Download Mitochondrial metabolite transport

© The Authors Journal compilation © 2010 Biochemical Society
Essays Biochem. (2010) 47, 37–52; doi:10.1042/BSE0470037
3
Mitochondrial metabolite
transport
Ferdinando Palmieri1 and Ciro Leonardo
Pierri
Department of Pharmaco-Biology, Laboratory of Biochemistry and
Molecular Biology, University of Bari, 70125 Bari, Italy
Abstract
The flux of a variety of metabolites, nucleotides and coenzymes across the
inner membrane of mitochondria is catalysed by a nuclear-coded superfamily
of secondary transport proteins called MCs (mitochondrial carriers). The
importance of MCs is demonstrated by their wide distribution in all eukaryotes,
their role in numerous metabolic pathways and cell functions, and the
identification of several diseases caused by alterations of their genes. MCs can
easily be recognized in databases thanks to their striking sequence features. Until
now, 22 MC subfamilies, which are well conserved throughout evolution, have
been functionally characterized, mainly by transport assays upon heterologous
gene expression, purification and reconstitution into liposomes. Given the
significant sequence conservation, it is thought that all MCs use the same basic
transport mechanism, although they exhibit different modes of transport and
driving forces and their substrates vary in nature and size. Based on substrate
specificity, sequence conservation and carrier homology models, progress has
recently been made in understanding the transport mechanism of MCs by new
insights concerning the existence of a substrate-binding site in the carrier cavity,
of cytosolic and matrix gates and conserved proline and glycine residues in each of
the six transmembrane α-helices. These structural properties are believed to play an
important role in the conformational changes required for substrate translocation.
1To
whom correspondence should be addressed (email [email protected]).
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Essays in Biochemistry volume 47 2010
Introduction
Mitochondria are essential not only for the metabolic pathways that take place
within these organelles, but also for many others occurring mainly outside
the mitochondrial matrix. Because of enzyme compartmentalization, many
metabolites produced outside the mitochondria must enter; for example,
ADP and phosphate (Pi) for oxidative phosphorylation, substrates of the
tricarboxylic acid cycle, fatty acid oxidation, mitochondrial replication and
repair of DNA, mitochondrial RNA and protein synthesis, and amino
acid degradation, coproporphyrinogen III and iron for haem biosynthesis,
group donors (S-adenosylmethionine and folate) and coenzymes [NAD+,
FAD, ThPP (thiamine pyrophosphate) and coenzyme A]. Likewise, other
metabolites produced inside the mitochondrial matrix must exit the organelles;
for instance, ATP, citrate, malate, phosphopyruvate, δ-aminolevulinic acid (the
product of the first step in haem biosynthesis), citrulline and ketone bodies
(acetoacetate). Besides metabolites, a variety of cations such as Ca2+, Mg2+,
Na+ and K+ must cross the inner mitochondrial membrane to regulate enzyme
activity and the volume of mitochondria.
