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The Plant Journal (2011) 66, 161–181
doi: 10.1111/j.1365-313X.2011.04516.x
Evolution, structure and function of mitochondrial
carriers: a review with new insights
Ferdinando Palmieri1,*, Ciro L. Pierri1, Anna De Grassi2, Adriano Nunes-Nesi3 and Alisdair R. Fernie4
Laboratory of Biochemistry and Molecular Biology, Department of Pharmaco-Biology, University of Bari,
Via Orabona 4, 70125 Bari, Italy,
European Institute of Oncology, IFOM-IEO Campus, Via Adamello 16, 20139 Milan, Italy,
Max-Planck Partner Group, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, 36570-000 Viçosa,
MG, Brasil, and
Max-Planck-Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany
Received 3 November 2010; accepted 25 January 2011.
For correspondence (fax +39 080 5442770; e-mail [email protected]).
The mitochondrial carriers (MC) constitute a large family (MCF) of inner membrane transporters displaying
different substrate specificities, patterns of gene expression and even non-mitochondrial organelle localization. In Arabidopsis thaliana 58 genes encode these six trans-membrane domain proteins. The number in other
sequenced plant genomes varies from 37 to 125, thus being larger than that of Saccharomyces cerevisiae and
comparable with that of Homo sapiens. In addition to displaying highly similar secondary structures, the
proteins of the MCF can be subdivided into subfamilies on the basis of substrate specificity and the presence of
specific symmetry-related amino acid triplets. We assessed the predictive power of these triplets by comparing
predictions with experimentally determined data for Arabidopsis MCs, and applied these predictions to the not
yet functionally characterized mitochondrial carriers of the grass, Brachypodium distachyon, and the alga,
Ostreococcus lucimarinus. We additionally studied evolutionary aspects of the plant MCF by comparing
sequence data of the Arabidopsis MCF with those of Saccharomyces cerevisiae and Homo sapiens, then
with those of Brachypodium distachyon and Ostreococcus lucimarinus, employing intra- and inter-genome
comparisons. Finally, we discussed the importance of the approaches of global gene expression analysis and
in vivo characterizations in order to address the relevance of these vital carrier proteins.
Keywords: Arabidopsis, comparative genomics, evolution, mitochondrial carrier, mitochondrial transporter,
plant genome.
Mitochondria are ubiquitous in eukaryotic cells and perform
a wide range of essential cellular functions. In plants, in
addition to respiration and cellular energy supply, they
are involved in further metabolic tasks including nitrogen
assimilation, photorespiration, C1 metabolism, photosynthesis in C4 plants, crassulacean acid metabolism and utilization of storage pools of carbon and nitrogen during seed
germination (Douce, 1985). They also play a role in the biosynthesis of amino acids, tetrapyrroles, fatty acids and
vitamin cofactors (Giege et al., 2003; Picault et al., 2004).
While the outer mitochondrial membrane is permeable to
solutes with a molecular mass of less than 4–5 kDa (Pfaff
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd
et al., 1968; Colombini, 1979; Ludwig et al., 1986; Benz et al.,
1990), the inner membrane is impermeable. Indeed, only
small and uncharged molecules such as O2 and CO2 can
readily pass through this membrane. The passage of
hydrophilic compounds across the inner mitochondrial
membrane is catalyzed mainly by a family of nuclear-coded
proteins known as the mitochondrial carrier family (MCF).
MCs are small proteins normally possessing a molecular
mass of about 30–34 kDa. All family members have common
structural features which are different from those of any
other known transporter family. Their primary structure
consists of three tandemly repeated homologous domains
162 Ferdinando Palmieri et al.
about 100 amino acids in length, and each repeat contains
two hydrophobic segments (spanning the membrane
as a-helices) and a characteristic amino acid sequence
motif PX[D/E]XX[K/R]X[K/R] (20–30 residues) [D/E]GXXXX
[W/Y/F][K/R]G (PROSITE PS50920, PFAM PF00153 and
IPR00193). Unlike the other members of the family, two
subfamilies, the aspartate/glutamate and ATP-Mg/Pi carriers, have additional N-terminal regulatory domains (more
than 150 amino acids), that usually contain Ca2+-binding
motifs. Molecules transported by the MCF proteins are
greatly variable in size and structure from H+ to NAD+ and
coenzyme A. Most of them are negatively charged, but
some are positively charged or zwitterions at physiological
pH values. Many MC subfamilies catalyse a 1:1 exchange
(antiport) reaction between substrates. However, other
modes such as unidirectional substrate transport (uniport)
and H+-compensated anion symport are also mediated
by some MCs. Furthermore, MCs can be subdivided on
the basis of the electrical nature of the reactions they catalyse with family members either being electrophoretic
(electrogenic) or electroneutral. The ADP/ATP and aspartate/
glutamate carriers, for example, drive electrogenic reactions
as their operation results in net charge transfer. By contrast,
the carrier subfamilies for Pi, glutamate and GTP/GDP as
well as for oxoglutarate and ornithine are electroneutral.
Considerable research has been conducted on characterizing members of the MCF in both yeast and animals (see
Klingenberg, 2008; Kunji and Robinson, 2010; Palmieri,
2004, 2006, 2008; Palmieri and Pierri, 2010a,b; Satrústegui
et al., 2007; for reviews). In recent years the advent and
exploitation of transcriptomic, proteomic and metabolomic
technologies as well as the availability of knock-out collections in Arabidopsis have greatly aided in increasing our
understanding of these proteins in plants (Picault et al.,
2004; Haferkamp, 2007).
In this article we have reviewed our current understanding of the structure, biochemical characteristics, expression pattern, subcellular localization and in planta function
of members of the MCF. In addition, intra- and inter-genome
comparisons have allowed a first assessment of the evolution of this protein family.
The atomic structure of the ADP/ATP carrier, a member of
the MCF, in complex with its powerfull inhibitor carboxyatractyloside, has been solved to 2.2 Å (Pebay-Peyroula et al.,
2003). This structure is composed of a six transmembrane
a-helix bundle (H1–H6) and three short a-helices (h12, h34,
h56) parallel to the membrane plane on the matrix side. H1–
H6 line a water-accessible cavity (occupied by the inhibitor)
which is open towards the cytosol and closed on the matrix
side by a salt-bridge network formed by the charged residues of the first part of the three signature motifs, PX[D/
E]XX[R/K]. The three-dimensional structure of the ADP/ATP
carrier is critical in our understanding of MCF proteins in
several ways. Firstly, it exhibits a three-fold pseudosymmetry in line with the three-fold sequence repeats
(Saraste and Walker, 1982), as was also observed by electron
microscopy of the 2D crystals of the yeast ADP/ATP carrier
(Kunji and Harding, 2003). Secondly, it roughly corresponds
to the ‘c’ (cytosolic)-state of the ADP/ATP carrier as carboxyatractyloside is an inhibitor that blocks the carrier in this
state (Klingenberg, 2008). Thirdly, this structure has been
highly used as a template for building homology models of
various carriers, thus greatly improving our understanding
of the MC structure/function relationships (Walters and
Kaplan, 2004; Wohlrab, 2004; Morozzo della Rocca et al.,
2005; Tonazzi et al., 2005; Cappello et al., 2006, 2007;
Robinson and Kunji, 2006; Robinson et al., 2008; Wibom
et al., 2009; Giangregorio et al., 2010).
