The elusive importance of being a mitochondrial Ca2+ uniporter

Abstract

The molecular components of the mitochondrial Ca2+ uptake machinery have been only recently identified. In the last months, in addition to the pore forming subunit and of one regulatory protein (named MCU and MICU1 respectively) other four components of this complex have been described. In addition, a MCU KO mouse model has been generated and a genetic human disease due to missense mutation of MICU1 has been discovered. In this contribution, we will first summarize the recent findings, discussing the roles of the different subunits of the mitochondrial Ca2+ uptake complex, pointing to the current contradictions in the published data, as well as possible explanations. Finally we will speculate on the recent, totally unexpected, results obtained in the MCU knock-out (KO) mice.

Full text

Accepted Manuscript
Title: The elusive importance of being a mitochondrial Ca2+
uniporter
Author: Diana Pendin Elisa Greotti Tullio Pozzan
PII: S0143-4160(14)00030-X
DOI: http://dx.doi.org/doi:10.1016/j.ceca.2014.02.008
Reference: YCECA 1542
To appear in: Cell Calcium
Received date: 6-2-2014
Accepted date: 7-2-2014
Please cite this article as: Diana PendinElisa GreottiTullio Pozzan The
elusive importance of being a mitochondrial Ca2+uniporter (2014),
http://dx.doi.org/10.1016/j.ceca.2014.02.008
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Accepted Manuscript
The elusive importance of being a mitochondrial Ca2+ uniporter
Diana Pendin1,2, Elisa Greotti1,2 and Tullio Pozzan1,2,3
1Inst. of Neuroscience, National Ressearch Council, Padova Italy
2Dept. Biomedical Sciences, University of Padova, Italy
3Venetian Institute of Molecular Medicine, Padova, Italy
Correspondence to:
Tullio Pozzan, Dept. Biomedical Sciences, Via G. Colombo 3, 35121, Padova, Italy
Email: tullio.pozzan@unipd.it
Abstract
The molecular components of the mitochondrial Ca2+ uptake machinery have been only recently
identified. In the last months, in addition to the pore forming subunit and of one regulatory protein
(named MCU and MICU1 respectively) other four components of this complex have been
described. In addition, a MCU KO mouse model has been generated and a genetic human disease
due to missense mutation of MICU1 has been discovered. In this contribution, we will first
summarize the recent findings, discussing the roles of the different subunits of the mitochondrial
Ca2+ uptake complex, pointing to the current contradictions in the published data, as well as
possible explanations. Finally we will speculate on the recent, totally unexpected, results obtained
in the MCU knock-out (KO) mice.
Mitochondria are organelles that form an elaborate network of tubules and single vesicles within the
cytoplasm that are in continuous dynamic equilibrium due to a highly controlled process of fission
and fusion among themselves. Several aspects of mitochondrial functions, from enzyme activity to
electron flow in the respiratory chain, from organelle movement to the release of pro-apoptotic
factors are controlled by cytosolic or matrix Ca2+ [1-5]. Ca2+ homeostasis in mitochondria is
controlled by three processes that regulate the Ca2+ content within the organelle: i) the
mitochondrial Ca2+ uniporter complex (MCUC) for uptake; ii) Ca2+ buffering in the mitochondrial
matrix, due to poorly characterized matrix protein and by the generation within the matrix of
insoluble xCa2+–xPO4x–xOH complexes [6];iii) Ca2+ extrusion, mediated by a xNa+/Ca2+
exchanger (mNCX) and a Ca2+ /H+ exchanger (mHCX)[3-4, 7]. Over the last two years, the proteins
involved in the Ca2+ uptake and release processes have been finally identified and the attention of
the researchers is now focused on unravelling their dynamic interactions and functional regulation

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(for a recent review on the topic see [8]).
Over 20 years ago, in a short paper aimed at characterizing the kinetic characteristics of
mitochondrial Ca2+ uptake, one of us proposed that the mitochondrial Ca2+ uniporter, MCU, was
most likely a gated channel [9] rather than a classical carrier, at that time the prevalent model. The
molecular identity of MCU (and, accordingly, its channel or carrier nature) remained a mystery
until recently, however. In 2004, Clapham and co-workers, based on electrophysiological
measurements on mitoplasts, confirmed the original intuition that MCU is a Ca2+ specific ion
channel [10]. In 2011, the molecular nature of this long searched protein was finally unravelled
simultaneously by two independent groups [11-12]. In less than 2 years the general picture has
become far more complex than anticipated from functional data: in addition to the pore-forming
subunits, named MCU, other proteins have been described that are capable of modulating the
activity, the Ca2+ affinity and (probably) the assembly of the channel in the mitochondrial
membrane. A protein structurally similar to MCU, named MCUb, has been discovered, that has an
inhibitory role on Ca2+ uptake by the organelles or on Ca2+ current measured in lipid bilayers using
recombinantly expressed MCU [13]. MICU1, identified about one year before MCU itself [14], was
more recently followed by another similar protein, MICU2 [15]. Both proteins are located on the
outer surface of the inner mitochondrial membrane where they appear to modulate the function of
MCU (see below). MCUR1 is another recently discovered member of the complex, whose deletion
results in abrogation of mitochondrial Ca2+ uptake [16]. The last identified member of
MCU-associated proteins is named EMRE, and its knock out completely blocks Ca2+ uptake by
mitochondria [17]. The exact stoichiometry of the different subunits forming MCUC and their
specific role is still debated (see below), but the present picture shows the mitochondrial Ca2+
uptake channel as one of the most, if not the most, sophisticated ion channel described thus far.
Such complexity in subunit composition and regulation, maintained and increased during evolution,
suggests an essential role of this mitochondrial function in cell physiology; indeed mitochondrial
Ca2+ uptake has been implicated in the regulation of oxidative phosphorylation, modulation of local
or general Ca2+ homeostasis, oxygen radical production and last, but not least, in programmed cell
death (for reviews see[4, 18-20]). In light of this, the recent paper by Finkel’s group [21]
demonstrating that MCU KO mice are not only born normally but that their phenotype is hardly
different from that of wild type animals was a major surprise for the majority of experts in the field;
we will broach this aspect in more detail below.
The molecular nature of the mitochondrial Ca2+ uniporter complex, MCUC