The mitochondrion is surrounded by an outer and inner membrane. While
the outer membrane is permeable to solutes with a molecular mass ≤ 4–5 kDa,
the inner membrane is very impermeable. Only some uncharged molecules
such as O2 and CO2 pass through. The passage of hydrophilic compounds
across the inner mitochondrial membrane is catalysed mainly by a superfamily of nuclear-coded proteins known as the MCF [MC (mitochondrial carrier) family]. Many MCs have isoforms encoded by different genes. So far, the
mammalian Pi carrier (known as PiC) is the only known member of the MCF
with two isoforms generated by alternative splicing; however, other isoforms
of this type may exist. The distribution of MCs in tissues varies considerably;
some are present in virtually all tissues, whereas others are tissue-specific,
reflecting their importance for special functions. The substrates transported
by MCs are mostly negatively charged, but some are positively charged or
zwitterions at physiological pH values and greatly vary in structure and size,
from H+ to NAD+. Furthermore, some carriers exert flux control on metabolic
pathways. All family members have common structural features; their primary
structures consist of three tandemly repeated homologous domains of approximately 100 amino acids in length, and each repeat contains two hydrophobic
segments (spanning the membrane as α-helices) and a characteristic sequence
motif PX[D/E]XX[K/R]X[K/R] (20–30 residues) [D/E]GXXXX[W/Y/F][K/
R]G. Notably, the Ca2+-binding aspartate/glutamate and ATP-Mg/Pi carriers have an additional extensive N-terminal domain which fulfils a regulatory
(non-transport) function. MC sequence features are different from those of
any other known transporter family. They have probably arisen after mitochondria had appeared in eukaryotes. Sequence conservation and intron position suggest that they may have evolved from two-tandem gene duplications of
an ancestral gene encoding the 100 amino acid repeat, and the repeat itself may
© The Authors Journal compilation © 2010 Biochemical Society
F. Palmieri and C.L. Pierri
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have evolved by duplication of a DNA sequence encoding a single transmembrane segment. Interestingly, the MCF is not restricted to mitochondria, since
some members are localized in other cell organelles, such as peroxisomes and
chloroplasts.
The present chapter attempts to summarize what is currently known about
the biochemical and molecular characterization of MCs. For detailed reviews
of prior literature on MCs, the reader is referred to references [1–14].
The function of mitochondrial carriers
The MCF is defined by the sequence features of its members: a tripartite
structure, a three-fold repeated signature motif and six transmembrane
α-helices. The term MCF was established after identifying its first members
which were all purified from mitochondria. In the post-genomic era,
with the availability of DNA databases, the MCF was extended by
searching for MCs of unknown function. 58 MCs are encoded by the
genome of Arabidopsis thaliana, 53 by that of Homo sapiens and 35 by the
Saccharomyces cerevisiae genome. Indeed, the MCF is the largest of the
known solute carrier families.
Figure 1 shows all MCs of S. cerevisiae and H. sapiens deduced from
genomic analysis. Approximately half of these carriers have now been characterized in terms of substrate specificity and kinetic parameters by transport assays upon gene expression, purification and reconstitution into liposomes. This procedure was first employed for the bacterial overproduction
and functional reconstitution of the bovine oxoglutarate carrier, which was
indeed the first eukaryotic membrane protein to be expressed in Escherichia
coli and refolded [15]. In addition, for some of the carriers characterized
biochemically, subcellular localization, tissue distribution and their physiological role in cell metabolism and specialized cell functions have been determined. Table 1 lists the main MC subfamilies identified so far according to
their substrate specificity. Notably, some substrates, and even certain defining
substrates, are transported by more than one subfamily. Generally, MC subfamilies are widespread in the eukaryotic kingdom indicating that they play
a role that has been conserved throughout the evolutionary process. There
are, however, some exceptions; for example, the GTP/GDP carrier subfamily
seems to be specific to fungi and protista. Moreover, MC subfamilies are characterized by specific triplets (Figure 3 and the section below on ‘Structure’)
besides the substrates transported.
Modes of transport and driving forces
MCs transport metabolites across the inner mitochondrial membrane in a
highly controlled manner, since it is essential for the H+ electrochemical
potential gradient (generated by the respiratory chain) to be preserved across
this membrane. Many MC subfamilies catalyse an obligatory 1:1 exchange
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Essays in Biochemistry volume 47 2010
Figure 1. Phylogenetic tree of the MCs found in genomic databases of H. sapiens (http://
www.ncbi.nlm.nih.gov/sites/entrez) and S. cerevisiae (http://www.yeastgenome.org/)
The tree was originated from an alignment performed by ClustalW implemented in Jalview
(http://www.jalview.org/); 53 carriers of H. sapiens (red underscore) and 35 of S. cerevisiae (blue
underscore) are shown. The bar indicates the number of substitutions per residue with 0.2 corresponding to a distance of 20 substitutions per 100 residues.