More recently further important structural information
based on the available biochemical characterization (substrate specificity) of MCs, 3D comparative models and
bioinformatics approaches has significantly contributed to
deepen our understanding of MC structure and function.
In addition to the salt-bridge network on the matrix side,
suggested by Nelson et al. (1998) and experimentally demonstrated by the resolution of the 3D structure of the ADP/
ATP carrier (Pebay-Peyroula et al., 2003), another saltbridge network has been hypothesized to exist on the
cytosolic side (Robinson et al., 2008). The latter is formed
by the charged residues of the sequence motif [F/Y][D/
E]XX[R/K] localized at the c-terminus of the even-numbered
transmembrane a-helices. These networks constitute the
cytosolic and matrix gates of MCs that close the protein
central cavity in the ‘m’ (matrix)-state (in which the internal
cavity is open towards the matrix and closed on the cytosolic
side) and the c-state, respectively. Moreover, multiple
sequence alignment of MCs of known function (substrate
specificity) revealed residues of the three even-numbered
transmembrane a-helices having the potentiality of discriminating the binding of three major classes of substrates:
nucleotides, carboxylates or amino acids (Robinson and
Kunji, 2006). Based on the 3D structure of the ADP/ATP
carrier, these important residues protrude into the carrier
cavity at approximately the midpoint of the membrane oneand-a-half helix turns above the matrix gate. They constitute
the substrate binding site or part of it when, depending on
the size, shape and chemistry of the substrate, residues of
the odd-numbered transmembrane a-helices located in the
cavity at the same level and/or other residues above and
below are also involved in binding. Finally, in the odd
transmembrane a-helices a well-conserved glycine is present nine residues before the prolines of the PX[D/E]XX[K/
R]X[K/R] motif; and in the even transmembrane a-helices
a conserved proline is present 10 residues after the glycine
corresponding to the last residue of the second part ([D/
E]GXXXX[W/Y/F][K/R]G) of the sequence motif (Palmieri and
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
Mitochondrial carriers 163
Figure 1. Mechanism of substrate translocation catalyzed by mitochondrial exchange carriers.
(a) Scheme depicting the transition of MCs from the c-state to the m-state and vice versa. Truncated cones on the left show 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); truncated cones on the right illustrate 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
truncated bicone-shaped solids depict the transition states of the carrier with the bound substrate entered from the cytosol (top) and from the matrix (bottom). Red
solid cuboids and green solid discs represent the substrate entering from the cytosol and from the matrix, respectively; red triangles indicate closed gates, and
dotted red triangles open/partially closed gates. All transport steps are fully reversibile. The positions of the cytosolic gate, P-G level 1, substrate binding site, P-G
level 2 and matrix gate are shown on the left.
(b) Crystal structure of the bovine ADP/ATP carrier first repeat, taken from the published 3D structure of the carboxyatractyloside–ADP/ATP carrier complex. The
conserved P and G positions in most MC odd and even transmembrane a-helices (Palmieri and Pierri, 2010a) are indicated by G1, P1, G2 and P2. In the ADP/ATP carrier
A19 is present instead of G. A19 (G1) and G73 (G2) are shown in yellow and in surf representation; P28 (P1) and P83 (P2) in red and surf representation. Panel (c) The
‘flexible hinged helix movements’ of MCs occurring during their catalytic exchange transport cycle. For sake of clarity, only the movements of a single repeat are
depicted. Subscripts 1 and 2 were added to the P and G to more easily identify the odd- and even-numbered transmembrane helix. The left side corresponds to the
c-state, the right side to the m-state and the middle part to the transition state; the grey oval in the transition state denotes the bound substrate; the yellow arrows
denote the ability of G to bend the helices; and the red arrows indicate kink/swivel at the P. The angle of observation is the same as that of panel (b). The carrier matrix
axis is closer to the reader. Panels (b) and (c) are reproduced with permission (from Palmieri and Pierri, 2010a; Figure 2).
Pierri, 2010a). It is interesting that: (i) the Gly and Pro of the
odd helices are aligned with the Gly and Pro of the even
helices in an antiparallel fashion; (ii) the above-mentioned
Pro and Gly are located strategically between the substrate
binding site and the gates on both sides (Palmieri and Pierri,
2010a); and (iii) as assessed for the Pro of the odd helices
(Pebay-Peyroula et al., 2003), the Gly of the odd helices and
the Gly and Pro of the even helices may also act as hinges
(Palmieri and Pierri, 2010a).
It is believed that during the catalytic exchange transport
cycle MCs undergo a conformational change from the
c-state to the m-state and vice versa (see Figure 1a) (Kunji
and Robinson, 2010; Palmieri and Pierri, 2010a,b). In brief, in
the c-state the substrate enters the carrier from the cytosolic
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
164 Ferdinando Palmieri et al.
side and binds to the carrier (Figure 1a). 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, according
to the ‘induced transition fit’ of carrier catalysis (Klingenberg, 2005). In the transition state: (i) the substrate is bound
in the cavity approximately at the center of the carrier, as
predicted by the ‘single binding center-gating pore’ mechanism (Klingenberg, 1976); and (ii) the carrier is compactly
structured around the substrate and almost entirely closed
on either side of the membrane (Figure 1a). The total
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). At this stage the substrate, which entered the
carrier from the cytosolic side, exits into the matrix and
the catalytic cycle continues with the entry of another
substrate from the matrix (Figure 1a). The above-reported
mechanism describes the MC-mediated antiport mode of
transport. However, some carriers may catalyze uniport,
besides antiport, although at lower rates. This means that
they are able to undergo a reversible transition between the
c-state and the m-state in the absence of the substrate,
because the activation energy barrier of the transition
between the two states of these carriers is much lower than
that of the obligatory 1:1 exchange carriers.
The large structural changes occurring during the transition from the c- to the m-state, and vice versa, are still
unknown, in particular because the 3D structure of MCs in
the m-state is not yet available. Based on current knowledge
as well as on the role of Pro and Gly of the transmembrane
a-helices, we have proposed that these conformational
changes are largely due to the movements of the even and
odd transmembrane a-helices (Palmieri and Pierri, 2010a).