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The search for the molecular component(s) responsible for mitochondrial Ca2+ uptake in energized
mitochondria started about 50 years ago and, until recently, produced only a long list of frustrating
failures. The keys for identifying such an elusive protein complex have been, on the one hand, the
novel screening techniques available (genome wide siRNA screening in particular) and, on the
other, the availability of the complete genome sequences of several eukaryotes that allowed the
identification of the phylogenetic development of the mitochondrial Ca2+ uptake properties.
Essential in the discovery of the first members of the mitochondrial Ca2+ uptake complex was a
rather old, and for a long time neglected, observation: the absence of energy-dependent
mitochondrial Ca2+ uptake in budding yeast [22].
Based on half a century of experiments and general consensus, the mitochondrial Ca2+
uniporter was predicted: i) to be expressed in practically all eukaryotes, but missing in budding
yeast; ii) to be an integral mitochondrial protein of the inner membrane; iii) to possess the
transmembrane domains necessary to form an ion channel; iv) its downregulation or overexpression
should inhibit or increase mitochondrial Ca2+ uptake respectively, without interfering with the
bioenergetic characteristics of the organelle (membrane potential in particular). The first two
proteins identified by using this type of rationale and suggested to play a role in Ca2+ uptake by
mitochondria were UCP2 and UCP3 [23]. These proteins belong to a family of H+
channels/transporters (their prototype is UCP1, i.e., the protein responsible for the physiological
uncoupling of brown adipose tissue mitochondria [24] (reviewed in [25]). Neither UCP2 nor UCP3
are expressed in budding yeasts and Trenker et al. found that, when they were downregulated by
specific RNAi, a substantial decrease in mitochondrial Ca2+ uptake occurred; on the contrary,
overexpression of UCP2 and 3 increased the efficacy of mitochondrial Ca2+ accumulation [23].
Neither of the two proteins, when expressed in budding yeast, however, conferred to their
mitochondria the capacity of accumulating Ca2+ in the matrix [23], leading the authors to suggest
that UCP2 and 3 are modulators of MCU and not MCU itself. A large set of experimental data,
however, cast doubts on a direct involvement of these proteins in mitochondrial Ca2+ uptake [26]
and their role in this process remains undefined.
Two years later, using genome-wide siRNA screening, Clapham’s group reached the
conclusion that the protein Letm1 is responsible for a novel Ca2+ uptake mechanism in
mitochondria [27]; in particular, they suggested that Letm1 is an electrogenic Ca2+/H+ antiport,
distinct from MCU. Downregulation of Letm1 resulted in inhibition of Ca2+ uptake, but its
overexpression hardly affected the process [27]; most relevant, and in clear contrast with the
rationale mentioned above, an homologue of Letm1 is present in yeast, named Yol027/Mdm38, and
its KO phenotype can be rescued by expression of mammalian Letm1 [28]. Their discoverers

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maintain that Letm1 is a Ca2+/H+ antiport that insures energy dependent mitochondrial Ca2+ uptake
at low cytosolic Ca2+ concentrations [29-30], though in their most recent contribution they conclude
that the exchange is electroneutral (Ca2+/2H+) and insensitive to Ruthenium Red (and accordingly it
should catalyse Ca2+ efflux and not influx). Other groups argued in favour of an indirect role of
Letm1 on Ca2+ uptake, due to its function as a K+/H+ antiport [31-33]. The debate and different
interpretation of the experimental data is not over yet, and this problem will not be further discussed
here. The interested reader could refer to some recent reviews for additional information on this
topic [34]
MCU and MICU1
A more convincing candidate component of MCUC was presented by Mootha’s group with the
functional characterization of MICU1, a protein of the Mitocarta [35] previously named CBARA1
or EFHA3: in their hands, KO of MICU1 abolished mitochondrial Ca2+ uptake, though
overexpression did not result in a major effect on the rate and extent of Ca2+ uptake by the organelle
[14]. MICU1, a 54-kDa membrane protein, has most, though not all, the other characteristics
mentioned above for being directly involved in mitochondrial Ca2+ uptake, if not MCU itself: i) it is
expressed in all eukaryotes investigated, but not in budding yeasts; ii) it is an intrinsic inner
mitochondrial membrane protein; iii) it possesses two classical EF-hand Ca2+ binding domains [14].
Mootha and coworkers argued that it was unlikely that MICU1 was the channel-forming component
of MCUC, as MICU1 is predicted to possess only one very short membrane-spanning domain,
unlike any other channel protein described thus far. Indeed, in 2012, Mootha’s and Rizzuto’s groups
described another protein, now named MCU, that had all the characteristics to be the long-searched
channel-forming uniporter subunit: it is not expressed in budding yeast, its downregulation inhibits
and overexpression increases Ca2+ uptake by mitochondria, it is located in the inner membrane and
possesses two predicted transmembrane -helixes domains. The nuclear gene encodes a 40-kDa
protein, while the mature form of MCU (after cleavage of the N-terminal import domain) is a
35-kDa protein, devoid of any recognizable Ca2+ binding motif, whose topology has been a matter
of discussion for some time [11-12]: the prevalent model today predicts that both its N- and C-
terminal domains face the mitochondrial matrix, with the two membrane-spanning domains
connected in the intermembrane space by a short loop containing the DIME motif, the most
conserved region among MCU orthologs [11-12]. Recently, employing structural bioinformatics
techniques and molecular dynamics, it has been shown that the putative pore region of MCU is
defined by eight helices and the negative electrostatic potential needed for cation permeability is
due to a cluster of negatively charged aminoacids found in the DIME region, in proximity of the

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pore [13]. Importantly, De Stefani et al. showed that when expressed recombinantly in E. coli or in
a wheat germ extract cell-free system, purified MCU forms channels permeable to Ca2+ and
inhibited by Ruthenium Red, the well-known blocker of mitochondrial Ca2+ uptake in isolated
organelles. Moreover, they showed that a point mutation in the predicted pore forming domain of
MCU abolishes the current of wt MCU in lipid bilayers and behaves as dominant negative when
transfected in living cells [11].
It has been argued that the kinetic characteristics of the reconstituted channel by purified
MCU are substantially different from those of the Ca2+ current in mitoplasts (IMiCa) [10]. This
observation is not surprising, as MCU in situ is associated with other regulatory subunits (see
below). Moreover, recent optimization of the MCU expression procedures yielded MCU activity in
bilayers with kinetics more similar to those of IMiCa [13]. These results have been confirmed
recently and extended by other groups [36]. To accommodate these findings, a first model was
proposed where MCU, arranged in oligomers, possibly tetramers [13], is postulated to be the
channel-forming subunits of MCUC, a supercomplex with a molecular weight around 480 kDa [12].
MCU and MICU1 physically interact and have a similar tissue expression pattern [12]. They
are both highly conserved during evolution: indeed, Mootha and co-workers analysed the
distribution of both proteins across 138 fully sequenced eukaryotic organisms and they found
homologs of MCU distributed widely across all major branches of eukaryotes, in nearly all
metazoan and plants and in some protozoa. A MCU homolog is also present in many fungi,
although with significant differences in the domain structure; moreover, fungi mostly lack a MICU1
homolog. A putative MCU homolog has been also found in three diverse bacterial species from the
Bacteroidetes/Chlorobi group. Functional studies are required to determine whether bacterial
homologs are also able to form channels. In that case they would be among the first identified
prokaryotic calcium channels [37].
While there is general agreement on the fact that MCU represents the pore forming subunit
of MCUC, the role of MICU1 is, on the contrary, less clear and contradictory data have been
published by different groups. In the original paper on MICU1, Perocchi et al. showed that MICU1
downregulation results in a potent inhibition of Ca2+ uptake in both live and
digitonin-permeabilized HeLa cells [14]. These data have been confirmed by Abell et al. in
Drosophila S2 cells [38]. On the contrary, Mallilankaraman and colleagues have shown that KO of
MICU1 leads to an increased mitochondrial Ca2+ accumulation at rest, suggesting a role for MICU1
in establishing a threshold that prevents Ca2+ uptake in basal conditions; they found, however, that
it has a marginal effect on the rate and extent of organelle Ca2+ accumulation upon large cytosolic
Ca2+ rises [39]. Csordas et al., on the one hand, confirmed the suggestion of Mallilankaraman et al.

