, the authors (115)
performed a whole-genome expression profiling and fingerprinting of the regulatory activity network.
The analysis of expression profiles observed for COX genes
in all of the experiments mentioned above have allowed us to
better understand the role of COX assembly during the metabolic reprogramming that occurs during the diauxic shift and
during several stress conditions. Taken together, these data
trace a clear carbon source-dependent expression profile of the
nuclear COX genes. Several conclusions emerge from the
systematic analyses mentioned before. 1) Nuclear genes encoding COX subunits, with the exception of COX5b, are
regulated by carbon sources; they are repressed by glucose and
induced during diauxic shift (61). However, the glucose repression does not abolish completely their transcription, and,
for example, COX6 mRNAs are still detectable in glucoserepressed cells (217). 2) The nuclear aerobic COX structural
genes present a temporal pattern of expression marked by an
induction coincident with the diauxic shift (glucose depletion)
(61) 3) Strains carrying null mutations of hap2 and hap4
present a significant decrease in the expression of COX aerobic
genes, ranging between 27% (COX4) and 52% (COX7) of the
wild type in a hap2 mutant and between 34% (COX4) and 62%
(COX7) of the wild type in a hap4 mutant (34). These results
indicate that the Hap complex mediates the regulation of the
nuclear COX genes, with the exception of COX5b (34, 76). In
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All nuclear COX structural genes are repressed by glucose
and induced during the diauxic shift (61). As an example, the
transcriptional regulation of COX6 gene has been extensively
studied (Fig. 3). COX6 is regulated by oxygen in a hemedependent way (194) and downregulated in the absence of
mtDNA (56), as described above. Because COX6 is also
repressed by glucose, cells grown in the presence of a respiratory carbon source such as lactate present four- to fivefold
higher levels of COX6 transcript than shown in cells grown in
glucose (216, 217). A peculiarity of COX6 expression is the
transcription of the gene in three mRNA species with a difference in length of ⬃150 bp from one to the other (217). The
three COX6 mRNAs show a microheterogeneity in the 5⬘ ends
and a major diversity in the 3⬘ ends, accounting for the
different lengths (217). More interesting is the fact that all
three mRNA species are subjected to glucose repression, but
with a different sensitivity. Glucose repression affects the two
larger mRNAs, and derepression occurs in a very short time
(⬃10 min). As a consequence, the relative ratio between
mRNA species is different in cells grown in repressing or
derepressing conditions. In glucose, the COX6 mRNAs population is enriched for the smallest mRNA class, whereas after
a shift to medium containing low glucose or respiratory carbon
sources, there are observed similar levels of the three different
COX6 transcripts. Nevertheless, the data available do not allow
discrimination between a different level of glucose-dependent
transcription and different stability of the mRNA species.
Transcriptional regulation of COX6 is mediated by at least four
different transcriptional factors: Hap complex, Snf1p, Ssn6p,
and Abf1p (Fig. 3 and Refs. 193, 194, 216). Snf1p is part of a
complex essential for the release of glucose-repressible genes
(46). When glucose in absent, Snf1p acts as a kinase, and it is
able to phosphorylate various activators and repressors, modulating their activity (40). One possible target to Snf1p is
Ssn6p, a factor involved in glucose repression (176). Because
in a snf1 mutant only a slight derepression of COX6 is observed
after the diauxic shift and because both a ssn6 single mutant
and a snf1-ssn6 double mutant are constitutively derepressed, it
is possible to conclude that both Snf1p and Ssn6p are involved
in COX6 transcriptional regulation and the action of Ssn6p is
epistatic to Snf1p-mediated regulation (216). Abf1p is a phosphoprotein that can have at least four different phosphorylation
states, correlating with the cellular growth conditions, higher in
cells grown in the presence of respiratory carbon sources. The
phosphorylation state of Abf1p is also affected by Snf1p/Ssn6p
because Ssn6p is essential for the dephosphorylation of Abf1p
in a Snf1p-dependent manner (179). Although dephosphorylated Abf1p does not bind DNA, the level of phosphorylation
determines the composition of Abf1p-containing protein-DNA
complex formed on the COX6 promoters (179, 193), which
modulates transcription of COX6 mRNA in a carbon sourcedependent way. At present, it is not clear whether Snf1p/Ssn6p
acts independently or in the same pathway as the Hap complex,
which mediates the induction of COX6 after diauxic shift in a
heme-dependent manner (34, 194).
C1135
Invited Review
C1136
REGULATION OF COX ASSEMBLY
TRANSLATIONAL REGULATION OF NUCLEAR COX
ASSEMBLY GENES
Although the regulation of nuclear-encoded COX subunits
expression is performed mostly at the transcriptional level,
there are some examples in the literature of translational
AJP-Cell Physiol • VOL
regulation. At this respect, it has been recently reported that the
expression of COX4 is regulated posttranscriptionally by the
mitochondrial content of cardiolipin, the major phospholipid
component of the mitochondrial membranes, and its precursor
phosphatidylglycerol in S. cerevisiae (185).
TRANSCRIPTIONAL REGULATION OF MITOCHONDRIAL
COX GENES
The three subunits forming the catalytic core of eukaryotic
COX are encoded in the mtDNA. As in the case of the
nuclear-encoded COX subunits, the expression of the mtDNAencoded COX subunits is affected by oxygen concentration
and by carbon source.