These ‘flexible hinged helix movements’ are schematically
depicted in Figure 1c for one repeat of MCs. For comparative
purposes Figure 1b illustrates the crystal structure of the
first repeat of the ADP/ATP carrier in the c-state, taken from
Pebay-Peyroula et al. (2003). The MC ‘hinged helix movements’ are the result of the substrate–protein interactions
and in summary consist of a tilt of the entire helical
segments and a kink/swivel of the helical termini at the level
of their Pro and Gly. Viewing the carrier from the cytosol,
even and odd helices would be seen rotating clockwise
during the c- to m-state transition, counter-clockwise during
the m- to c-state transition, and vice versa viewing the carrier
from the matrix side. Furthermore, during the transition
from the c-state to the m-state, the kink of the Pro in the even
helices towards the cavity axis brings together the [F/Y][D/
E]XX[R/K] portions closing the cytosolic salt-bridge network;
the kink/swivel of the Gly in the odd helices towards the
cavity axis rotates their N-termini behind the cytosolic saltbridge network (Figure 1c, right side, upper segments),
whereas the matrix termini of the even and odd helices
move apart away from the cavity axis hinging on their Pro
and Gly residues (Figure 1c, right side, lower segments). The
opposite movements take place during the transition from
the m- to the c-state (Figure 1c). In this way, during the
transition from the c- to the m-state the matrix gate breaks
and the cytosolic gate closes, and vice versa during the
transition from the m- to the c-state (Figure 1).
Only six MCs were sequenced after their purification from
mitochondria by direct amino acid analysis or by DNA
sequencing (Indiveri et al., 1997 and references therein).
These early studies led to the conclusion that they all belonged
to the same protein family which was named MCF. In the
genomic era, many proteins of unknown function with the
characteristic sequence features of the MCF have emerged
from genome sequencing of various organisms. The genome
of Saccharomyces cerevisiae encodes 35 MCs (Palmieri
et al., 1996), that of Arabidopsis thaliana 58 (Picault et al., 2004)
and the Homo sapiens genome about 50 (Palmieri, 2004).
The first step in elucidating MC function is to search for the
substrate(s) transported by a particular carrier. Phylogenetic
clustering, genetic information, knowledge of cell metabolism and complementation of phenotypes have often provided clues about the transported substrate. However, in this
respect these methods are not conclusive. Until now, the
best strategy employed to identify the substrate specificity of
new MCs includes searching databases for carriers of
unknown function, heterologous gene expression in Escherichia coli and reconstitution of purified recombinant carriers
into liposomes, in which substrate transport is assayed by
direct measurements (Fiermonte et al., 1993). In a few cases
putative MCs were expressed in S. cerevisiae, purified from
isolated mitochondria and reconstituted into liposomes
(Palmieri et al., 1999a, 2001a,b). When using such gene-tofunction strategies about half of the MCs in S. cerevisiae
(Palmieri et al., 2006 and references in this work), a third in
H. sapiens (Palmieri, 2004 and references in this work) and a
quarter in A. thaliana (Picault et al., 2004 and references in
this work) were identified.
Table 1 lists the main subfamilies into which MCs can be
divided according to their specificity. Four considerations
should be made. First, some substrates, and even certain
defining substrates, are transported by more than one
subfamily. Second, the best transported substrate in reconstituted liposomes might not always be the most important
substrate under physiological conditions. This situation is
particularly true in different tissues, or specialized cells, in
which the transported substrates may well be present in the
cytosol and/or in the mitochondrial matrix at different
concentration ratios. Third, some subfamilies may transport
additional, yet untested substrates (see Marobbio et al.,
2008). Fourth, most of the subfamilies reported in Table 1
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
Mitochondrial carriers 165
Table 1 MC subfamilies defined by substrate specificity and symmetry-related amino acid triplets
1. For nucleotides and dinucleotides
Coenzyme A/PAP
Main substrates
Klingenberg (2008) and
Fiore et al. (1998)
Coa, PAP,
dephospho-coa, AXP
Prohl et al. (2001) and
Fiermonte et al. (2009)
Fiermonte et al. (2004) and
Traba et al. (2008, 2009)
Dolce et al. (2001),
Marobbio et al. (2002)
and Lindhurst et al. (2006)
Marobbio et al. (2006)
and Floyd et al. (2007)
11(DNS), 19(AGT),
23(KL[G/S]), 84(TYG),
85(QRX), 88(NYV)
23 (K[V/A]Q), 34 (IVR),
88 ([K/Q]SS)
23 (RQ[Q/A]),
30(DE[A/T/N]), 84(EYA),
23(R[T/S]K), 34(IT[K/R]),
80 (L[A/T]K), 85(GAT)
Thpp, thmp;
(d)NDP, (d)NTP
Folates, FAD
NAD+, (d)AMP, (d)GMP
GTP, GDP, dgtp,
dgdp, ITP, IDP
2. For di-/tri-carboxylates and keto acids
Tzagoloff et al. (1996),
Titus and Moran (2000)
and Bedhomme et al. (2005)
Palmieri et al. (2001b)
Todisco et al. (2006) and
Palmieri et al. (2009)
Vozza et al. (2004)
Malate, succinate,
phosphate, sulfate,
Palmieri et al. (1996),
Fiermonte et al. (1998b)
and Palmieri et al. (2008a)
Oxoglutarate, malate
Indiveri et al. (1987) and
Fiermonte et al. (1993)
Oxoglutarate, citrate
Picault et al. (2002)
Succinate, fumarate
Citrate, malate, isocitrate,
cis-aconitate, PEP
Oxoadipate, oxoglutarate
Palmieri et al. (1997b) and
Catoni et al. (2003)
Kaplan et al. (1993, 1995)
Oxaloacetate, sulfate,
Palmieri et al. (1999b) and
Marobbio et al. (2008)
Fiermonte et al. (2002)
Aspartate, glutamate,
Palmieri et al. (2001c) and
Cavero et al. (2003)
3. For amino acids
Palmieri et al. (2001a) and
Fiermonte et al. (2001)
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
19 (G[G/A]K), 27 (CNY),
30 ([D/E]WE), 37 (QQR),
83 ([PEP), 85 (R[I/V][S/T])
19 (GGK), 27 (HNY),
30 (DWQ)
19 (SAK), 30 (DAI),
33 (KAK), 37 (QKR)
19 (GGK), 27 (CNY),
30 (DWE), 89 (FP[L/F])
22 (EGS), 23 (IEL),
84 (QGK), 85 (RSL),
88 (KLS)
26 (TG[C/S]),
27 (H[N/T][S/Q/N]),
33 (K[N/M]K),
88 (RQ[I/L/T])
26 (VGS), 27 (QTM),
33 (KLK), 35 (RRR),
77 (GTY), 84 (YVH),
88 (RQT), 93 (TSE)
26 (IGS), 27 (QSL),
33 (KLK), 35 (RRQ),
77 (GTY), 84 (YLH),
88 (RMT), 93 ([K/R]DN)
22 (EAG), 84 (KNG),
88 (RNT)
22 (E[A/S][S/T]),
84 (KN[S/D]), 88 (RRV)
22 (EE[A/G]), 77 (PTK),
81 (E[H/N]L) 84 (K[F/W]G),
85 (RNG), 88 (KY[M/L])
23 (VAA), 26 (TGM),
30 (E[F/Y]D), 80 (YRR),
84 ([L/M]GH), 88 (RQ[C/S])
22 (GQA), 77 (NTR),
80 (LRV), 84 (EFL),
85 (KSF), 88 (KYA)
22 (GQA), 77 (QCR),
84 (EFQ), 85 (KSF), 88 (KYT)