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that MICU1 somehow reduces the Ca2+ affinity of the MCUC at low Ca2+ concentration, but also
found that its downregulation strongly inhibits mitochondrial Ca2+ uptake in response to
agonist-induced Ca2+ rises [40]. Very recently, Alvarez and coworkers showed that in HeLa cells
silenced for MICU1, the mitochondrial Ca2+ uptake is increased at cytosolic Ca2+ concentration < 2
uM, while it is decreased at cytosolic Ca2+ concentration > 4 uM [41]. Moreover, they observed that
at low cytosolic Ca2+, after a prolonged MCU opening, an inactivation mechanism occurs in
MICU1 knocked down HeLa cells, independently from ROS production and phosphorylation
processes [41]. Finally, in a recent paper, Hoffman et al. confirmed that at low Ca2+ concentrations
MICU1 exerts an inhibitory effect on MCU-dependent Ca2+ uptake, showing that: i) in HeLa cells
expressing a MICU1 variant (lacking the ability to bind MCU or mutated in EF-hand domains, thus
acting as a dominant negative), mitochondria are more effective at buffering cytosolic Ca2+
increases than their wt counterparts; ii) mitoplasts from HeLa cells expressing MICU1 lacking the
ability to bind MCU or mutated in EF-hand domains, exhibit larger MCU currents (IMCU) compared
to wt; the same result is obtained in HeLa cells with a stable knockdown of MICU1 [42]. Hoffman
et al. also suggested that MICU1 could have pathogenic role in some forms of cardiovascular
diseases (CVD): in particular in endothelial cells from CVD patients, they showed that MICU1
levels are drastically reduced and that the consequent mitochondrial Ca2+ phenotype can be reversed
by the reintroduction of MICU1 [42].
The reasons for such experimental discrepancies are not entirely clear yet and our biased
opinion is that they may depend in part on differences in the experimental conditions (buffer
composition and probes for Ca2+ measurement, cell model employed, etc.) and, possibly more
interesting, in a larger complexity of the control of MCUC functions, for example by protein
paralogs of MICU1 (see below).
No consensus has been reached yet also on the topology of MICU1 in the inner membrane.
Perocchi et al. suggested that the largest part of MICU1 faces the intermembrane space [14];
Csordas et al. [40] confirmed that the two EF-hands of MICU1 are localized on the outer surface of
the inner mitochondrial membrane, and thus are ideally located to sense the changes in cytosolic
Ca2+ concentration (the outer mitochondrial membrane being freely permeable to Ca2+). This
topology is thus consistent with the models in which MICU1 senses cytosolic Ca2+ and controls the
Ca2+ affinity of the MCUC. Hoffman et al., on the contrary, concluded that MICU1 is
compartmentalized primarily in the matrix side of the IMM. Moreover, they also identified the
interaction domains with MCU, showing that the N-terminal polybasic domain of MICU1 interacts
with the two coiled-coil domains of MCU and it is necessary, together with the two EF-hand
domains, to exert the MICU1 effects on MCU [42]. It is our biased opinion that the model of

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Perocchi et al., Csordas et al. and Patron et al. [43] (see below) is better consistent with the
available experimental data.
Futher complexity in the regulation of mitochondrial Ca2+ uptake by MICUs and MCUs paralogs
Our understanding of the complexity of MCUC regulation increased enormously in the last year.
Mootha’s group revealed the expression in most eukaryotic cells of two paralogs of MICU1,
MICU2 (EFHA1) and MICU3 (EFHA2) [15]. The authors suggested that these other two genes
arose by gene duplication of MICU1, but they exhibit a distinct pattern of tissue expression. MICU2
and 3 share about 25% sequence identity with MICU1 and, similarly to it, possess two EF-hands
Ca2+ binding sites. MICU2 is a 45-kDa protein ubiquitously expressed in mammalian tissues,
exclusively localized in the inner mitochondrial membrane. MICU3 expression seems to be mostly
expressed in the nervous system and up to now it has not been functionally characterized. On the
contrary, an extensive characterization of the role of MICU2 in the process of organelle Ca2+
accumulation and in the formation of the MCUC has been carried out by Mootha and coworkers
[15]. In particular, they showed that in vivo silencing of MICU1 and MICU2 caused an important
reduction in mitochondrial Ca2+ uptake in response to large Ca2+ pulses, at least in part due to a
decreased MCU expression. They proposed a reciprocal regulation of MICU1 and 2, that appears to
be cell specific, as: in HEK293T cells MICU1 siRNA reduces MICU2 protein expression level
(although mRNA levels remain unchanged), but not vice versa; in the mouse liver loss of MICU2
results in decreased expression of MICU1, while MCU protein levels are reduced by silencing
MICU1, MICU2 and the combination of the two; in HeLa cells, siRNA against one of the MICUs
results in the downregulation of the other isoform but it does not affect MCU levels. Moreover, they
demonstrated that MCU overexpression increases both the levels of MICU1 and 2, and MICU1
overexpression increases the levels of MICU2. Finally, they demonstrate that MICU1 and 2 co-
precipitate in a complex with MCU [15].
Recently, Rizzuto and coworkers demonstrated that: (i) MICU1 and MICU2 are localized in
the intermembrane space; (ii) MICU1 and MICU2 heterodimerize in a 95 kDa dimer through the
formation of a disulphide bond (between MICU1-Cys465 and MICU2-Cys410); (iii) MICU1 and
MICU2 interact with MCU through the short DIME loop of MCU that faces the intermembrane
space; (iv) MICU2 exerts an inhibitory effect on MCU channel activity in planar lipid bilayers and
in intact HeLa cells; (v) MICU1 shows a stimulatory effect on MCU activity in both
electrophysiological analyses of purified MCU inserted in planar lipid bilayers and in agonist-
challenged intact cells; (vi) the protein levels of MICU1 and 2 (but not the mRNA) are strongly