Oxygen concentration affects expression of the three
mtDNA genes in different manners. The levels of COX3
mRNA decrease two- to threefold under anaerobic growth, but
the levels of COX1 mRNA are maintained constant under
different oxygen concentrations. Interestingly, expression of
Cox1p and Cox2p is affected by oxygen tension at the posttranscriptional level (51, 158).
Mitochondrial COX genes are repressed by glucose, even in
the presence of oxygen, and induced during diauxic shift (137,
201). This regulation can be assigned to the general glucose
repression of mitochondrial transcription. In fact, a lower total
mitochondrial RNA synthesis was observed in repressed cells
than in derepressed cells both in isolated mitochondria and in
vivo (39, 137, 201, 220). A general glucose repression mechanism of mitochondrial transcription is also supported by the
fact that mitochondrial RNAs present the same kinetics of
derepression during a shift from medium containing glucose to
medium supplemented with a respiratory carbon source such as
glycerol (201). After release from glucose repression, the
abundance of mitochondrial transcripts increases very slowly
and coincides with active cell growth (201). However, the final
change in RNA abundance varies significantly between different RNA species (201). In derepressing conditions (growth in
medium containing galactose), the level of COX1 mRNA is
increased almost 5-fold (137); in inducible conditions (growth
in medium containing glycerol), it is increased 20-fold,
whereas COX2 and COX3 mRNAs are increased ⬃3-fold
(201). The final mitochondrial RNA abundance after derepression is dependent on three additional regulatory mechanisms.
First, mitochondrial mRNAs have a half-life considerably
shorter than the rRNAs (132), and they do accumulate differently. Second, mitochondrial promoters of diverse transcriptional units can sustain expression with different strength, with
up to 20-fold of difference between weak and strong ones (25,
207). Finally, mitochondrial transcription is also influenced by
the existence of polycistronic transcription units. Probably
resulting from attenuation of RNA polymerase elongation,
within the polycistronic transcription units, proximal genes are
more transcribed than distal genes, with a difference of up to
17-fold (136).
Two mechanisms were proposed to be involved in general
derepression of mitochondrial transcription after diauxic shift:
the increase of mtDNA copy number and a fivefold induction
of the mitochondrial RNA polymerase encoded by the nuclear
gene RPO41 (73, 210). Nevertheless, the magnitude of variation of mtDNA copy number, from zero- to threefold between
repressing and derepressing conditions, is not enough to ac-
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addition, nuclear aerobic COX structural genes are upregulated
to approximately two times in wild-type cells overexpressing
HAP4 (115). 4) HAP4 gene, encoding the catalytic subunit of
the Hap complex, is also induced ninefold during diauxic shift,
and it is one of the two transcriptional activators, among the
149 genes encoding putative transcriptional factors, to be
induced more than threefold during diauxic shift (61). However, it has been demonstrated that the active Hap complex is
formed by only one copy of each subunit (131), and HAP2,
HAP3, and HAP5 are constitutively expressed (61, 76). 5) The
Hap target sequence is present in the promoter (between ⫺800
and ⫺150 bp) of all the nuclear aerobic COX structural genes
(34). However, binding of Hap2p/Hap3p to the Hap binding
site present in the promoter of COX6 was not observed in vitro,
suggesting an indirect effect of Hap2/3/4/5 on COX6 transcription (193). 6) The expression of nuclear COX structural genes
changes less than twofold in response to environmental stress
changes other than diauxic and oxygen concentration shift
(45).
Although the analysis of the expression profiles observed for
COX genes indicates clear lines of coregulation, this observation seems not to be extended to nuclear-encoded COX assembly factors. The regulation by diauxic shift of these genes is
quite heterogeneous, and there are no clear traces of a common
regulation pattern. For example, COX15 (involved in hydroxylation of heme o during heme A biosynthesis) and COX20 (a
chaperon of Cox2p) are induced more than three times,
whereas COX10 (involved in the farnesylation of protoheme
during heme A biosynthesis) and COX11 (involved in the
insertion of copper into the CuB center of Cox1p) are unaffected (61) The expression of some of these genes, such as
COX10, COX17 (involved in the delivery of copper to mitochondria), OXA1 (necessary for the membrane insertion of the
COX core subunits), and PET117 (coding for an assembly
factor of unknown specific function), is practically unaffected
during diauxic shift but highly induced in cells overexpressing
HAP4. The promoter region of these genes does not contain the
consensus Hap target sequence, suggesting an indirect effect of
the Hap complex on their regulation by different environmental
stimuli (115). It is possible that additional transcriptional
factors play an important role in the regulation of the COX
assembly factors. For example, analysis of the expression
regulation of the nuclear gene PET494, coding for a translational activator of COX3 mRNA (129), showed that PET494 is
repressed by glucose and induced by respiratory carbon
sources such as ethanol, but its regulation is not mediated
either by the Hap complex or by Snf1p, Snf2p, Ssn6p, and
Hxk2p, typical transcriptional factors controlling glucose repression of many nuclear genes (129).
Although there is a constant flux of information available,
further analyses are necessary to understand which are the
limiting factors and regulatory key points of the COX assembly
process and to obtain a global view about how COX biogenesis
is regulated.