(antiport) reaction between substrates (Figure 2). Some carry out unidirectional
substrate transport (uniport) as the exclusive transport mode [e.g. H+ transport
by the UCP (uncoupling protein)] and others a slow uniport besides antiport
(e.g. the carnitine-acylcarnitine carrier).
Concerning the electrical nature of their transport reactions, MCs are
either electrophoretic (electrogenic) or electroneutral (Figure 2). To date, three
well-characterized subfamilies are known to be electrophoretic. The ADP/ATP
carrier and aspartate/glutamate carrier subfamilies catalyse exchanges across the
mitochondrial membrane that result in charge imbalances because they transport ADP3− for ATP4− and glutamate− plus an H+ for aspartate−, respectively.
The third subfamily is the UCP mentioned above. Electroneutral balance can
© The Authors Journal compilation © 2010 Biochemical Society
F. Palmieri and C.L. Pierri
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Figure 2. Modes of transport catalysed by MCs and their driving forces
Typical examples of reaction mechanisms and driving forces are shown.
be achieved by co-transport (symport) and countertransport of solutes and by
uniport of electroneutral metabolites (Figure 2). In some cases electroneutrality is imposed by simultaneous carrier-mediated H+ movement (Figure 2). The
carrier subfamilies for Pi and glutamate, and in yeast for oxaloacetate, mediate
the transport of anions together with an equivalent amount of H+ (anion/H+
symport). Moreover, the human ornithine carrier can transport ornithine+
against citrulline plus an H+, the yeast GTP/GDP carrier exchanges GTP4−
plus an H+ against GDP3−, and the tricarboxylate (citrate) carrier citrate2− plus
an H+ against malate2−. Other subfamilies catalyse an exchange of anions or
cations. The oxoglutarate carrier, for instance, exchanges oxoglutarate2− for
malate2− and the ornithine carrier ornithine+ for lysine+, arginine+ or an H+.
As their driving force MCs utilize either the electrical or the chemical
component, or both, of the H+ electrochemical potential gradient across the
inner mitochondrial membrane and/or the concentration gradient of the solutes (Figure 2). Because the electrical component of the protonmotive force
is rather high, the electrical nature of the ADP/ATP and aspartate/glutamate
carriers provides a powerful means of ejecting ATP4− and aspartate− against
the concentration gradient from the mitochondrial matrix to the cytosol. In
the case of H+ symport or H+ exchange, the transmembrane pH gradient
regulates the distribution of anionic and cationic solutes across the membrane.
© The Authors Journal compilation © 2010 Biochemical Society
© The Authors Journal compilation © 2010 Biochemical Society
ATP-Mg, Pi, ADP, AMP
ThPP, ThMP, dNDP, dNTP, ADP, ATP
Pyrimide (deoxy)nucleotides
Folates, FAD
ATP, ADP, AMP
ATP-Mg/Pi
ThPP
Pyrimidine nucleotides
Folate/FAD
Adenine nucleotides in
FA and sterol biosynthesis, gluconeogenesis from lactate, isocitrate/oxoglutarate
shuttle.
Krebs cycle, gluconeogenesis from pyruvate, urea synthesis, sulfur metabolism.
Gluconeogenesis.
Citrate, malate, phosphoenolpyruvate,
isocitrate, cis-aconitate
Malate, succinate, Pi, sulfate, thiosulfate
Succinate, fumarate
Dicarboxylates
Succinate/fumarate
Redox reactions, redox balance of mitochondria and chloroplasts.
Mitochondrial protein and RNA synthesis, DNA replication and repair.
synthesis.
Mitochondrial DNA and RNA synthesis and catabolism.
Mono-carbon unit donor, redox reactions.
Peroxisomal FA β-oxidation.
synthase.
Modulation of the matrix adenine nucleotide content, enzyme regulation.
Regulation of mitochondrial ThPP-dependent enzymes, mitochondrial DNA
catabolism, acetylglutamate synthesis, protein acetylation, mitochondrial FA
Oxidative phosphorylation.