166 Ferdinando Palmieri et al.
Table 1 (Continued)
Main substrates
Ornithine, (lysine,
citrulline, arginine,
Palmieri et al. (1997a),
Hoyos et al. (2003) and
Fiermonte et al. (2003)
Carnitine, acylcarnitines
Indiveri et al. (1990) and
Palmieri et al. (1999a)
Marobbio et al. (2003),
Agrimi et al. (2004),
Palmieri et al. (2006a) and
Bouvier et al. (2006)
23 ([V/I][A/S]W) but
(KSN) in S. cerevisiae,
26 (GL[V/C]) but (ELI) in
S. cerevisiae, 84 (EGA), but
(QAV) in atbac2
23 (VTW), 85 (FSN)
4. For other substrates
Wohlrab and Briggs (1994)
and Fiermonte et al. (1998a)
19 (G[E/G]G),
23 ([D/E][C/S][A/G]),
26 ([L/F]RT), 80 ([G/A]RW),
85 ([A/S][S/T/D]X), 88 (FQF)
19 (CEG), 23 (HDA),
80 (G[R/K]M), 88 (KKQ)
23 ([D/E][V/I/S/Q][A/V/T/S]),
88 ([R/K] [D/E][F/M])
19 (GTG), 22 (E[S/A/H][A/C]),
23 (HDA), 27 ([F/Y][T/N]T)
With a few exceptions, the substrates transported by each carrier subfamily were identified in liposomes reconstituted with the recombinant
protein. The characterizing triplets of each carrier subfamily are the triplet sets present in the functionally identified MCs of each family. The
acronyms of the indicated subfamilies are: AAC, ADP/ATP carrier; AGC, aspartate/glutamate carrier; ANT, peroxisomal adenine nucleotide
translocator; APC, ATP-Mg/Pi carrier; CAC, carnitine carrier; CoA/PAP, coenzyme A /adenosine 3¢,5¢-diphosphate carrier; CTP, citrate carrier; DIC,
dicarboxylate carrier; DTC, di-/tri-carboxylate carrier; FAD, FAD carrier; GC, glutamate carrier, GGC, GTP/GDP carrier; NDT, NAD+ carrier; OAC,
oxaloacetate/sulfate carrier; ODC, oxodicarboxylate carrier; OGC, oxoglutarate carrier; ORC, ornithine carrier; PiC, phosphate carrier; PNC,
pyrimidine nucleotide carrier; SAMC, S-adenosylmethionine carrier; SFC, succinate/fumarate carrier; TPC, thiamine pyrophosphate carrier; UCP,
uncoupling protein. AXP, adenine nucleotides; dNDP, deoxynucleoside diphosphates; dNTP, deoxynucleoside triphosphates; PEP, phosphoenolpyruvate; Pi, phosphate; ThMP, thiamine monophosphate; ThPP, thiamine pyrophosphate.
are present in all eukaryotes. Furthermore, the carrier
subfamilies defined on the basis of their function (substrate
specificity) are also characterized by specific amino acid
triplets (Figure 2 and Table 1). As mentioned above, MCs
are three-fold pseudo-symmetric. Therefore, each carrier
displays symmetry-related triplets of amino acids when its
three repeat sequences are aligned (Robinson et al., 2008).
Figure 2 shows the triplets protruding into the carrier
cavity of all MCs present in A. thaliana, H. sapiens and
S. cerevisiae. These residues belong to the odd-numbered
(triplets 11–34) and even-numbered (triplets 73–96) transmembrane a-helices of the above carriers. The Pro and Gly
triplets (19, 28, 73 and 83) are also shown in Figure 2,
although they do not protrude into the cavity. Each subfamily is characterized by the complete set of triplets listed
in Table 1 that are not present, altogether, in any other
subfamily. For example, all the members of the AAC
subfamily exhibit triplets 11 (DNS), 19 (AGT), 23 (KL[G/S]),
84 (TYG), 85 (QRX) and 88 (NYV); and all the members of the
AGC subfamily triplets 22 (GQA), 77 (QCR), 84 (EFQ), 85
(KSF) and 88 (KYT). The number of characterizing triplets
ranges from two to eight in the various subfamilies
(Table 1). As can be seen in Figure 2, related subfamilies
transporting structurally related substrates may share some
triplets. For example the NAD+, PyC and FAD subfamilies
share triplet 19 (GGK) amongst their three to six character-
izing triplets. It is worth mentioning that the NAD+ and PyC
subfamilies, which have been characterized in greater depth
than the FAD subfamily, share some substrates as well
(Marobbio et al., 2006; Palmieri et al., 2009; Todisco et al.,
2006; S. Todisco and M.A. Di Noia, personal communication). The OGC and DTC subfamilies also share two triplets
(KLK and GTY) of the eight characterizing triplets, as well as
some transported substrates (Fiermonte et al., 1993; Picault
et al., 2002). Given the importance of these structural
features some additional remarks should be made. Although
all five biochemically characterized members of the ornithine carrier subfamily (ORC1 and ORC2 in H. sapiens, BAC1
and BAC2 in A. thaliana and Ort1p in S. cerevisiae) transport
ornithine, some differences in substrate specificity have
been noticed. It is likely that this subfamily will be divided
into additional subfamilies as there are significant differences in triplets 23, 26 and 84 (see Figure 2 and Table 1),
in particular with respect to Ort1p and BAC2. The GGC
subfamily is defined by transporting GTP and GDP specifically and by a specific set of triplets (Figure 2 and Table 1).
Surprisingly, however, certain GGC triplets are very similar
to some of three subfamilies transporting carboxylates
(citrate, SFC and ODC). Moreover, the UCP subfamily and
an ‘unnamed’ family have been included in Table 1 for their
relevance, although the substrate specificity of several of
their members is yet to be determined.
ª 2011 The Authors
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Mitochondrial carriers 167
Figure 2. Alignment of symmetry-related amino acid triplets of all the MCs of H. sapiens (53), S. cerevisiae (35) and A. thaliana (58).
Each triplet is formed by the three aligned residues of each carrier, which are derived from the inter-repeat multiple sequence alignment of the MCs indicated above.