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correlated: the decrease of MICU1 protein expression decreases also the level of MICU2 and results
in the absence of gatekeeping on MCU [43].
The model proposed by Rizzuto’s group can be thus summarized: MCU activity needs a
very fine tuning, necessary to prevent an excess of its activity at resting Ca2+ concentration, to avoid
massive energy dissipation and possibly the triggering of cell death; an efficient activation
mechanism is also necessary to permit fast Ca2+ uptake when increases in cytosolic Ca2+ are
triggered by cell stimulation. These two essential control mechanisms are provided, respectively, by
MICU2, which exerts its inhibitory activity at low Ca2+ level, and by MICU1, which plays its
activating role during cytosolic Ca2+ rises. The authors also showed that MCU can
immunoprecipitated only with the dimeric form of MICU1 and 2.
It is not easy to rationalize all these data, that appear in some cases clearly contradictory. A
possible unifying model that explains most, but perhaps not all, the experimental findings described
above is the following: MCUC is composed of at least 3 proteins that interact with each other:
MCU (that forms the channel), MICU1 and 2 (that regulate the Ca2+ affinity of the channels,
MICU2 reducing and MICU1 increasing the activity). The level of expression of these three
proteins is under reciprocal control, that may vary in different cell types, though. Accordingly, in
some model systems, downregulation of MICU1 or 2 results not only in a decrease in the other
paralog, but also of MCU, resulting in a net inhibition of Ca2+ uptake; under different conditions or
cell type, downregulation of MICU1 results also in a reduction of MICU2 protein levels, but not of
MCU, leading to a paradoxical activation of Ca2+ uptake at rest (and possibly its inhibition at higher
Ca2+ levels). In conclusion, the precise roles of MICU1 and 2 remains in part undetermined, with
very complex roles on the affinity of the overall mechanism of Ca2+ uptake, composition of the
MCUC, and regulation at the gene expression level and protein stability.
Not only does MICU1 have paralogs, but a similar situation appears to be true for MCU; in
particular, Raffaello et al. have recently demonstrated the existence of an isoform of MCU
(CCDC109B), named MCUb, whose primary sequence is 50% homologous to that of MCU, but
with key mutations in the predicted pore forming region [13]. MCUb is a 35-kDa protein with two
transmembrane domains, encoded by a gene located on Homo sapiens chromosome 4. Rather than
an isoform of MCU, MCUb appears to be a subunit of the complex with inhibitory characteristics:
its overexpression in intact cells reduces Ca2+ uptake of mitochondria in response to cytosolic Ca2+
increases, while in lipid bilayers the coexpression of MCU and MCUb dramatically inhibits the
Ca2+ current due to MCU alone [13]. Similarly to MICU2, also the expression of MCUb is quite
variable in mouse tissues, and in general lower than that of MCU. Concerning mRNA levels,
MCUb transcript is abundant in heart and lung, while it is scarce in skeletal muscle, and it is in

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general lower than MCU. As a consequence, the ratio MCU/MCUb varies in different tissues and
this variability seems not correlated to MCU expression levels. These data are also confirmed in
terms of Ca2+ uptake efficacy, that is greater in skeletal muscle than in heart [44]. Structurally, the
main differences between MCU and MCUb concern the DIME domain. In particular an E256V
substitution makes MCUb less electronegative than MCU and that could affect the kinetics of Ca2+
permeability. Moreover, substitution in MCU of the two aminoacids R251 and D256 of the putative
pore-forming domain with the corresponding aminoacids of MCUb (W and V, respectively) results
in the inhibition of the Ca2+ channel activity of MCU, both when expressed in living cells or in
reconstituted lipid bilayer experiments [13]. The existence of an endogenous negative modulator of
MCU, however, offers a potentially unprecedented mechanism for regulating the activity of an ion
channel through the expression of a physiological dominant negative subunit. MCUb can be co-
immunoprecipitated with MCU [17]; this information, together with the data about the physical
interaction between MCU and MICU1 and 2 suggests the existence of an heteromeric complex
containing all these proteins, though their relative stoichiometry is presently unknown.
More subunits of the MCUC
If all that has been discussed above were not complex enough, in the last few months two other
proteins have been discovered that appear to be strongly associated with the Ca2+ transporting
complex made by MICUs and MCUs, i.e., MCUR1 [16] and EMRE [17]. MCUR1 (Mitochondrial
Ca2+ Unipoter Regulator 1, also named CCDC90A) was identified by using siRNA screening in
HEK293T. It is a 40-kDa protein with two transmembrane domains and one coiled-coil region. The
N- and C-terminus face the intermembrane space, while the major part of the protein is exposed to
the matrix (Rhee et al., 2013). The striking phenotype of cells deprived of MCUR1 is the lack of
any energy dependent Ca2+ uptake, without effects on mitochondrial membrane potential. Cells
deprived of MCUR1 present several bioenergetics defects typical of a block of mitochondrial Ca2+
uptake, such as increase autophagy and reduced sensitivity to apoptosis [16]. The authors showed
that MCUR1 coprecipitates with MCU, but not with MICU1, and that its overexpression results in
an increase of mitochondrial Ca2+ uptake, but only when MCU is expressed. MCUR1 appears also
to regulate the expression of MCU, as downregulation of MCUR1 results in a significant increase
of both the mRNA and the protein levels of MCU. Although MCUR1 possesses two predicted
transmembrane domains, no evidence was obtained in support of its channel forming activity,
suggesting that MCUR1 affects either the opening of MCU or the assembly of the complex or both.
Most relevant, however, an MCUR1 analogue is expressed in budding yeast, named FMP32 [45],
unlike what is predicted for proteins specifically involved in the regulation of Ca2+ uptake by

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mitochondria. Accordingly, one would be tempted to speculate that MCUR1 is not a specific
regulator of MCUC, but has additional important functions (yet unknown) in the organelles that are
independent of its effects on Ca2+ uptake.
The most recently discovered member of the mitochondrial uniport complex subunits is the
protein called EMRE (Essential MCU REgulator) or C22ORF32. EMRE was identified by
quantitative mass spectrometry (SILAC) as a component of a high MW complex of inner
mitochondrial membrane isolated by blue native electrophoresis after immunoprecipitating MCU
[17]. EMRE is a 10-kDa single-pass membrane protein with a highly conserved C-terminus and
enriched in aspartate. In the isolated complex, MCUs and MICUs are found together with EMRE,
while MCUR1 was not co-immunoprecipitated with the other proteins. In particular, EMRE has
been shown to interact with MCU at the IMM and with MICU1 in the intermembrane space,
possibly acting as a bridge between MCU and MICUs. Furthermore, the authors demonstrate that
EMRE is necessary for the association of MCU with MICU1/2, since, in absence of EMRE, MCU
can still co-precipitate with MCUb, MICU1 and 2 can dimerize, while MCU cannot co-precipitate
with MICU1/2 [17]. The reason for the discrepancy in relation to the data of Mallilankaraman et
al., who could co-immunoprecipitate MCUR1 with MCU [16], and the latter data on EMRE is
presently unknown. No EMRE homologs exist in plants, fungi or protozoa suggesting that it most
likely arose in the metazoan lineage. As far as EMRE mRNA expression is concerned, it was found
in all mouse tissues and is predicted to be broadly expressed in mammalian tissues. Downregulation
of EMRE results in complete inhibition of energy dependent Ca2+ uptake in intact cells and isolated
organelles; moreover, overexpression of MCU in cells silenced for EMRE does not allow the
recovery of mitochondrial Ca2+ uptake. EMRE thus appears to be a necessary regulator, similar to
MCUR1, of the MCU transport, rather than a pore-forming unit; in addition, as EMRE is not
expressed in plants and given that MCU per se (either expressed in E. coli or synthetized in vitro
and inserted in lipid bilayers) results in Ca2+ permeable channel [11], a conservative interpretation
of these apparently contradictory data is that EMRE is involved in the assembly of the Ca2+ uptake
machinery in the inner mitochondrial membrane of mammalian cells. This is, for the time being, a
pure speculation, but the hypothesis is clearly experimentally testable.
The contributions by Mallilankaraman et al. on MCUR1 and of Sancak et al. on EMRE are
the first, and thus far the only, studies on the role of these proteins on Ca2+ uptake by mitochondria.
Thus, the impact of these observations on the overall control mechanism of the process awaits
further and more detailed studies. Despite this caveat, taken together, the data indicate that Ca2+
import in mitochondria involves an extremely complex molecular machinery, highly conserved in