Krebs cycle, FA β-oxidation, haem biosynthesis, branched-chain amino-acid
Main metabolic roles
oxo acids
Citrate
peroxisomes
NAD+, d(AMP), (d)GMP
NAD+
GTP/GDP
GTP, GDP, dGTP, dGDP
For di-/tri-carboxylates and
ADP/ATP
CoA, PAP, dephospho-CoA, ADP, ATP
Main substrates
ADP/ATP
CoA/PAP
For nucleotides/dinucleotides
MC subfamilies
S-adenosylmethionine; ThMP, thiamine monophosphate; ThPP, thiamine pyrophosphate; UCP, uncoupling protein.
Coenzyme A; dNDP, deoxynucleoside diphosphates; dNTP, deoxynucleoside triphosphates; FA, fatty acid; PAP, adenosine 3´,5´-diphosphate; Pi, phosphate; SAM,
The substrates transported by each carrier subfamily were identified in liposomes reconstituted with the recombinant protein, with a few exceptions. CoA,
Table 1. MC subfamilies identified according to substrate specificity
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Essays in Biochemistry volume 47 2010
For amino acids
Glutamate
Aspartate/glutamate
Ornithine
Carnitine
SAM
For other substrates
UCP
Phosphate
Oxoglutarate
Oxodicarboxylates
Oxaloacetate/sulfate
in yeast.
α-lsopropylmalate
Urea synthesis, amino acid degradation.
Malate/aspartate shuttle, urea synthesis, gluconeogenesis, cysteine degradation.
Urea synthesis, basic amino acid metabolism, polyamine biosynthesis.
FA β-oxidation.
Mitochondrial DNA, RNA and protein methylation.
Thermogenesis.
Oxidative phosphorylation, counter ion for malate.
Aspartate, glutamate, cysteine sulfinate
Ornithine, citrulline, lysine, arginine (histidine)
Carnitine, acylcarnitines
SAM, S-adenosylhomocysteine
H+
Pi
L-Glutamate
Malate/aspartate shuttle, isocitrate/oxoglutarate shuttle, nitrogen metabolism.
Lysine and tryptophan catabolism and synthesis in yeast.
Krebs cycle, sulfur metabolism, transfer of reducing equivalents, leucine synthesis
Oxoglutarate, malate
Oxoadipate, oxoglutarate
Oxaloacetate, sulfate, thiosulfate,
F. Palmieri and C.L. Pierri
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Essays in Biochemistry volume 47 2010
For example, with a higher pH inside, the carrier-mediated H+-compensated
uptake of Pi− or Pi2− (by the PiC) or of glutamate− (by the glutamate carrier) is
stimulated, as is also the efflux of cationic solutes such as export of ornithine+
in exchange for an H+ (by the ornithine carrier).
Structure
A breakthrough was achieved in 2003 when the atomic structure of the
bovine carboxyatractyloside-inhibited ADP/ATP carrier was solved to
2.2 Å (1 Å = 0.1 nm) [16]. Basically, this structure is composed of six
transmembrane α-helices (H1–H6) lining a funnel-shaped cavity (occupied
by the inhibitor) which is open towards the cytosol and closed on the matrix
side by a salt-bridge network. This network is formed by the charged residues
of the first part of the three signature motifs, PX[D/E]XX[R/K], which
are located at the C-terminus of the odd transmembrane α-helices. The
crystal structure of the ADP/ATP carrier–carboxyatractyloside complex has
proven previous data and hypotheses, i.e. that the above-mentioned network
constitutes the closed gate of monomeric MCs in the ‘c’ (cytosolic)-state [5],
that carriers possess a three-fold pseudosymmetry based on the three-fold
sequence repeats [17] and on electron microscopy observations [18] and
display six transmembrane α-helices with the N- and C-termini exposed
toward the cytosolic side of the membrane ([19] and references therein).
Moreover, the three-dimensional structure of the ADP/ATP carrier greatly
aided the interpretation of experimental results (e.g. site-directed mutagenesis
[20]) and stimulated further research (e.g. in the monomeric/dimeric state of
MCs [21]).