Invited Review
REGULATION OF COX ASSEMBLY
count for the full increase in RNAs level (136, 201). Moreover,
20 –50 times overexpression of the genes encoding the core of
mitochondrial RNA polymerase (RPO41) and its specificity
factor (MTF1) in repressed cells do not result in release of
mtDNA genes from glucose repression (201).
Derepression of mtDNA genes is affected indirectly by some
nuclear transcription factors: Snf1p, Reg1p, and Urr1p. Mutations in SNF1 are known to prevent derepression, whereas
mutations in REG1 or URR1 lead to a constitutive derepression
state (201). Interestingly, overexpression of the catalytic subunit Hap4p increases 2.3-fold the COX2 mRNA level and
1.45-fold the COX1 mRNA level in repressed cells (115),
suggesting that the Hap complex could also indirectly affect
the expression of mtDNA genes.
Mitochondria contain the translational machinery necessary
for the synthesis of the mtDNA-encoded COX subunits. The
genes encoding for the ribosomal RNA of the large and small
ribosomal subunits and a set of tRNAs are also located in the
mtDNA in yeast and higher eukaryotes. The proteins forming
the mammalian mitoribosome are all encoded in the nuclear
genome, whereas in the yeast S. cerevisiae, the Var1 protein, a
component of the small ribosomal subunit, is encoded in the
mtDNA. The remaining proteins necessary for expression of
mtDNA genes are nuclear encoded, synthesized in cytoplasmic
ribosomes, and imported into mitochondria.
Mitochondrial translation can be studied in whole cells in the
presence of inhibitors of the cytoplasmic ribosomes such as
emetine and cycloheximide and in isolated mitochondria (in
organello) systems that we and others have used extensively
(13, 15). However, a good portion of details concerning expression of mtDNA genes cannot be conveniently approached
because of the lack of a true in vitro system for mitochondrial
translation. Fox and colleagues (26) developed a valuable in
vivo genetic system with a reporter ARG8 gene. In this system,
a mitochondrial gene is replaced by the ARG8 gene in strains
carrying a null mutation in the chromosomal arg8 gene. Arg8p
is a soluble protein that is normally synthesized in the cytosol
and transported into the mitochondrial matrix. A good portion
of the elements that control mitochondrial translation and are
described below have been found by using this methodology
combined with classical in organello protein synthesis.
Most yeast genes involved in the expression of the three
mtDNA-encoded proteins forming the core of the enzyme
seem to be absent in high eukaryotes, particularly in humans
(reviewed in Ref. 181). This can be easily explained from
qualitative differences between the two species in their mtDNA
and mRNA. First, yeast COX1 contains introns, whereas all
human mtDNA genes are intronless. Yeast introns must be
spliced before translation by specific intron-encoded maturases
with the assistance of helicases and DEAD-box proteins that
function as RNA chaperones to destabilize kinetic traps in
RNA folding of introns in vivo (96, 128, 173, 177). Splicing of
COXI introns is a fascinating process, but its description is out
of the scope of this review. A second and even more significant
difference resides in the mechanism of specific mitochondrial
mRNA processing and translation. In yeast, translation is
governed by mRNA-specific activators, which interact with the
AJP-Cell Physiol • VOL
ribosomes (77) and recognize targets in the 5⬘-untranslated
regions (5⬘-UTR) of the mRNAs (167), whereas in human,
mitochondrial mRNAs lack 5⬘-UTRs (9), and the existence of
specific translational activators seems to be uncertain.
Expression of the mitochondrially encoded COX subunits
also requires one or more translational activator factors specific
for each mRNA. Translation of COX1 mRNA requires Pet309p
(125) and Mss51p (60), translation of COX2 mRNA involves
the action of Pet111p (156), and translational activation of
COX3 mRNA requires Pet54p, Pet494p, and Pet122p (54,
107). The mRNA-specific translational activators recognize the
5⬘-UTR of their target mRNAs to promote translation (53).
These translational activators are inner membrane proteins,
either integral (Pet309p, Mss51p, Pet111p, Pet494p, and
Pet122p) or peripheral (Pet54p), a localization that would
facilitate a coupling of mitochondrial translation with the inner
membrane, allowing for cotranslational insertion of newly
synthesized hydrophobic mitochondrial gene products into the
membrane (141). As explained below, most of these activators,
with the exception of Mss51p, have been shown to interact
among them (29, 141), suggesting some level of regulation of
the expression of the different subunits forming the catalytic
core of COX (65). The network of interactions among translational activator proteins is very extensive, suggesting a high
level of regulation of the mitochondrial translational machinery. The physical interactions among the different translational
activators are not essential for their activity because they retain
it in the absence of one of the partners. However, the amount
of the different activators is probably important, as suggested
by the fact that overexpression of the COX2 translational
activator, Pet111p, prevents translation of COX1 mRNA (65).