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its basic features with regulatory mechanisms that were added during evolution. Together with the
enormous set of data indicating the involvement of mitochondrial Ca2+ uptake in many key aspect
of cell physiology and pathology (from Ca2+ buffering to modulation of metabolism, from control
of specialized functions to the control of apoptotic or necrotic cell death), it was almost obvious to
anticipate that functional KO of MCU would result in an embryonically lethal phenotype. Indeed, a
dramatic phenotype was obtained in a study carried out in zebrafish using morpholinos against
MCU [46].
The paradox of the MCU KO mouse
The paper by Finkel and co-workers, published a few weeks ago, came as a shocking result for most
students in the field: MCU KO mice obtained using the gene trap technique are not only regularly
born, but their phenotype is very mild: the animals are slightly smaller than their wild type
littermates and they have modest defects in skeletal muscle strength and some alterations of
metabolic functions (in particular in the control of pyruvate dehydrogenase) [21]. Thus, while in
less developed animals MCU plays a non-dispensable role, this appears not to be the case in more
complex animal species such as the mouse. The obvious explanation, i.e., the existence in mammals
of alternative pathways for Ca2+ uptake in mitochondria does not appear to be easily tenable (see
also below), based on the published data: mitochondria isolated from both skeletal and cardiac
muscles of MCU KO mice are totally incapable of accumulating Ca2+ in an energy dependent way
and in MEFs from the same transgenic mice Ca2+ mobilization from intracellular stores results in no
detectable increase in mitochondrial Ca2+ levels [21]. Surprisingly, Pan et al. found that the Ca2+
content in mitochondria of MCU KO cells is only partially reduced compared to that in wild type
animals. The authors themselves thus hypothesized the existence of alternative Ca2+ uptake
pathways, capable of catalysing only a slow Ca2+ accumulation, though [21]. The nature of this
(these) pathway(s), if any, remains unknown. On the existence of alternative mitochondrial Ca2+
uptake pathways of interest are the recent observations by Graier and co-workers. In one of these
studies they argued that, depending on the cell type and the methodology employed, up to 5
different Ca2+ currents can be identified in mitoplasts by electrophysiology (see [47]). In a more
recent study, the same group investigated, using the patch clamp technique, the inward cation
currents and single Ca2+ channel activities in mitoplasts from stable MCU knockdown HeLa cells
[48]. Reduction of MCU levels resulted in a strong reduction in the frequency of single Ca2+
channel activity in patched mitoplasts (they named this current i-MCC). However, ablation of MCU
resulted in a strong elevation of a high conductance mitochondrial Ca2+ current (that they named xl-
MCC). They suggested the existence in mitochondria of at least two different Ca2+ channels, one

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MCU dependent (and responsible for the i-MCC current) and another, MCU-independent. As much
as the existence of multiple mitochondrial Ca2+ influx pathways is fascinating (and could explain
the mild phenotype of MCU KO mice), it needs stressing that, in MCU KO animals, either in
isolated organelles or in living cells, Pan et al. did not found any evidence for energy dependent
Ca2+ uptake.
Pan et al. also showed that the cardiac infarct size (caused by 20 min of no-flow global
ischaemia) in wild type and MCU KO animals is indistinguishable, but differently sensitive to the
permeability transition pore (PTP) inhibitor Cyclosporin A (CsA): the KO animals are totally
insensitive to the drug, while the wild type are strongly protected by the same treatment [21]. The
latter experiment is of special relevance as it confirms that the effects of CsA on ischemic death of
cardiac cells is directly related to the inhibition by the drug of Ca2+ activated Permeability
Transition Pore, PTP, opening. Most relevant for what discussed here, the data indicate that the
default death pathway caused by ischemia in wild type hearts is to a large extent mediated by
Ca2+-activated PTP opening, but an alternative death pathway can be activated if mitochondrial
Ca2+ accumulation is genetically blocked. No information is yet available on whether the wt and
KO mice have the very same sensitivity to ischemic death, for example whether the extent of the
infarcted zone is affected by MCU KO using different protocols (e.g., duration of the ischemic
protocol, sensitivity to O2- radical production, etc.) or whether the death of cardiac cells in MCU
KO is primarily due to necrosis rather than apoptosis. As far as PTP and cell death are concerned, it
needs stressing that, as clearly demonstrated by Bernardi et al., the inhibitory effect of CsA on PTP
opening is not a generic effect on the activation of that channel, but rather it is due to the CsA-
dependent reduced sensitivity of PTP to Ca2+-dependent opening [49]. In other words, the lack of
CsA effect does not exclude the involvement of PTP in the death of cardiac myocytes of KO
animals (as the PTP may be opened by other mechanisms related to ischemia), but confirms that the
default pathway of ischemia-dependent cell death in the hearts of wild type animals is mediated
through MCU and Ca2+ activation of PTP. What other pathway might so efficiently compensate the
lack of MCU remains totally unexplained. Along the same line of reasoning, it is quite remarkable
that the embryonic development of all organs, requiring massive apoptosis to reach completion, is
essentially unaffected in the MCU KO mice. Plenty of evidence, obtained both in vitro and in vivo,
suggests that apoptosis is highly dependent on mitochondrial Ca2+ accumulation and PTP opening.
Again, as suggested above for ischemic cell death, one may postulate that normal embryogenesis
takes advantage of the classical, Ca2+- and PTP-dependent, pathway, while mammals have
developed alternative signals that can efficiently substitute the default mechanism, if necessary.
Alternatively, one must assume that the MCU-dependent mechanism is either irrelevant or easily