The triplets are ordered horizontally according to the number of the first-repeat amino acids of the bovine ADP/ATP carrier sequence (NP_777083). Amino acids are
coloured according to the default Jalview–Zappo style. The carriers are listed according to the following major groups of substrates: nucleotides (panel a); carboxylic
acids including keto acids (panel b); amino acids and other substrates (panel c).
The MCF is probably the largest family of membrane
metabolite transport proteins. MCs are also highly abundant in the genome of several species of dicots, monocots and algae ranging in number from 37 to 125
(Table 2). This number is comparable with or higher than
that found in S. cerevisiae (35) and H. sapiens (53). In
general, in the plant species whose genomes have been
assembled, MCs are distributed almost uniformly in all
the chromosomes (Table 2). A high number of MCs is
also detectable in the genomes of dicots, monocots and
algae species which are currently being assembled: 66
MCs in Arabidopsis lyrata, 57 in Carica papaya, 81 in
Morchella esculenta, 61 in Vitis vinifera, 65 in Mimulus
guttatus, 65 in Ricinus communis, 61 in Cucumis sativus
for dicots; 64 in Oryza sativa, 91 in Populus trichocarpa
for monocots; 60 in Selaginella moellendorfi and 93 in
Physcomitrella patens for algae.
To further characterize plant MCs we investigated their
symmetry-related triplets and compared these to MC subfamilies for which substrate specificity was determined in
H. sapiens, S. cerevisiae and A. thaliana (Table 3). In other
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168 Ferdinando Palmieri et al.
Figure 2. (Continued)
words, after having identified numerous MC subfamilies on
the basis of substrate specificity and symmetry-related
triplets, we predicted a function (e.g. the transported substrates) for many MCs of A. thaliana, B. distachyon and
O. lucimarinus. To the best of our knowledge, none of
the B. distachyon and O. lucimarinus MCs have been
functionally characterized until now. In A. thaliana the
function of several members of the family has been experimentally investigated. In Table 3 the conclusions of these
studies have been compared to the function predicted in this
review. In many cases there is a perfect or partial agreement,
in others a disagreement, between the experimentally tested
and predicted functions. The transporters which displayed
unexpected functions can be split into two groups; those
displaying novel substrate specificity and those demonstrating similar substrate specificity to that expected but
which reside at different organellar locations. In the case of
the former it is, however, important to note that substrate
specificity is difficult to determine in the absence of highly
comprehensive experiments offering a wide range of
potential substrates under appropriate conditions.
A phylogenetic tree of all the MCs of A. thaliana, H. sapiens
and S. cerevisiae deduced from genomic analysis is
presented in Figure 3a (the protein sequences can be found
in Supporting Information). This figure shows that the MCs
of these three evolutionarily distant species (146 carriers
altogether), based on their sequence similarity, cluster into
many different clades suggesting a large variety of specialized functions. Indeed, the MCF is highly divergent, full
alignment showing only five identical amino acids (with a
frequency between 92 and 99% of the analyzed samples) and
only 11 highly conserved amino acids. However, nearly all
clades include members of A. thaliana, H. sapiens and
S. cerevisiae, with the following exceptions: the GTP/GDP
carrier belongs only to fungi, the AT3G20240 and
AT4G32400 gene products only to plants, the succinatefumarate clade to plants and fungi, and the clade of UCP to
animals and plants. The fact that the great majority of closely
related sequences are present in all three kingdoms shows
that the common ancestor of all eukaryotes already pos-
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Mitochondrial carriers 169
Figure 2. (Continued)
sessed many MC functions that have been retained in animals, plants and yeast. In other words, many MC functions
existed before the speciation events that have produced the
three kingdoms.
Several other interesting features became apparent from
this analysis. The most recent branches of the tree cluster
species-specific carriers. These intraspecies parologs are
more similar to each other than to their interspecies
potential/verified orthologs. For example, AtSAMC1 is
closer to AtSAMC2 than to human SAMC1 or yeast Sam5p.
This suggests that intraspecies paralogs originated from
duplications which occurred independently in each of the
three lineages. Interestingly, in several clades A. thaliana
exhibits a larger number of paralogs than do H. sapiens and
S. cerevisiae. The duplication, retention and differentiation
of the A. thaliana paralogs could reflect specific functional
requirements of plants as compared to fungi and animalia
(which could, for example, be due to the presence of
different plastid types in plants).
A phylogenetic tree of all the MCs of A. thaliana, B. distachyon and O. lucimarinus (Figure 3b; the protein
sequences can be found in Tables S2–S6) shows that the
possibility to correctly detect orthologs between plants is
greater than between plants and animalia or yeast, which is
in agreement with the fact that the three analyzed plant
species are more similar to each other than to species of
different kingdoms. Therefore, at this level of resolution,
once a MC is characterized in a model species (e.g. A. thaliana), the one-to-one ortholog may most likely be traced in
another member of the green lineage such as B. distachyon
or O. lucimarinus. Further points of interests concern the
repertoire of MCs which is not identical in different plants as
independent duplications occurred in all the species (less in
A. thaliana as confirmed by synteny analysis; see below).
Furthermore, like A. thaliana (Figure 3a), B. distachyon and
O. lucimarinus also display more paralogs than H. sapiens
and S. cerevisiae, particularly in the clades grouping carriers
for nucleotides and amino acids. Therefore, this trait is
common in plants as compared with animals and fungi.
By contrast to Figure 3a, Figure 3b shows that the most
recent branches frequently group carriers from different
plant species although some lineage-specific duplications
are detectable. Finally, to summarize, the repertoire of MCs
in the existing eukaryotes derives from a set of carriers
which were already functionally specialized in the ancestral
eukaryote and later, after several speciation events, underwent independent rounds of duplication in all the kingdoms
including plants.
A few additional remarks concerning the early MC evolution are worth mentioning. MCs are unambiguously recog-
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170 Ferdinando Palmieri et al.
Table 2 MCs present in each chromosome of plant genomes
A. thaliana
M. truncatula
G. max
B. distachyon
S. bicolor
Z. mays
O. lucimarinus
mbp MC No. mbp MC No. mbp MC No. mbp MC No. mbp MC No. mbp MC No. mbp
755 0
73 14
77 5
74 10
68 9
62 2
62 5
64 3
55 1
59 7
60 4
– –
– –
– –
– –
– –
– –
– –
– –
– –
– –
– –
– –
1414 60
14 0
300 15
234 4
230 7
247 6
216 9
169 8
170 4
174 7
152 10
149 3
– –
– –
– –
– –
– –
– –
– –
– –
– –
– –
– –
– –
2061 73
C. reinhardtii
MC No. mbp MC No.