Expression of Cox1p is regulated at the translational level
depending on the availability of the other COX assembly
partners (13, 15, 152). As explained above, COX1 expression
in yeast is under the control of two genes, MSS51 and PET309,
which products are involved in the maturation and translation
of COX1 mRNA (60, 125). We have recently reported that
mutant forms of mss51 or overexpression of the wild-type gene
act as suppressors of shy1-null mutants by increasing the levels
of newly synthesized Cox1p (13). The function of Shy1p,
related to maturation and/or assembly of Cox1p (13, 152, 180),
is of high interest because mutations in its human homologue,
Surf1p, have been shown to be responsible for most diagnosed
cases of Leigh’s syndrome presenting with COX deficiency
(191, 224). Screens of a collection of strains carrying null
mutations of COX biogenesis factors showed that the amount
of newly synthesized Cox1p is not only reduced in shy1
mutants but in most COX assembly mutants and restored by
the mss51 suppressors of shy1 or by mutations in cox14,
another COX assembly factor. The mechanism of action of
Mss51p in translation of COX1 mRNA differs from classical
translational activators. In common with all translational activators, including Pet309p, Mss51p acts on the 5⬘-UTR to
initiate translation (152); however, mss51 mutants, unlike
pet309 mutants, cannot be suppressed by changes in the 5⬘UTR of COX1 mRNA (15, 152). In addition, Mss51p is
required for Cox1p synthesis (i.e., translation elongation) by
acting on a target coded by the protein sequence itself (152).
Mss51p and Cox1p form a transient complex (15, 152) that is
stabilized by Cox14p by interacting with Cox1p (15) and could
function to downregulate Cox1p synthesis when COX assem-
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TRANSLATIONAL REGULATION OF MITOCHONDRIAL
COX GENES
C1137
Invited Review
C1138
REGULATION OF COX ASSEMBLY
bly is impaired (15). In the model that we have proposed, the
release of Mss51p from the complex to make it available for
Cox1p synthesis occurs at a downstream step in the assembly
pathway, most likely catalyzed by Shy1p (Fig. 4) (15).
The expression of Cox2p in yeast is one of the better
characterized steps of COX metabolism. Translation of yeast
COX2 mRNA is initiated by Pet111p (138, 156), which is
present in the mitochondrial membranes at a low concentration, limiting the synthesis of Cox2p (74). The yeast protein is
synthesized as a precursor (pCox2p), containing an NH2terminal sequence that plays a role in targeting Cox2p into the
inner membrane and is subsequently proteolytically removed
(explained below). The first 14 codons in NH2-terminal leader
peptide of Cox2p are also involved in the regulation of downstream translation of COX2 mRNA. Interestingly, the secondary structure of the mRNA rather than the amino acid sequence
is more important for the function of the positive element
(209). Three additional sequences located in the Cox2p NH2terminal region of the mature protein have been described to
play an inhibitory role in the absence of the positive element
(211). Details about the maturation of the Cox2p precursor are
described below.
The synthesis of Cox3p in yeast is governed by three specific
translational activators, Pet54p, Pet122p, and Pet494p (29, 53),
which interact with the 5⬘-UTR sequences of COX3 mRNA
(52). As mentioned above, it has been shown that COX3specific activators interact to form a COX3 mRNA-specific
activator complex (141).
Although most studies on the control of mitochondrial translation concern initiation, elongation and termination are in
some cases limiting steps. An interesting review on the subject
can be found elsewhere (192).
REGULATION OF COFACTOR AVAILABILITY
Mitochondrial copper homeostasis, heme A biosynthesis,
and insertion of both prosthetic groups into the corresponding
newly synthesized subunits forming the catalytic core of the
enzyme involve several pathways that must be precisely coordinated.
AJP-Cell Physiol • VOL
Copper is a metal prosthetic group essential for COX assembly and function (19). The enzyme contains three copper
atoms. Two are bound to Cox2p, constituting the CuA site, and
the third, referred to as CuB, is associated with the heme A
group of heme a3 in Cox1p. In living cells, copper ions are
mainly bound to small soluble proteins, called copper chaperones, which are responsible for intracellular copper trafficking
and compartmentalization. In yeast, mitochondrial copper acquisition and insertion into the apoenzyme have been shown to
depend on at least five evolutionary conserved proteins. Three
of these are copper chaperones necessary for the delivery of
copper to mitochondria: Cox17p, Cox19p, and Cox23p. Yeast
Cox17p is a small hydrophilic protein containing a CCxC
metal-binding motif (70) that binds copper (20, 81, 150).
Involvement of COX17 in copper homeostasis was originally
proposed because the respiratory-deficient phenotype of cox17null mutant strains can be rescued by supplementation of
copper to the medium (70). The protein localizes in both the
cytoplasm and the mitochondrial intermembrane space (70), a
localization that is consistent with a function as a soluble
copper chaperone, probably shuttling copper from the cytoplasm into the mitochondrial intermembrane space (20, 70).
This hypothesis has been challenged by the observation that,
although apo-Cox17p is predominantly a monomer with a
simple hairpin structure, the protein-copper complex exists in a
dimer-tetramer equilibrium (82), making unlikely its free passage through the outer membrane protein channel. In addition,
it has been recently shown that tethering of Cox17p to the inner
mitochondrial membrane does not affect COX assembly (50).
Two homologues of Cox17p, the small soluble proteins
Cox19p and Cox23p, exhibit a cellular distribution similar to
Cox17p and contain a twin Cx9C metal-binding motive (17,
145). The recombinant form of Cox19p was reported to bind
copper (50), but the COX assembly defect of cox19-null
mutant could not be rescued by supplementation of copper to
the medium (145). Mutants of cox23 also fail to assemble
COX, but their respiratory-deficient phenotype is complemented by exogenous copper supplementation, albeit only with
concomitant overexpression of COX17 (17). Although Cox23p
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Fig. 4. Coupling between COX assembly and translation of mitochondrial COX1 mRNA in S. cerevisiae.