Unfortunately, the difficulties in crystallizing uninhibited MCs have prevented further progress in defining their structure and, in particular, their
conformation in the ‘m’ (matrix)-state. However, important findings have been
recently obtained by multiple sequence alignment analysis of MCs of known
function (substrate specificity) as an extension of the early and fundamental discovery of the three-fold repeated signature motif in the primary structure of the
ADP/ATP carrier [17]. The first of these recent findings regards the existence
of a common, or a similarly located, substrate-binding site [22]. Thus Robinson
and Kunji [22] proposed three contact points, corresponding to residues of the
three even-numbered transmembrane α-helices, that protrude into the cavity at
approximately the midpoint of the membrane one-and-a-half helix turns above
the matrix gate. For example, point II on helix 4 is defined by G[IVLMT] in
nucleotide carriers, by R[QHNTV] in carboxylic acid carriers and by R[D/E] in
amino acid carriers. Furthermore, on the basis of inter-repeat multiple sequence
alignment of MCs of known function, Robinson et al. [23] found that specific
triplets protruding into the carrier cavity are distinct features of the various MC
subfamilies. These triplets are either asymmetrical or symmetrical. The asymmetrical ones (i.e. aligned residues of each carrier formed by different amino acids) are
important for substrate binding. The amino acid pair located at each contact point
© The Authors Journal compilation © 2010 Biochemical Society
F. Palmieri and C.L. Pierri
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Figure 3. Alignment of amino acid triplets of at least one isoform of MCs with known
substrate specificity of H. sapiens and S. cerevisiae plus some of A. thaliana
Each triplet of a single carrier is formed by the three aligned residues derived from the
inter-repeat multiple sequence alignment. Only those triplets of residues protruding into the
carrier cavity are shown with the exception of triplets 28, 73 and 83. The triplets are ordered
horizontally according to the number of the first-repeat amino acids of the bovine ADP/ATP
carrier sequence (GenBank® accession number NP_777083). The carriers are subdivided into
groups based on their substrate specificity.
(I, II and III) are typical asymmetrical triplets (numbers 80 and 81 in Figure 3).
Other asymmetrical triplets are, for instance, 84 and 88 in the ATP-Mg/Pi carrier subfamily and 84, 85 and 88 in the aspartate/glutamate and glutamate carrier
© The Authors Journal compilation © 2010 Biochemical Society
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Essays in Biochemistry volume 47 2010
subfamilies. In analysing the symmetrical triplets (i.e. formed by three identical
amino acids), Robinson et al. [23] revealed the existence of two triplets (93 and
96, which are the charged residues of the motif [F/Y][D/E]XX[R/K]; Figure 3)
located at the C-terminus of the even-numbered transmembrane α-helices. These
triplets would form a salt-bridge network on the cytosolic side which would
close the carrier in the m-state constituting the cytosolic gate of MCs.
Interhelical multiple sequence alignment showed the presence of well-conserved proline and glycine residues in both odd- and even-numbered transmembrane α-helices (see triplets 28 and 83 for proline and 19 and 28 for
glycine; Figure 3). The proline and glycine residues of the even helices are
aligned with the proline and glycine residues of the odd helices in an antiparallel fashion (Figure 2 of [24]). Of note, in both the odd and even helices,
a proline or a glycine residue is located above (one helix turn) and below
(one-and-a-half helix turns) the residues of the common substrate-binding
site. In addition, the proline residues of the odd helices and the proline
residues of the even helices are located above (one-half helix turn) and below
(two-and-a-half helix turns) the matrix and cytosolic gates, respectively. It has
been proposed that the glycine and proline residues of the even helices and the
glycine residues of the odd helices act as hinges to open or close the carrier on
the matrix or cytosolic side [24], analogous to what has been asserted for the
proline residues of the odd helices [16].