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Accepted Manuscript
dispensable for programmed cell death activation in mammalian embryos. A last, but at present
totally speculative hypothesis, would be the existence of embryo specific spliced variants of MCU
that bypass the stop codon inserted in the first intron (the strategy used to generate the KO mouse of
Finkel and coworkers). Spliced variants of MCU are indeed predicted, but whether or not they
actually result in functional proteins and, more important, whether the stop codon could be
bypassed by the splicing mechanism is presently unknown. In addition, this hypothesis is
contradicted by the total lack of fast Ca2+ uptake in MEF from MCU KO animals. Admittedly,
MEFs are cells cultured in vitro and the possibility that they loose the embryonic MCU spliced
variants, if they exist, cannot be excluded. The last surprising finding of the MCU KO mouse is that
live KO mice were obtained only in a mixed genetic background of C57BL/6 and CD1, while the
absence of mitochondrial Ca2+ uptake appears to be embryonically lethal in a pure background
(unpublished observations).
Clearly, the very mild phenotype of MCU KO mice was totally unexpected for most
investigators in the field. However, before playing a requiem for the relevance of MCU in cell
physiology and pathology of mammals, more information needs however to be obtained. Among
them: i) tissue specific and inducible KO animals must be generated and thoroughly investigated; ii)
the mystery of the genetic background difference needs to be explained; iii) the existence of
alternative Ca2+ influx pathways must be further investigated. It is however rather surprising and
evolutionary contradictory that a highly sophisticated gated channel such as the mitochondrial Ca2+
influx mechanism, with at least 6 different subunits controlling its activity and assembly (not to
mention an isoform specific mitochondrial subunit of the Na+/Ca2+ exchanger), would turn out to
be a key property of mitochondria of lower eukaryotes and almost completely dispensable in
mammals. An additional, and most important caveat, in downgrading the MCUC to a dispensable
complex in mammals comes from the recent identification of a genetic human disease, in which
mutations of MICU1 were associated with proximal myopathy, learning difficulties and a
progressive extrapyramidal movement disorder [50]. Evidence has been provided indicating that the
phenotype of the patients is caused by a primary defect in mitochondrial Ca2+ signalling, arguing
for a critical role of mitochondrial Ca2+ uptake in several human tissues.
It is worth mentioning that MCU is not the sole example in the recent literature where the
KO in mice of genes that were believed to be essential turned out to be devoid of phenotype (or
with a very mild one). Just to name a few: KO of creatine kinase [51] or interleukin-1a/b double
knock out [52] induced no obvious phenotype in mice. Strikingly, myoglobin KO mice were
created in 1998 [53] and they too showed no obvious phenotype. In the years that followed, several

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Accepted Manuscript
compensatory mechanisms that include increases in cardiac capillary density, coronary flow,
hemoglobin, changes in fatty acid metabolism and resistance to hypoxic stress [54-59] were
reported. One is tempted to conclude that, on the one hand, the compensating capabilities to the lack
of a single gene are far more efficient than we had anticipated, on the other that the functional
phenotyping presently employed are still too simplified to reveal the importance of a protein or of a
signalling pathway. It is thus easy to predict that the next few years will be full of surprising
findings in the field of mitochondrial Ca2+ homeostasis. This topic, for many years investigated
primarily with phenomenological approaches, is now based on strong molecular and genetic basis
and we are looking forward to a fascinating future.
Aknowledgments
We are grateful to Dr. Paulo Magalhaes for critically reading the manuscript. The original work of
the Authors has been supported by a grant FIRB from the Italian Ministry of University and
Research (MIUR), by the Veneto Region and by the National Research Council (CNR) to T.P.
References
[1] J.G. McCormack, R.M. Denton, The role of mitochondrial Ca2+ transport and matrix Ca2+ in
signal transduction in mammalian tissues, Biochim. Biophys. Acta, 1018 (1990) 287-291.
[2] P. Pizzo, I. Drago, R. Filadi, T. Pozzan, Mitochondrial Ca2+ homeostasis: mechanism, role,
and tissue specificities, Pflugers Archiv : European journal of physiology, 464 (2012) 3-17.
[3] I. Drago, P. Pizzo, T. Pozzan, After half a century mitochondrial calcium in- and efflux
machineries reveal themselves, EMBO J., 30 (2011) 4119-4125.
[4] R. Rizzuto, T. Pozzan, Microdomains of intracellular Ca2+: molecular determinants and
functional consequences, Physiol. Rev., 86 (2006) 369-408.
[5] L. Contreras, I. Drago, E. Zampese, T. Pozzan, Mitochondria: the calcium connection,
Biochim. Biophys. Acta, 1797 (2010) 607-618.
[6] D.G. Nicholls, Mitochondria and calcium signaling, Cell Calcium, 38 (2005) 311-317.
[7] R. Palty, I. Sekler, The mitochondrial Na(+)/Ca(2+) exchanger, Cell Calcium, 52 (2012) 9-
15.
[8] S. Marchi, P. Pinton, The mitochondrial calcium uniporter complex: molecular components,
structure and physiopathological implications, J Physiol, (2013).
[9] M. Bragadin, T. Pozzan, G.F. Azzone, Activation energies and enthalpies during Ca2+
transport in rat liver mitochondria, FEBS Lett., 104 (1979) 347-351.
[10] Y. Kirichok, G. Krapivinsky, D.E. Clapham, The mitochondrial calcium uniporter is a highly
selective ion channel, Nature, 427 (2004) 360-364.
[11] D. De Stefani, A. Raffaello, E. Teardo, I. Szabo, R. Rizzuto, A forty-kilodalton protein of the
inner membrane is the mitochondrial calcium uniporter, Nature, (2011).
[12] J.M. Baughman, F. Perocchi, H.S. Girgis, M. Plovanich, C.A. Belcher-Timme, Y. Sancak, X.R.
Bao, L. Strittmatter, O. Goldberger, R.L. Bogorad, V. Koteliansky, V.K. Mootha, Integrative
genomics identifies MCU as an essential component of the mitochondrial calcium uniporter,
Nature, 476 (2011) 341-345.