0.895 2
0.982 4
0.930 0
0.847 0
0.818 1
0.783 3
0.701 1
0.670 1
0.613 3
0.593 2
0.538 4
0.528 3
0.708 1
0.468 1
0.428 2
0.366 2
0.149 0
0.154 0
0.549 1
0.321 3
The data were retrieved from Ensembl-plants ( for A. thaliana and B. distachyon, NCBI and PGDD (http:// for O. lucimarinus, and Phytozome ( for the other species. The ID limit used with
Biomart/Ensembl was the IPR-ID ‘IPR001993’ and with Biomart/Phytozome the PFAM-ID ‘PF00153’. All the sequences were validated by protein
blast analysis on the non-redundant database ( The number of MCs refers to sequences which are longer
than 265 amino acids and non-redundant. chr, chromosome; mbp, mega base pairs.
nized by their sequence features: a tripartite structure, a
three-fold repeated signature motif, and six transmembrane
a-helices (two in each of the three repeats) separated by
hydrophilic loops. These structural features clearly indicate
that MCs result from the tandem triplication of a primordial
100 amino acid two-helix domain (Kuan and Saier, 1993;
Palmieri, 1994; Fiermonte et al., 1999). Moreover, given that
a low-grade sequence similarity is also detectable in the two
helices of each repeat, it might be hypothesized that the
primordial repeat may itself have evolved by duplication of a
DNA sequence encoding a single transmembrane segment.
It should be noted, however, that in the MCs identified in
plants, fungi and animalia there is insufficient evidence to
assess whether this similarity results from homology or
convergent evolution.
In the past, besides sequence similarity, the location of
the introns in MC genes has also been exploited to
investigate the evolution of MCF members. It was
observed that introns tend to interrupt the coding
sequence of the human citrate, carnitine and dicarboxylate
carrier genes at positions corresponding to protein folding
in or near the hydrophilic loops in the MC amino acid
sequences (Iacobazzi et al., 1997, 1998; Fiermonte et al.,
1999). To verify this tendency at a genome-wide level, we
extended the analysis to the entire set of 58 A. thaliana
MC genes. After having generated the multiple protein
sequence alignment of the 58 MCs, assigned the predicted
membrane folding and the position of the intron sites
relative to the aligned residues, the intron density (i.e. the
number of introns per residue) in protein regions with
distinct structure assignment was measured. The results
revealed that hydrophilic loops host a notable 1.8-fold
excess in intron density (0.021 intron sites/residue) compared with transmembrane helices (0.012 intron sites/
residue, chi-squared test P-value <10–4). The most straightforward interpretation of this finding is that intron gain
events, which may also modify the coding sequence, are
more frequently retained in loop-coding sequences than in
helix-coding sequences. In other words, given that transmembrane helices are the most conserved regions in MCs,
intron sites can be under-represented in sequences encoding transmembrane helices due to negative selection.
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Mitochondrial carriers 171
Table 3 Function prediction of plant MCs based on symmetry-related triplets of the MC subfamilies defined by substrate specificity in
H. sapiens, S. cerevisiae or A. thaliana
Experimentally tested
A. thaliana
B. distachyon
O. lucimarinus
In (partial)
Unknown1 (AAC?)
Unknown2 (AAC?)
Unknown3 (AAC?)
Unknown4 (CoA/PAP?)
Unknown5 (CoA/PAP?)
Unknown6 (Nt)
Unknown7 (Nt)
Unknown8 (Nt)
Unknown9 (Ant1?)
Unknown10 (Ant1?)
Unknown11 (Nt)
Unknown12 (Nt)
Unknown13 (Nt)
Unknown14 (CoA/PAP?)
Unknown15 (CoA/PAP?)
Unknown16 (CoA/PAP?)
Unknown17 (CoA/PAP?)
Unknown18 (Nt)
Unknown19 (Nt)
Unknown20 (Nt)
Unknown21 (TPC?)
Unknown22 (TPC?)
FAD1 (folate?)
Unknown23 (NT)
Folate (folt1)
Unknown24 (NT)
Unknown25 (DIC?)
Unknown26 (DTC?)
Unknown27 (acids)
Unknown28 (acids)
Unknown29 (acids)
Unknown30 (acids)
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Millar and Heazlewood (2003)
and Haferkamp et al. (2002)
Millar and Heazlewood (2003)
and Haferkamp et al. (2002)
Haferkamp et al. (2002)
Leroch et al. (2008)
Thuswaldner et al. (2007)
Arai et al. (2008) and
Linka et al. (2008)
Arai et al. (2008) and
Linka et al. (2008)
Kirchberger et al. (2008)
Palmieri et al. (2008b)
Bedhomme et al. (2005)
Palmieri et al. (2009)
Palmieri et al. (2009)
Fukao et al. (2001) and
Linka et al. (2008)
Palmieri et al. (2008a)
Palmieri et al. (2008a)
Palmieri et al. (2008a)
Picault et al. (2002)
Catoni et al. (2003)
Hoyos et al. (2003)
Hoyos et al. (2003) and
Palmieri et al. (2006b)
172 Ferdinando Palmieri et al.
Table 3 (Continued)
Experimentally tested
A. thaliana
B. distachyon
O. lucimarinus
In (partial)
Unknown31 (ornithine)
Unknown32 (amino acid)
Carnitine (CAC)
Unknown33 (amino acid)
Unknown34 (amino acid)
Unknown35 (amino acid)
Unknown36 (samc?)
Unknown37 (samc?)
Unknown38 (samc?)
Unknown39 (amino acid)
Unknown40 (phosphate?)
Unknown41 (phosphate?)
Unknown42 (phosphate?)
Lawand et al. (2002) and
Millar and Heazlewood (2003)
Palmieri et al. (2006a)
and Bouvier et al. (2006)
Palmieri et al. (2006a)
and Bouvier et al. (2006)
Borecký et al. (2001) and
Hanak and Jezek (2001)
Hanak and Jezek (2001)
Hamel et al. (2004) and
Millar and Heazlewood (2003)
Hamel et al. (2004) and
Millar and Heazlewood (2003)
Hamel et al. (2004) and
Millar and Heazlewood (2003)
The function of A. thaliana, B. distachyon and O. lucimarinus MC genes was predicted on the basis of the symmetry-related triplet sets reported in
Table 1, and the predicted function was compared with the experimentally tested function if available. When the function could not be predicted,
the most likely function based on similarity to the triplet sets of Table 1 was given in parenthesis with a question mark; in some cases only the most
probable class of substrates (nucleotides (Nt), acids or amino acids) was indicated. For the acronyms under the heading ‘predicted’ see Table 1; for
the acronyms under the heading ‘experimentally tested’ see references.