COX1 mRNA translation requires the action of 2 proteins, Pet309p and Mss51p. Both act on the 5⬘-untranslated region (UTR) to initiate translation (145). In addition, Mss51p is required for Cox1p synthesis (i.e.,
translation elongation) by acting on a target coded by
the protein sequence itself (145). Mss51p and Cox1p
form a transient complex (13, 145) that is stabilized by
Cox14p (13) and could function to downregulate Cox1p
synthesis when COX assembly is impaired (13). In this
model, the release of Mss51p from the complex to make
it available for Cox1p synthesis occurs at a downstream
step in the assembly pathway, most likely catalyzed by
Shy1p (13).
Invited Review
REGULATION OF COX ASSEMBLY
AJP-Cell Physiol • VOL
known at present. The biosynthesis of heme A is regulated by
downstream events in the COX assembly process. Barros and
Tzagoloff (19) measured the amount of heme A and heme O in
different yeast COX mutants and observed that all of the
mutants analyzed showed a drastic reduction of steady-state
levels of heme A, with the exception of shy1-, cox20-, and
cox5a-null mutant, in which heme A was still detectable at
10 –25%, probably associated with residual assembled COX.
The overexpression of COX15 significantly increased the
amount of heme A in COX mutants, including mutants in
which Cox1p was not synthesized, suggesting that the absence
of heme A in the mutants is not due to a rapid turnover of the
cofactor in the absence of COX subunit one but due to a
feedback regulation of the heme A synthesis when the COX
assembly process is blocked. The COX mutants analyzed, with
the obvious exception of cox10, showed also an accumulation
of heme O, indicating that this compound is stable. In addition,
the cox15-null mutant presented a very low amount of heme O,
a phenotype that was not rescued by the overexpression of
COX10. This observation suggested that the first step of the
heme A biosynthesis is also positively regulated in a Cox15pdependent manner (19).
Because copper and heme A are COX cofactors and together
form the metal active centers of the enzyme, it is possible to
hypothesize that copper is involved in the regulation of heme
A biosynthesis. In fact, some circumstantial evidence has
pointed to a possible specific link between copper and heme A.
To address this question, an interesting study recently showed
that the presence of copper does not affect expression, stability,
and activity of Cox10p and Cox15p (135), concluding that the
biosynthesis of heme O and heme A are not regulated by
copper.
COORDINATION OF THE DIFFERENT STEPS IN THE COX
ASSEMBLY PROCESS OCCURRING INSIDE MITOCHONDRIA
In the previous sections, we have exposed that COX biogenesis involves the intrinsic coordination of a large number of
pathways, including mitochondrial transcription, translation,
import, and metal metabolism, which are precisely localized in
a limited number of foci in a defined mitochondrial area, the
matrix face of the inner membrane. Yeast two-hybrid analyses
and coimmunoprecipitation experiments have allowed for the
description of a network of multiple protein-protein interactions necessary for the coordination of the events in COX
assembly. First of all, the efficient expression of the mtDNAencoded genes COX1, COX2 and COX3 requires a tight coordination of transcription and translation, which is achieved
through the localization of active transcription complexes to
the inner membrane, where translational factors are present
(Fig. 5). The coordination between transcription and translation
results from protein-protein interactions among the RNA polymerase Rpo41p, the matrix protein Nam1p, involved in RNA
processing and translation, and the mitochondrial membrane
protein Sls1p, a component of the nucleoids, structures that
facilitate the interaction of mtDNA with the inner face of the
inner membrane (162, 163). Yeast two-hybrid analyses have
shown additional interactions between Nam1p and the COX
translational factors Pet309p, Pet111p, and Pet494p, which
further interact with Pet54p and Pet122p. The interaction
between Nam1p and Pet309p was confirmed by coimmuno-
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does not physically interact with Cox17p in a stable complex,
recent data showed that it is also required for mitochondrial
copper homeostasis, functioning in a common pathway with
Cox17p acting downstream of Cox23p. (81). In the same line,
Cox19p would participate in a different portion of the copper
distribution pathway (17). Two additional chaperones facilitate
copper insertion into the COX CuA and CuB active sites,
respectively, Sco1p and Cox11p, which directly receive copper
from Cox17p (93). In vitro experiments have shown that a
soluble truncated form of both Cox11p and Sco1p are able to
bind copper (41, 144); however, it is still not clear how the
transfer of copper occurs between these proteins because physical interactions among them have not been detected (93).
However, both Cox11p and Sco1p are anchored to the mitochondrial inner membrane through a transmembrane ␣-helix
and expose the copper binding side in the intermembrane space
(20, 42) where they could receive copper directly from the
copper chaperones. The metallochaperone for the formation of
the CuB site of Cox1p is the product of COX11 (41, 88), which,
at least in the prokaryote Rhodobacter sphaeroides, also participates in the formation of the Mg/Mn centers present at the
interface of subunits 1 and 2 (88). The metallochaperone for
the formation of the CuA center of Cox2p is the product of
SCO1 (71), which transfers copper from Cox17p to Cox2p.