Transport mechanism
As schematically represented in Figure 4, MCs possess a central cavity, in which
the substrate is bound, and two gates alternately open on the cytosolic or the
matrix side. When the matrix gate is closed and the c-gate is opened (c-state),
the substrate is released toward the cytosol and another substrate enters. As
the substrate binds to the carrier, the protein rearranges until the transition
state is reached in which a maximum of interactions between the protein and
the substrate take place, in agreement with the ‘induced transition fit’ of carrier
catalysis [14]. In the transition state (i) the substrate is bound at the centre of
the carrier to residues located at the level of the common binding site and to
others above and below, according to its size and shape, and (ii) the carrier is
almost entirely closed on either side of the membrane (Figure 4). The binding
energy of the optimum fit interactions between the carrier and the substrate in
the transition state triggers additional structural changes leading to the matrix
conformation (in which the c-gate is closed and the m-gate is opened) (Figure 4).
At this stage the substrate, which entered the carrier from the cytosolic side,
exits into the matrix and the cycle continues with the entry of another substrate
from the matrix (Figure 4). In summary, during the transition from the c- to the
m-state, on the matrix side the even and odd transmembrane helices move apart
and the matrix gate breaks; on the cytosolic side the helices come together to
close the c-gate. The opposite occurs during the transition from the m- to the
c-state.
© The Authors Journal compilation © 2010 Biochemical Society
F. Palmieri and C.L. Pierri
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Figure 4. Schematic representation of the transition from the c-state to the m-state,
and vice versa, which MCs undergo in the catalytic exchange transport cycle
Trapezoids (left-hand side) illustrate the c-state after the release of the substrate towards the
cytosol (bottom) and immediately after the entry of the substrate from the cytosolic side (top);
trapezoids (right-hand side) show the m-state after the release of the substrate into the matrix
(top) and immediately after the entry of the substrate from the matrix side (bottom); and the
two central rounded rectangles depict the transition states of the carrier with the bound substrate entered from the cytosol (top) and from the matrix (bottom). The box in the centre of the
carrier indicates the ‘presence’ of an internal cavity without reference to its shape (which varies
in the different states depicted). Green ovals and dark blue rectangles represent the substrate
entering from the cytosol and from the matrix respectively; solid triangles indicate closed gates,
and dotted triangles open/partially closed gates.
The conformational changes occurring during the transition from the
c- to the m-state, and vice versa, are still unknown. Recently, we have proposed that the conformational changes are caused by a tilt of the even and
odd helices and by the ability of proline and glycine residues of both the
even and odd helices (mentioned in the ‘Structure’ section) to kink or swivel
towards the cavity axis [24]. Specifically, after the substrate enters the carrier in the c-state and is bound in the transition state, a tilt of the even and
odd helices takes place, which would be seen from the cytosol as occurring
clockwise from the c- to the m-state and counter-clockwise during the m- to
c-state. Consequently, the [F/Y][D/E]XX[K/R] portions of the even helices
are brought together by a swivel or kink at the level of the proline residues
of these helices (and the c-gate closes) while the N-termini of the odd helices
are rotated behind the c-gate by the kink or swivel of the glycine residues
of these helices. Analogously, after the substrate enters the carrier in the mstate and is bound in the transition state, the PX[D/E]XX[K/R] segments are
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brought together by a kink of the proline residues in the odd helices (forming
the m-gate), while the N-termini of the even helices are rotated behind
the m-gate, as seen in the crystal structure of the carboxyatractylosideinhibited ADP/ATP carrier [16]. The above-reported mechanism describes
the MC-mediated antiport mode of transport. However, some carriers are
able to undergo a reversible transition between the c-state and the m-state in
the absence of substrate. Therefore they catalyse uniport, besides antiport,
although at a lower rate. This means that the activation energy barrier of the
transition between the two states of these carriers is much lower than that of
strict 1:1 exchange carriers.
Regulation of mitochondrial carrier activity
MCs are generally present in minute amounts and must ensure a sufficient
rate of flux to fulfil the needs of the respective metabolic pathways.