Whole genome comparisons reveal the existence of collinear regions, which consist of genetic loci that co-localize in
the same or similar order between distinct genomic
portions. We exploited the presence of MC genes in collinear
regions of the alga O. lucimarinus, the dicot A. thaliana and
the monocot B. distachyon as model systems of green
plants (Figure 4). In O. lucimarinus six MC genes (FAD1–
FAD2, unk21–unk22, unk36–unk37) are traced in collinear
regions found between chromosomes 13 and 21 (Figure 4a).
It is known that O. lucimarinus chromosome 21 derives from
a duplication event and subsequent fusion of the ancestral
chromosomes 9 and 13 and that this event post-dates the
divergence between O. lucimarinus and O. tauri (Palenik
et al., 2007). Therefore, the chromosome 21-inserted FAD2,
unk22 and unk37 are a recent acquisition of O. lucimarinus,
while their counterparts on chromosome 13 are the one-toone orthologs of O. tauri. These three gene pairs in O. lucimarinus are identical both at nucleotide and amino acid
levels. Therefore the increase in gene dosage should produce higher levels of gene products unless mechanisms of
dosage compensation silence one of the two paralogs.
In angiosperms recurrent events of whole genome
duplications (WGDs) occurred at different evolutionary
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Mitochondrial carriers 173
Figure 3. Phylogenetic trees of MCs.
The trees of H. sapiens (53), S. cerevisiae (35) and A. thaliana (58) MCs (a) and of B. distachyon (55), O. lucimarinus (40) and A. thaliana (58) MCs (b) originated from
ClustalW multiple-sequence alignments by using the neighbor-joining method implemented in MEGA4 (Tamura et al., 2007). Bootstrap values for 1000 replicates
are reported on each node; gene names and aliases describing the function on each terminal node. MC subgroups are coloured in yellow (transporting nucleotides),
orange (dinucleotides), red (acids), green (UCP), blue (amino acids) and purple (phosphate).
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174 Ferdinando Palmieri et al.
Figure 4. MCs in collinear regions.
(a–c) Intra-genome comparison. Genes encoding MCs are superimposed on the chromosomes of O. lucimarinus (a), A. thaliana (b) and B. distachyon (c) according
to the genomic coordinates (chromosome size is in scale). Lines connect paralogs in collinear regions duplicated by segmental duplication (red lines) and whole
genome duplication (WGD) (blue lines). Regions duplicated by WGD were gathered from literature data on A. thaliana (Van de Peer et al., 2009) and B. distachyon
(International Brachypodium Initiative., 2010). (d) Inter-genome comparison. The circle represents the chromosomes of A. thaliana (yellow) and B. distachyon
(blue). Genes in regions with conserved gene order between the two genomes (synteny blocks) with lines pairing orthologs are reported. One-to-one relationships
between orthologs are indicated by gray lines and one-to-two relationships by green lines. For all panels, circles were generated with Circos (Krzywinski et al., 2009);
gene pairs in collinear regions were recovered from PLAZA (; Proost et al., 2009) for O. lucimarinus and from PGDD (http:// for A. thaliana and B. distachyon; for gene names and colors see Table 3 and Figure 3, respectively (unk, unknown).
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Mitochondrial carriers 175
Figure 5. Co-expression analysis of genes encoding MCs found in genomic databases of A. thaliana.
Heatmap of gene expression data and clustering analysis of the corresponding gene expression dataset obtained from the expression browser tool at the Bioarray
Resource (BAR;; Toufighi et al., 2005) were performed using MULTEXPERIMENT VIEWER software (Saeed et al., 2003). The analysis was
performed with 56 carriers of A. thaliana. The conditions tested are according to the AtGenExpress Plus-Extended Tissue Series dataset defined by BAR including
both a developmental series and a wide variety of tissue types. Abbreviations: VR, vegetative rosette; d, development; X/C, xylem/cork; O þ S, ovary þ stigma;
E þ WS, epidermis þ whole stem; G þ MC, guard þ mesophyll cells.
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176 Ferdinando Palmieri et al.
times (see Soltis et al. (2008) and Freeling (2009) for
reviews). In spite of several WGD events, only three pairs
of MC paralogs (MRS3–MRS4, unk9–unk10, unk18–unk20)
are detectable in collinear regions of A. thaliana (Figure 4b). This apparent discrepancy can be explained by
the high rate of gene loss and gene rearrangements in
A. thaliana that reduced the total number of genes and
degenerated homologous segments (Vandepoele et al.,
2002; Thomas et al., 2006). According to a model of the
ancestral A. thaliana genome (Van de Peer et al., 2009), the
location of MRS3–MRS4 and unk9–unk10 on chromosome
pairs 1–2 and 3–5, respectively, should indicate that these
genes represent a lineage-specific acquisition not shared
with other dicots. By contrast, the location of the unk18–
unk20 pair on chromosomes 2–3 should be the result of a
segmental duplication.
The genome of B. distachyon hosts more pairs (six) of MC
paralogs in collinear segments compared to the A. thaliana
genome (Figure 4c), although the latter underwent a higher
number of WGDs. The higher conservation of collinearity
in B. distachyon should reflect, at least in part, a lower rate of
gene loss and chromosome rearrangement in B. distachyon
than in A. thaliana. Among the B. distachyon MC paralog
pairs, three (DIC1–DIC2, DTC–unk26 and unk12–unk13) are
located in regions duplicated by the WGD at the base of
cereal diversification (International Brachypodium Initiative
2010). These paralogs are therefore a common characteristic
of cereals that is not expected in more evolutionary distant
angiosperms. The expansion of the PiC subfamily and the
acquisition of unk11–unk12 in B. distachyon post-dates the
divergence of cereals, showing that they are recent gains
of the B. distachyon lineage. In summary, the intra-genome
comparisons discussed above show that WGDs and segmental duplications have contributed to expand the gene
repertoire of MCs in green plants. The observation that some
MC paralogs escaped the large-scale gene loss following
WGD events (Freeling, 2009) reinforces the idea that these
paralogs are functionally important and contribute to plant
complexity and diversification.