Sco1p has a metal binding CxxxC motif analogous to the
copper binding motif of Cox2p, and this motif is essential for
its function, as demonstrated by site-directed mutagenesis
(159). SCO1 was originally identified as a multicopy suppressor of a cox17-null mutant (70) and has been shown to directly
interact with Cox2p (122). In addition, because the CuA center
is formed by a Cu(I) ion and a Cu(II) ion, it remains to be
elucidated whether Sco1p mediates the transfer of both different valent ions or, alternatively, whether two Cu(I) are inserted
in Cox2p by Sco1p and the active site is successively oxidized.
It was demonstrated that Sco1p is able to bind both Cu(I) and
Cu(II), although it is not clear whether Sco1p receives both
cations or only the monovalent copper Cu(I) from Cox17p
(94). Although the experimental data could support a role for
Sco1p in copper insertion, considering its structural similarity
with the protein family of disulfide reductases, it has been
considered that it is more likely to be involved in the reduction
of cysteines in the Cox2p copper binding site. This reduction is
necessary for the cofactor incorporation (1, 219).
Heme a is a unique heme compound present exclusively in
COX. It differs from protoheme (heme b) because it has a
farnesyl instead of a vinyl group at carbon C2 and a formyl
instead of a methyl group at carbon C8 (44). The first step of
the heme A biosynthetic pathway is the conversion of heme b
in heme O catalyzed by the farnesyl-transferase Cox10p (200).
The subsequent oxidation of heme O to heme A occurs in two
discrete monooxygenase steps. The first consists on a monooxygenase-catalyzed hydroxylation of the methyl group at carbon
position 8, resulting in an alcohol that would then be further
oxidized to the aldehyde by a dehydrogenase. The first step is
catalyzed by Cox15p, in concert with ferredoxin (Yah1p) and
the putative ferredoxin reductase Arh1p, which are probably
necessary for the electron supply to the oxygenase Cox15p (16,
18, 28). The identity of the putative gene product involved in
the oxidation of the alcohol resulting from the Cox15p action
to the corresponding aldehyde to yield heme A remains un-
C1139
Invited Review
C1140
REGULATION OF COX ASSEMBLY
precipitation experiments (141). These series of contacts
among factors involved in transcription and translation of
mtDNA-encoded COX genes ensure an unbroken flux of information in which mRNAs transcribed from the mitochondrial
genome are processed and delivered to the mitochondrial
translational machinery. As described above, physical associations between the COX-specific translational activators
Pet309p, Pet122p, Pet54p, Pet111p, and Pet494p also occur. In
this way, the colocalization of mtDNA-encoded COX subunit
translation facilitates the coordinated assembly of the core of
the enzyme (141). The evolutionary conservation of a complex
system in which several specific translational factors are organized together, instead of a single specific activator, probably
results from the necessity to avoid competition between mRNAs species and to maintain a homogeneous rate of synthesis
(141).
Cox2p is synthesized as a precursor and requires further
maturation before assembly into the functional complex. After
synthesis and before the cleavage of its NH2-terminal by a
peptidase complex present in the inner mitochondrial membrane (IMP-complex), the precursor form of Cox2p is exported
across the inner membrane by the Oxa1p machinery (80, 84)
and the product of COX18 (182). Oxa1p is a member of a
conserved protein family that mediates the export of NH2terminal domains of membrane proteins, such as Cox2p, across
the inner membrane to the intermembrane space of mitochondria. The function of Oxa1p is not limited to Cox2p insertion
but extends to the insertion of other proteins such as the
mitochondrially encoded Cox1p and Cox3p proteins (84, 85).
The product of COX18 has been associated with the export of
the C-tail of yeast Cox2p, although a direct interaction between
Cox2p and Cox18p has not yet been shown (168). Once
exported, pCox2p is presented to the IMP-complex by Cox20p
that chaperones pCox2p through the proteolytic process and
maybe also the mature Cox2p through its insertion into the
nascent COX assembly intermediates (86). The IMP-complex
is formed by three subunits of which Imp1p and Imp2p have
catalytic functions with different substrate specificities (148,
174) and Som1p stabilizes the complex (99). Imp1p is responsible for the proteolytic cleavage of pCox2p, but its function
AJP-Cell Physiol • VOL
requires the stabilizing presence of the other two subunits (99,
148).
The three mtDNA-encoded COX subunits are highly hydrophobic. To avoid their unproductive aggregation in the matrix
hydrophilic environment, the synthesis of these proteins is
strictly coupled to membrane insertion (Fig. 5). The COOHterminal domain of Oxa1p interacts with the large subunit of
the mitoribosome, promoting the contact between the translational apparatus and the membrane insertion machinery to
facilitate the insertion of the nascent hydrophobic COX subunits into the mitochondrial inner membrane right after their
synthesis (100, 187). Oxa1p cooperates with the mitochondrial
membrane protein Mba1p to recruit mitochondrial ribosomes
to the inner membrane. Yeast strains carrying null mutations in
mba1 exhibit a respiratory-deficient phenotype, similar to
oxa1-null mutants, due to the severe impairment on the membrane insertion of mtDNA-encoded proteins (85, 149). Mba1p
functions as a ribosome receptor and helps Oxa1p in the
orientation of the ribosome exit site toward the inner membrane insertion machinery. Mba1p was shown to interact with
the large ribosomal subunit, interaction that does not require
Oxa1p or the neosynthesized protein (149). However, both
ribosomal subunits remain bound to the inner membrane in the
absence of Oxa1p and Mba1p, indicating that other factors
collaborate to the tethering of ribosomes to the membrane
(149). One of these additional factors was recently identified as
Cox11p, necessary for the formation of the CuB center in
Cox1p (105). This observation is particularly interesting, taking into account that the two metal active sites of Cox1p, a
protein containing 12 transmembrane helices, are buried in a
hydrophobic pocket deeply embedded below the membrane
surface (98, 198). Considering the localization of the heme a/a3
and copper cofactors in Cox1p, it is reasonable to think that
they are cotranslationally inserted (reviewed in Refs. 43, 50).