Thus their activity has to be adapted to different tissues under various
physiological conditions and in varied metabolic and energetic states. Carrier
activity can be regulated in a number of ways. (i) Modulation of driving
forces, kinetic parameters and concentrations of the substrate transported,
counter-substrate and competing substrates. The relevance of the kinetic
parameters of carrier isoforms can be exemplified by the rate of phosphate
import into mitochondria (controlling the rate of ATP production by
oxidative phosphorylation). Thus during muscle contraction, the capacity of
the ubiquitous PiC-B, which has a higher affinity for Pi, is overwhelmed and
the muscle-/heart-specific PiC-A, with its lower substrate affinity, becomes
operative with increased concentrations of cytosolic P i. (ii) Interaction
with allosteric inhibitors or activators. The ornithine carrier, for example,
is inhibited by spermine and spermidine and stimulated by malate and
phosphate, and the aspartate/glutamate and ATP-Mg/Pi carriers are activated
by Ca2+. In this mechanism of Ca2+ signal transduction, Ca2+ exerts metabolic
effects by reacting with the N-terminal domains of these transporters without
entering the organelles. (iii) Modulation of carrier gene expression at the
transcriptional or translational level as shown, for example, for the citrate
carrier.
Diseases
In recent years, the rapid advance in the identification of MC function has
led to the disclosure of several diseases associated with defective carriers ([25]
for a review; [26,27]). These disorders are rare errors of metabolism caused
by alterations of nuclear genes encoding MCs. Their symptoms correlate
with the metabolism affected and its significance in tissues. The 11 known
MC-related diseases are listed in Table 2; they are inherited in an autosomal
recessive manner, except for adPEO (autosomal dominant progressive external
ophthalmoplegia).
© The Authors Journal compilation © 2010 Biochemical Society
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Table 2. Diseases caused by defects of MCs
Disease and carrier acronyms are defined in [25] and [9] respectively, except for TPC (thiamine
pyrophosphate carrier). ALA, aminolevulinic acid.
Disease
Gene
Carrier
Substrate
CAC deficieny
AGC2 deficiency
SLC25A20
SLC25A13
CAC
AGC2
Carnitine/acylcarnitine
Aspartate/glutamate
(NICCD/CTLN2)
HHH syndrome
adPEO
Senger’s syndrome
Congenital Amish
SLC25A15
SLC25A4
?
SLC25A19
ORC1
AAC1
AAC1
TPC
Ornithine/citrulline
ADP/ATP
ADP/ATP
ThPP
microcephaly
AAC1 deficiency
Neonatal myoclonic
SLC25A4
SLC25A22
AAC1
GC1
ADP/ATP
Glutamate
epilepsy
PiC (isoform A)
SLC25A3
PiC
Phosphate
deficiency
AGC1 deficiency
Congenital
SLC25A12
SLC25A38
AGC1
–
Aspartate/glutamate
? (glycine/ALA)
sideroblastic anaemia
Structure–function relationships
To obtain information on the structure and function of MC proteins,
extensive site-directed mutagenesis of several carriers has been performed.
The results indicate that a relatively limited number of amino acid
substitutions affect transport activity. Notably, nearly all amino acid
substitutions causing loss of function and the missense mutations found in
patients with MC-related diseases are located in areas of carrier structure
that are key for their function, i.e. carrier cavity surface corresponding to the
substrate-binding site, matrix and cytosolic gates, and proline-glycine areas
of the transmembrane α-helices [24].
Carrier import into mitochondria
MCs are synthesized on cytosolic ribosomes and must be imported into
mitochondria. Based on data obtained using the ADP/ATP carrier, a general
model of MC import has been proposed [28]. Unlike most nuclear-coded mitochondrial proteins, MCF members contain targeting information in their mature
sequence. Several short targeting sequences are distributed over the MCF
sequence, and it is currently believed that recognition by the mitochondrial
import machinery involves the simultaneous binding of several signals [29]. A
few MCs possess cleavable presequences which are not essential for targeting.