Page 15 of 18
Accepted Manuscript
[13] A. Raffaello, D. De Stefani, D. Sabbadin, E. Teardo, G. Merli, A. Picard, V. Checchetto, S.
Moro, I. Szabo, R. Rizzuto, The mitochondrial calcium uniporter is a multimer that can include
a dominant-negative pore-forming subunit, EMBO J., 32 (2013) 2362-2376.
[14] F. Perocchi, V.M. Gohil, H.S. Girgis, X.R. Bao, J.E. McCombs, A.E. Palmer, V.K. Mootha,
MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake, Nature, 467 (2010)
291-296.
[15] M. Plovanich, R.L. Bogorad, Y. Sancak, K.J. Kamer, L. Strittmatter, A.A. Li, H.S. Girgis, S.
Kuchimanchi, J. De Groot, L. Speciner, N. Taneja, J. Oshea, V. Koteliansky, V.K. Mootha, MICU2, a
paralog of MICU1, resides within the mitochondrial uniporter complex to regulate calcium
handling, PLoS One, 8 (2013) e55785.
[16] K. Mallilankaraman, C. Cardenas, P.J. Doonan, H.C. Chandramoorthy, K.M. Irrinki, T.
Golenar, G. Csordas, P. Madireddi, J. Yang, M. Muller, R. Miller, J.E. Kolesar, J. Molgo, B.
Kaufman, G. Hajnoczky, J.K. Foskett, M. Madesh, MCUR1 is an essential component of
mitochondrial Ca2+ uptake that regulates cellular metabolism, Nat. Cell Biol., 14 (2012) 1336-
1343.
[17] Y. Sancak, A.L. Markhard, T. Kitami, E. Kovacs-Bogdan, K.J. Kamer, N.D. Udeshi, S.A. Carr,
D. Chaudhuri, D.E. Clapham, A.A. Li, S.E. Calvo, O. Goldberger, V.K. Mootha, EMRE is an
essential component of the mitochondrial calcium uniporter complex, Science, 342 (2013)
1379-1382.
[18] T. Pozzan, R. Rizzuto, P. Volpe, J. Meldolesi, Molecular and cellular physiology of
intracellular calcium stores, Physiol. Rev., 74 (1994) 595-636.
[19] R. Rizzuto, P. Bernardi, T. Pozzan, Mitochondria as all-round players of the calcium game,
J. Physiol., 529 Pt 1 (2000) 37-47.
[20] D.E. Clapham, Calcium signaling, Cell, 131 (2007) 1047-1058.
[21] X. Pan, J. Liu, T. Nguyen, C. Liu, J. Sun, Y. Teng, M.M. Fergusson, Rovira, II, M. Allen, D.A.
Springer, A.M. Aponte, M. Gucek, R.S. Balaban, E. Murphy, T. Finkel, The physiological role of
mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter, Nat Cell
Biol, 15 (2013) 1464-1472.
[22] E. Carafoli, A.L. Lehninger, A survey of the interaction of calcium ions with mitochondria
from different tissues and species, Biochem. J., 122 (1971) 681-690.
[23] M. Trenker, R. Malli, I. Fertschai, S. Levak-Frank, W.F. Graier, Uncoupling proteins 2 and 3
are fundamental for mitochondrial Ca2+ uniport, Nat. Cell Biol., 9 (2007) 445-452.
[24] D.G. Nicholls, E. Rial, A history of the first uncoupling protein, UCP1, J Bioenerg
Biomembr, 31 (1999) 399-406.
[25] D. Ricquier, F. Bouillaud, The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP
and AtUCP, Biochem. J., 345 Pt 2 (2000) 161-179.
[26] P.S. Brookes, N. Parker, J.A. Buckingham, A. Vidal-Puig, A.P. Halestrap, T.E. Gunter, D.G.
Nicholls, P. Bernardi, J.J. Lemasters, M.D. Brand, UCPs--unlikely calcium porters, Nat. Cell Biol.,
10 (2008) 1235-1237; author reply 1237-1240.
[27] D. Jiang, L. Zhao, D.E. Clapham, Genome-wide RNAi screen identifies Letm1 as a
mitochondrial Ca2+/H+ antiporter, Science, 326 (2009) 144-147.
[28] K. Nowikovsky, E.M. Froschauer, G. Zsurka, J. Samaj, S. Reipert, M. Kolisek, G.
Wiesenberger, R.J. Schweyen, The LETM1/YOL027 gene family encodes a factor of the
mitochondrial K+ homeostasis with a potential role in the Wolf-Hirschhorn syndrome, J. Biol.
Chem., 279 (2004) 30307-30315.
[29] D. Jiang, L. Zhao, C.B. Clish, D.E. Clapham, Letm1, the mitochondrial Ca2+/H+ antiporter,
is essential for normal glucose metabolism and alters brain function in Wolf-Hirschhorn
syndrome, Proc Natl Acad Sci U S A, 110 (2013) E2249-2254.
[30] M.F. Tsai, D. Jiang, L. Zhao, D. Clapham, C. Miller, Functional reconstitution of the
mitochondrial Ca2+/H+ antiporter Letm1, J Gen Physiol, 143 (2014) 67-73.

Page 16 of 18
Accepted Manuscript
[31] E. Froschauer, K. Nowikovsky, R.J. Schweyen, Electroneutral K+/H+ exchange in
mitochondrial membrane vesicles involves Yol027/Letm1 proteins, Biochim Biophys Acta,
1711 (2005) 41-48.
[32] K. Nowikovsky, E.M. Froschauer, G. Zsurka, J. Samaj, S. Reipert, M. Kolisek, G.
Wiesenberger, R.J. Schweyen, The LETM1/YOL027 gene family encodes a factor of the
mitochondrial K+ homeostasis with a potential role in the Wolf-Hirschhorn syndrome, J Biol
Chem, 279 (2004) 30307-30315.
[33] A.G. McQuibban, N. Joza, A. Megighian, M. Scorzeto, D. Zanini, S. Reipert, C. Richter, R.J.
Schweyen, K. Nowikovsky, A Drosophila mutant of LETM1, a candidate gene for seizures in
Wolf-Hirschhorn syndrome, Hum. Mol. Genet., (2010).
[34] K. Nowikovsky, T. Pozzan, R. Rizzuto, L. Scorrano, P. Bernardi, Perspectives on: SGP
symposium on mitochondrial physiology and medicine: the pathophysiology of LETM1, J Gen
Physiol, 139 (2012) 445-454.
[35] M.J. Falk, E.A. Pierce, M. Consugar, M.H. Xie, M. Guadalupe, O. Hardy, E.F. Rappaport, D.C.
Wallace, E. LeProust, X. Gai, Mitochondrial disease genetic diagnostics: optimized whole-
exome analysis for all MitoCarta nuclear genes and the mitochondrial genome, Discov Med, 14
(2012) 389-399.
[36] D. Chaudhuri, Y. Sancak, V.K. Mootha, D.E. Clapham, MCU encodes the pore conducting
mitochondrial calcium currents, Elife, 2 (2013) e00704.
[37] A.G. Bick, S.E. Calvo, V.K. Mootha, Evolutionary diversity of the mitochondrial calcium
uniporter, Science, 336 (2012) 886.
[38] E. Abell, R. Ahrends, S. Bandara, B.O. Park, M.N. Teruel, Parallel adaptive feedback
enhances reliability of the Ca2+ signaling system, Proc. Natl. Acad. Sci. U.S.A., 108 (2011)
14485-14490.
[39] K. Mallilankaraman, P. Doonan, C. Cardenas, H.C. Chandramoorthy, M. Muller, R. Miller,
N.E. Hoffman, R.K. Gandhirajan, J. Molgo, M.J. Birnbaum, B.S. Rothberg, D.O. Mak, J.K. Foskett,
M. Madesh, MICU1 is an essential gatekeeper for MCU-mediated mitochondrial Ca(2+) uptake
that regulates cell survival, Cell, 151 (2012) 630-644.
[40] G. Csordas, T. Golenar, E.L. Seifert, K.J. Kamer, Y. Sancak, F. Perocchi, C. Moffat, D. Weaver,
S. de la Fuente Perez, R. Bogorad, V. Koteliansky, J. Adijanto, V.K. Mootha, G. Hajnoczky, MICU1
controls both the threshold and cooperative activation of the mitochondrial Ca(2)(+)
uniporter, Cell Metab, 17 (2013) 976-987.
[41] S. de la Fuente, J. Matesanz-Isabel, R.I. Fonteriz, M. Montero, J. Alvarez, Dynamics of
mitochondrial Ca2+ uptake in MICU1-knockdown cells, Biochem. J., (2013).
[42] N.E. Hoffman, H.C. Chandramoorthy, S. Shamugapriya, X. Zhang, S. Rajan, K.
Mallilankaraman, R.K. Gandhirajan, R.J. Vagnozzi, L.M. Ferrer, K. Sreekrishnanilayam, K.
Natarajaseenivasan, S. Vallem, T. Force, E.T. Choi, J.Y. Cheung, M. Madesh, MICU1 Motifs Define
Mitochondrial Calcium Uniporter Binding and Activity, Cell Rep, 5 (2013) 1576-1588.
[43] M. Patron, D. De Stefani, V. Checchetto, A. Raffaello, E. Teardo, D. Vecellio Reane, M.
Mantoan, V. Granatiero, I. Szabò, R. Rizzuto, MICU1 and MICU2 finely tune the mitochondrial
Ca2+ uniporter by exerting opposite effect on MCU activity, Molecular Cell, Accepted (2014).
[44] F. Fieni, S.B. Lee, Y.N. Jan, Y. Kirichok, Activity of the mitochondrial calcium uniporter
varies greatly between tissues, Nat. Commun., 3 (2012) 1317.
[45] A. Sickmann, J. Reinders, Y. Wagner, C. Joppich, R. Zahedi, H.E. Meyer, B. Schonfisch, I.
Perschil, A. Chacinska, B. Guiard, P. Rehling, N. Pfanner, C. Meisinger, The proteome of
Saccharomyces cerevisiae mitochondria, Proc Natl Acad Sci U S A, 100 (2003) 13207-13212.
[46] J. Prudent, N. Popgeorgiev, B. Bonneau, J. Thibaut, R. Gadet, J. Lopez, P. Gonzalo, R.
Rimokh, S. Manon, C. Houart, P. Herbomel, A. Aouacheria, G. Gillet, Bcl-wav and the
mitochondrial calcium uniporter drive gastrula morphogenesis in zebrafish, Nat. Commun., 4
(2013) 2330.