This model is supported by the observation of a weak interaction between Cox11p and the ribosome that would facilitate the
insertion of copper concomitantly with the translation of
Cox1p. Because heme a3 forms the binuclear active site of
Cox1p with CuB, a cotranslational mechanism of heme insertion could also occur. In any case, there is no doubt that the
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Fig. 5. Coupling between mitochondrial transcription,
translation, and posttranslational events in S. cerevisiae.
Transcription and translation are coupled at the MIM
through interactions nucleated by the mitochondrial RNA
(mtRNA) polymerase. Membrane association of the transcription machinery is proposed to involve the MIM protein
Sls1p and the matrix protein Nam1p. Nam1p associates
with multiple gene-specific translational activators, including Pet309p, a specific translational activator of COX1
mRNA. Pet309p interacts with COX2 and COX3 mRNAspecific translational activator proteins on the inner surface
of the MIM (IMS) (135). The mitoribosomes interact with
the membrane protein Mba1p and with Oxa1p, which drives
the membrane insertion of the nascent polypeptides in a
cotranslationally manner (149). On the basis of the finding
that also the Cu binding protein Cox11p binds the mitoribosomes (105), a cotranslational mechanism has also been
proposed for the cofactor insertion in Cox1p. Modified from
Ref. 172.
Invited Review
REGULATION OF COX ASSEMBLY
AJP-Cell Physiol • VOL
ribosomal particles and completion of ribosome assembly in
close proximity to the inner membrane (147). The dual role of
the m-AAA protease in protein quality control and ribosome
assembly in mitochondria suggest the possibility that mitochondrial translation is regulated by a negative-feedback loop
(147). Accumulating nonassembled or altered proteins, the
substrates of the m-AAA protease may compete with MrpL32
for binding to the m-AAA protease and thereby impair MrpL32
processing and mitochondrial protein synthesis (147). In this
way, mitochondrial protein synthesis, quality-control, and ultimately MRC assembly, including COX biogenesis, are tightly
coordinated.
RETROGRADE REGULATION: COMMUNICATION FROM
MITOCHONDRIA TO NUCLEUS
COX is the final product to the coordinate expression of the
nuclear and mitochondrial genomes. To achieve this coordination a bidirectional communication is necessary between the
two genomes: an anterograde signaling from nucleus to mitochondria and a communication in the opposite direction, called
retrograde regulation. The retrograde regulation acts as a sensor of mitochondrial functionality. In fact, it consists on the
transfer of information from mitochondria to the nucleus in
response to changes in the functional state of the organelle,
which allows the cells to appropriately adjust their metabolism
(35). For example, the drastic reduction of the expression of
nuclear COX genes in mutants lacking fragments or all of
mtDNA strongly argues in favor of a retrograde regulation of
these genes (56). The description of the current knowledge
regarding retrograde regulation is beyond the purpose of this
paper but extensive reviews on the subject can be found
elsewhere (35). Briefly, several factors that mediate retrograde
regulation have been characterized in yeast, including three
positive regulators, the RTG1, RTG2 and RTG3 genes (101,
118), and four negative regulators (120, 121, 189). The RTG1
and RTG3 products are transcription factors that bind to an
upstream activation sequence called R box of target genes
while the role of the RTG2 product is unknown although it is
necessary for the function of Rtg1p/Rtg3p (reviewed in Ref.
35). Because not all the genes encoding for mitochondrial
proteins are regulated by the RTG genes it has been suggested
that other factors are probably involved (63).
COX AND THE ORGANIZATION OF THE MRC
The assembly of monomeric COX is just the first step
toward the building of a maximally efficient functional enzyme. It is known that COX acts as a dimer to maximize its
efficiency (158). In addition, its arrangement with respect to
other complex and mobile electron carriers forming the respiratory chain (MRC) at a higher organizational level probably
also influences the efficiency at which electrons transfer from
one to the other finally reaching the pocket in the COX
complex where oxygen is bound and sequentially reduced to
water.
In general, two alternative models for the arrangement of the
MRC complexes in the membrane have been proposed. Chance
and Williams (47) proposed in 1955 their “solid-state” model
in which the MRC was acting as a single unit and the substrate
is channeled directly from one enzyme to the next in the MRC.
This concept changed gradually because the enzymes of the
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insertion of heme a3 into Cox1p takes place before the addition
of subunit 2, because the farnesyl group of heme a3 is packed
between subunits 1 and 2 (43). The factors involved in the
insertion of heme a/a3 into Cox1p have not yet been characterized in eukaryotes. Heme A has been shown to auto-insert
in a four helix bundle protein mimicking the hydrophobic
pocket of Cox1p (69). However, recent studies performed in R.
sphaeroides have shown that the homologue of yeast Shy1p is
necessary for the insertion of heme a3 and stability of the
binuclear center (180).