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Of late, it has been found that the presequences of the Pi and citrate carriers
improve import through different mechanisms, by providing a binding site for
the chaperone Hsc70 (heat-shock cognate 70 stress protein) (Pi carrier) and by
acting as an internal chaperone (citrate carrier) [30].
Conclusions
Despite the substantial effort of many laboratories and the significant progress
achieved in the field, as described herein, further research is still needed to
complete our understanding of mitochondrial metabolite transport.
One obvious goal in the study of MCs is the identification of the substrate
specificity of family members that have remained functionally unidentified.
Notably, many important transport activities across the mitochondrial membrane, such as of pyruvate, acetoacetate, α-oxo acids of branched-chain amino
acids, glutamine, choline, γ-aminobutyric acid, N-acetyl glutamate and several
cations, have yet to be associated with specific proteins; it may be that proteins other than MCs are responsible for some of these activities. This is very
likely to be the case for cations, since Mg2+ has been found to be translocated
by a member of the Mg2+ transporter family comprising CorA in bacteria and
Alr1p in the plasma membrane of lower eukaryotes. Another important aspect
of MCs that warrants further in-depth investigation is their physiological role
in cell metabolism and special functions. The recent findings that citrate carrier
plays a role in insulin secretion and histone acetylation demonstrate that this
area of research is expanding and may reveal relevant data.
The determination of the crystal structure of the ADP/ATP carrier
(roughly corresponding to the c-conformation) and the exciting novel insights
into the MC structural properties reported herein allow us to build a realistic
schematic model of the MC-catalysed transport mechanism. However, the
molecular details of substrate translocation through MCs will be elucidated
when the difficulties in crystallizing the highly hydrophobic MC proteins are
overcome and the three-dimensional structure of uninhibited carriers in various conformations (including the m-state) becomes available.
The ongoing functional identification of other MCF members will help
detect other carrier-related diseases and to comprehend the molecular basis
of their symptoms. Understanding their pathogenetic mechanism will probably lead to new therapeutic approaches. For example, patients affected by
carnitine-acylcarnitine carrier deficiency with some residual carrier activity and a
mild phenotype might benefit from treatment with statins and/or fibrates acting
via stimulation of carrier gene expression. Another interesting finding concerns
the potential significance of aspartate/glutamate carrier gene polymorphisms in
autism. Therefore the possible role of MC gene polymorphisms in complex disorders is a promising area for future research. Lastly, carriers that are specific to
fungi and/or protista (e.g. the GTP/GDP carrier subfamily) may be suitable targets for novel drugs effective against pathogenic fungi and protista of H. sapiens
(e.g. Candida albicans and Trypanosoma brucei) and plants (e.g. Gibberella zeae).
© The Authors Journal compilation © 2010 Biochemical Society
F. Palmieri and C.L. Pierri
51
Summary
•
•
•
•
•
MCs are nuclear-coded proteins that transport numerous metabolites
across the mitochondrial matrix. To date, 22 MC subfamilies have
been characterized according to their substrate specificity.
Important recent findings concern the existence of a similarly located
substrate-binding site, a cytosolic gate (besides the known matrix gate)
and of conserved proline and glycine residues in the odd and even
transmembrane α-helices.
The conformational changes occurring after substrate binding and
leading to substrate translocation involve a tilt of even and odd transmembrane α-helices and a kink/swivel of the helix proline and glycine
pairs that are strategically located between the substrate-binding site in
the centre of the carrier and each of the two gates.
MC activity is regulated in different ways to meet the demands of the
respective metabolic pathways.
Several diseases are caused by alterations of nuclear genes encoding
specific MCs.
We thank the Ministero dell’Università e della Ricerca (MIUR), the Center of
Excellence in Genomics (CEGBA) and the Italian Human ProteomeNet no.
RBRN07BMCT_009 for supporting our work.
This chapter was received on 24 October 2009 and accepted on 23 November 2009.
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