Page 17 of 18
Accepted Manuscript
[47] C. Jean-Quartier, A.I. Bondarenko, M.R. Alam, M. Trenker, M. Waldeck-Weiermair, R. Malli,
W.F. Graier, Studying mitochondrial Ca(2+) uptake - a revisit, Mol. Cell. Endocrinol., 353
(2012) 114-127.
[48] A.I. Bondarenko, C. Jean-Quartier, R. Malli, W.F. Graier, Characterization of distinct single-
channel properties of Ca(2)(+) inward currents in mitochondria, Pflugers Arch., 465 (2013)
997-1010.
[49] E. Basso, L. Fante, J. Fowlkes, V. Petronilli, M.A. Forte, P. Bernardi, Properties of the
permeability transition pore in mitochondria devoid of Cyclophilin D, J. Biol. Chem., 280
(2005) 18558-18561.
[50] C.V. Logan, G. Szabadkai, J.A. Sharpe, D.A. Parry, S. Torelli, A.M. Childs, M. Kriek, R. Phadke,
C.A. Johnson, N.Y. Roberts, D.T. Bonthron, K.A. Pysden, T. Whyte, I. Munteanu, A.R. Foley, G.
Wheway, K. Szymanska, S. Natarajan, Z.A. Abdelhamed, J.E. Morgan, H. Roper, G.W. Santen,
E.H. Niks, W.L. van der Pol, D. Lindhout, A. Raffaello, D. De Stefani, J.T. den Dunnen, Y. Sun, I.
Ginjaar, C.A. Sewry, M. Hurles, R. Rizzuto, M.R. Duchen, F. Muntoni, E. Sheridan, Loss-of-
function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations
in mitochondrial calcium signaling, Nat. Genet., (2013).
[51] K. Steeghs, A. Heerschap, A. de Haan, W. Ruitenbeek, F. Oerlemans, J. van Deursen, B.
Perryman, D. Pette, M. Bruckwilder, J. Koudijs, P. Jap, B. Wieringa, Use of gene targeting for
compromising energy homeostasis in neuro-muscular tissues: the role of sarcomeric
mitochondrial creatine kinase, J Neurosci Methods, 71 (1997) 29-41.
[52] R. Horai, M. Asano, K. Sudo, H. Kanuka, M. Suzuki, M. Nishihara, M. Takahashi, Y. Iwakura,
Production of mice deficient in genes for interleukin (IL)-1alpha, IL-1beta, IL-1alpha/beta,
and IL-1 receptor antagonist shows that IL-1beta is crucial in turpentine-induced fever
development and glucocorticoid secretion, J Exp Med, 187 (1998) 1463-1475.
[53] D.J. Garry, G.A. Ordway, J.N. Lorenz, N.B. Radford, E.R. Chin, R.W. Grange, R. Bassel-Duby,
R.S. Williams, Mice without myoglobin, Nature, 395 (1998) 905-908.
[54] P.P. Mammen, S.B. Kanatous, I.S. Yuhanna, P.W. Shaul, M.G. Garry, R.S. Balaban, D.J. Garry,
Hypoxia-induced left ventricular dysfunction in myoglobin-deficient mice, Am J Physiol Heart
Circ Physiol, 285 (2003) H2132-2141.
[55] A. Molojavyi, A. Lindecke, A. Raupach, S. Moellendorf, K. Kohrer, A. Godecke, Myoglobin-
deficient mice activate a distinct cardiac gene expression program in response to
isoproterenol-induced hypertrophy, Physiol Genomics, 41 (2010) 137-145.
[56] G. Schlieper, J.H. Kim, A. Molojavyi, C. Jacoby, T. Laussmann, U. Flogel, A. Godecke, J.
Schrader, Adaptation of the myoglobin knockout mouse to hypoxic stress, Am J Physiol Regul
Integr Comp Physiol, 286 (2004) R786-792.
[57] R.W. Grange, A. Meeson, E. Chin, K.S. Lau, J.T. Stull, J.M. Shelton, R.S. Williams, D.J. Garry,
Functional and molecular adaptations in skeletal muscle of myoglobin-mutant mice, Am J
Physiol Cell Physiol, 281 (2001) C1487-1494.
[58] A.P. Meeson, N. Radford, J.M. Shelton, P.P. Mammen, J.M. DiMaio, K. Hutcheson, Y. Kong, J.
Elterman, R.S. Williams, D.J. Garry, Adaptive mechanisms that preserve cardiac function in
mice without myoglobin, Circ Res, 88 (2001) 713-720.
[59] A. Godecke, U. Flogel, K. Zanger, Z. Ding, J. Hirchenhain, U.K. Decking, J. Schrader,
Disruption of myoglobin in mice induces multiple compensatory mechanisms, Proc Natl Acad
Sci U S A, 96 (1999) 10495-10500.

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Figure 1. Schematic representation, based on our interpretation of published data, of the
Mitochondrial Calcium Uniporter Complex (MCUC) organization.
IMM: Inner Mitochondrial Membrane, IMS: Inter Membrane Space.