The CuA active site is located above the inner membrane
surface, in a domain of Cox2p folded in a ␤-barrel and
protruding into the mitochondrial intermembrane space. The
Sco1p-mediated insertion of copper into Cox2p probably takes
place after the extrusion of the Cox2p copper binding domain
in the intermembrane space and before the addition of this
subunit to a COX assembly intermediate (50). However, the
cleavage of the precursor form of Cox2p by Imp1/2p is not
affected by copper insertion (86).
Another interesting question is how the nuclear-encoded
structural subunits and assembly factors find their way to the
intramitochondrial site of COX assembly. The localization of
the nuclear-encoded COX subunits and COX assembly factors,
such as Cox10p, Cox11p, Cox15p, Cox19p, and Cox23p, is
facilitated by the fact that they are synthesized by cytoplasmatic ribosomes tightly associated to the mitochondrial outer
membrane (126, 127). Because COX assembly takes place at
discrete foci on the matrix face of the mitochondrial inner
membrane, it is possible to imagine that these sites are located
close to the nucleoids that anchor the mtDNA to the membrane
and close to the TOM/TIM import translocation contacts to
allow for the coordination between import of the cytosolic
subunits and biogenesis of the core subunits.
Finally, unassembled and misfolded membrane protein subunits, which could have deleterious effects if accumulated in
the mitochondrial inner membrane, are degraded by energydependent proteases, which include the conserved AAA ATPdependent proteases (7, 49, 114). Their function has been
extensively studied in S. cerevisiae, where pleiotropic phenotypes have been observed in strains carrying inactive variants
of these enzymes (7, 49, 114). In general, the AAA proteases
combine proteolytic and chaperone-like activities, forming a
membrane-integrated quality-control system. Cells lacking an
AAA protease loose COX activity and respiratory competence
(186, 203). Two AAA proteases are present in the inner
membrane of mitochondria, the i-AAA and m-AAA proteases
(147). They both are membrane bound and formed by homologous subunits with ATP-dependent metallopeptidase activity
but are exposed to opposite membrane surfaces. The i-AAA
protease is composed of Yme1 subunits and is active on the
intermembrane side (117). The m-AAA proteases constitute
hetero-oligomeric complexes that are composed of Yta10
(Afg3) and Yta12 (Rca1) subunits in yeast and expose their
catalytic sites to the matrix.(6). Strains carrying mutations in
the i- and m-AAA protease subunits are COX and respiratory
deficient (5, 38, 190). Proteolysis by AAA proteases is modulated by another membrane-protein complex that is composed
of prohibitins in eukaryotic cells (142, 183). Recently, a second
important role for the mAAA protease has been described. The
mitochondrial ribosomal protein MrpL32 is processed by the
m-AAA protease, allowing its association with preassembled
C1141
Invited Review
C1142
REGULATION OF COX ASSEMBLY
AJP-Cell Physiol • VOL
complexes because, even when experimentally removed, it
does not affect the stability of supercomplexes (172). Supercomplexes have been identified in P. denitrificans (184), in
which only homologues of the mitochondrial-encoded subunits
exist, suggesting that these subunits could play a key role in the
assembly of supercomplexes (170). On the other hand, studies
performed in yeast cardiolipin mutants have shown that this
mitochondrial membrane phospholipid plays an essential role
in the assembly of bc1 and COX complexes (222, 223). Studies
performed in yeast have shown that cardiolipin does not seem
to be essential for the formation of MRC supercomplexes, but
it is important for the stability of supercomplexes containing
functional COX as well as the individual complex (154). In
addition, cardiolipin is required to prevent formation of the
resting state of COX in the membrane (154). Finally, it has
been recently shown that cardiolipin biosynthesis and mitochondrial MRC function are interdependent. Cardiolipin biosynthesis is regulated at the level of cardiolipin synthase
activity by the pH component change of the proton-motive
force generated by the functional electron transport chain (72).
Supercomplex assembly and its regulation are other examples of the large number of players in the network of
pathways that leads to the biogenesis of functionally efficient mitochondrial COX.
In conclusion, most of the ATP used for the different
processes performed by eukaryotic cells are derived from the
metabolic pathways housed in mitochondria. The function of
COX, the terminal enzyme of the MRC, is essential for the
regulation of ATP production within mitochondria. Alterations
in COX assembly have a great impact in cell performance and
survival and, although not reviewed here, also in human health.
A better understanding of COX biogenesis is essential for
elucidating the molecular basis of aerobic energy production in
normal and disease conditions. Although there is a large body
of information concerning the biogenesis of COX, our knowledge about how the specific pathways involved and the process
as a whole are regulated is still fragmentary. The list of genes
involved in COX assembly in yeast is probably close to
completion, but their products are still mostly uncharacterized.
Many laboratories are making a continuous effort to understand the specific function of the many factors involved and to
discover the remaining ones. This effort must combine classical biochemistry and molecular biology approaches with genome-wide analyses to uncover the effect of altering specific
pathways on the whole process of COX assembly. In this
regard, a systems biology approach can be foreseen as the key
to obtain such knowledge.
ACKNOWLEDGMENTS
We thank Karine Gouget for critically reading the manuscript.
GRANTS
This study was supported by National Institute of General Medical Sciences
Grant GM-071775A and a research grant from the Muscular Dystrophy
Association (both to A. Barrientos).
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