A major goal of inter-genome comparisons is to unveil
orthology relationships. Conserved synteny (i.e. detection of
collinear segments between different genomes) is a reliable
tool to identify one-to-one orthologs, especially in the case
of large gene families such as that of MCs. Extensive
conserved synteny is reported within dicots and monocots,
but much less between the two clades because breaks in
collinearity are the natural effect of a greater evolutionary
distance (Tang et al., 2008). Even though approximately 500
million years separate monocots and dicots from the
common ancestor of the angiosperm, 15 MCs in A. thaliana
and 13 in B. distachyon are present in conserved synteny
blocks (Figure 4d). Most of these genes are related by a
one-to-one relationship that connects the (two most likely)
orthologs in the two species. In addition, one-to-two
relationships were also found, which associate 10 MCs in
the two genomes, nine of which are specific for nucleotides
(Figure 4d). The preferential retention of nucleotide carriers
suggests that their expansion is tolerated in angiosperms or,
more likely, that they functionally contribute to angiosperm
Expression and localization
Despite the fact that only a relatively limited number of plant
transporters have been characterized at the biochemical
level, a wealth of information is available in databases such
as ATGenExpress, Aramemnon and SUBA. Figure 5 displays
a heatmap documenting the relative expression levels of 56
members of this family in A. thaliana within a wide range of
tissues. When these data are evaluated from a functional
perspective the following conclusions can be drawn. Several of the transporters (for example AT5G66380_FOLT1,
AT5G01340_SFC1, AT4G39460_SAMC2, AT1G25380_NDT2
and AT4G27940) are expressed constitutively across tissue
types indicating that they perform essential housekeeping
functions. However, the vast majority of the biochemically
characterized members of the MCF are differentially expressed
among different cell types. Notably, AT4G39460_SAMC1
(Bouvier et al., 2006; Palmieri et al., 2006a) is highly
expressed in mature pollen and developing seeds commensurate with the acknowledged import of the precursor
methionine to these tissues (Gallardo et al., 2002; Palmieri
et al., 2006a). Of the other transporters three separate classes were distinguishable. Those which were predominantly
expressed in pollen, seeds and vegetative rosettes were
AT5G46800_CAC, AT2G22500_DIC1, AT4G24570_DIC2,
AT2G47490_NDT1, AT3G08580_AAC1, AT5G13490_AAC2,
AT1G79900_BAC2 and a few, as yet, uncharacterized proteins, whilst those which were predominantly expressed in
embryo and seedling stages as well as those expressed in
heterotrophic root and stem tissue generally corresponded
to genes of unknown function. The only clear exception to
this observation is the fact that the functionally characterized dicarboxylate transporters (Palmieri et al., 2008a)
display differential behavior, with AT2G22500_DIC1 and
AT4G24570_DIC2 being highly expressed in pollen and
seeds but not in roots or stems whereas AT5G09470_DIC3
is massively expressed in roots and stems but not in
pollen, demonstrating the importance of both tissue specific
expression and plant-tissue communication.
Having studied the tissue specificity of the MCF protein
expression we next turned our attention to their levels of
expression under a range of stresses by evaluating data
from AtGenExpress concerning exposure to cold, osmotic,
salt, drought, genotoxic, oxidative, UV-B, wounding and
heat stress (Figure S1). Interestingly, of the 28 A. thaliana
MCF members whose function has been experimentally
ª 2011 The Authors
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Mitochondrial carriers 177
investigated, relatively few (six or seven) are strongly
transcriptionally regulated (either in root or shoot tissue).
These MCs were AT5G09470_DIC3, AT5G13490_AAC2,
AT3G48850_PiC2, AT1G79900_BAC2, AT2G22500_DIC1,
AT4G24570_DIC2 and AT2G47490_NDT1 with all other
members largely exhibiting constitutive patterns of expression. Of the seven transporters highlighted above, four
(AT3G48850_PiC2, AT1G79900_BAC2, AT2G22500_DIC1 and
AT4G24570_DIC2) display broadly similar expression profiles being highly upregulated under conditions of cold,
osmotic and salt stress whereas the other three were
generally characterized as displaying fewer and more specific changes. Indeed, they were generally downregulated.
The transcript levels of AT3G54110_UCP1 and AT5G58970_UCP2 were not induced under the different stress
conditions tested (Figure S1). This finding is in agreement
with a recent study by Van Aken et al. (2009) that suggests
that UCP proteins are not amongst the most widely stressresponsive mitochondrial proteins.
Notably, the expression profile in roots under different
stress conditions seems to be independent of the changes
observed in shoots (Figure S1). For example, when the
expression profile of AT3G48850_PiC2 is compared distinct
patterns are apparent in roots and shoots. While in roots
only a moderate increase in expression was observed after
6-h exposure to salt stress, in shoots gene expression was
highly upregulated by cold, osmotic, salt, oxidative, UV-B
and wounding stresses. Differential expression patterns
between roots and shoots are also apparent for
AT2G22500_DIC1, AT4G24570_DIC2 and AT5G27520_PNC2
under cold and salt stresses in roots and cold, osmotic,
salt, oxidative, UV-B and wounding stresses in shoots. Thus,
under the stress conditions tested the expression responses
in shoots seem to be more affected by environmental
changes than in roots.
Whilst these database resources are highly useful in
obtaining an overview of transcriptional regulation of the
MCs, further information can be obtained from focused
studies. For example GUS staining of Arabidopsis plants
transformed with a AT4G39460_SAMC1 promoter-GUS
fusion revealed a clear upregulation of its expression
following wounding (Palmieri et al., 2006a), whereas GUS
staining and RT-PCR analysis indicated that transcription
of AT1G79900_BAC2 is upregulated by stress (Toka et al.,
2010). It thus seems likely that the sensitivity of targeted
studies may well be necessary to elucidate subtle differences in the level and site of expression of at least some of
the plant MCs. Additionally, as mentioned above, Van Aken
et al. (2009) demonstrated that the MCs are one of
the most represented families of mitochondrial proteins
under conditions of stress. The MCF members found to be
upregulated in response to stress were the phosphate
transporter (AT3G48850_PiC2), ADP/ATP exchanger (AT4G28390_AAC3), dicarboxylic acid transporter (AT2G22500_
DIC1, AT4G24570_DIC2, AT4G27940), the peroxisomal
adenine nucleotide carrier (AT5G27520_PNC2) and a
mitochondrial substrate carrier of unknown function
(AT5G61810). However, it is clear that these studies
only cover a minority of members of plant MCs and that
much research is required to improve our understanding
of the precise function of each and every member of the
Despite the difficulties encountered in studying isolate,
intact and highly purified organelles, several proteomics
studies have been performed with isolated organelles;
the results obtained have been used to generate databases, such as SUBA (
request.ep). These studies have revealed the presence of
MCF members in several different plant organelles (Millar
and Heazlewood, 2003; Eubel et al., 2008; Linka et al., 2008).
We have summarized all available information concerning
the subcellular location of MCF proteins in plants (Table S1).
There exists a minority of MCs that are not localized to
mitochondria as their name would suggest (Palmieri, 1994;
Haferkamp, 2007). Recently it has been demonstrated that
MCF proteins are localized in plant plastids (Bedhomme
et al., 2005; Bouvier et al., 2006; Kirchberger et al., 2008;
Palmieri et al., 2009), peroxisomes (Fukao et al., 2001;
Palmieri et al., 2001b; Arai et al., 2008; Eubel et al., 2008;
Linka et al., 2008) and the endoplasmic reticulum of Arabidopsis (Leroch et al., 2008). In many cases these findings
were in contrast to their bioinformatically predicted locations and thus strongly support the contention that the
determination of subcellular localization of MCF proteins
should be confirmed experimentally by methods beyond
those afforded by potentially ambiguous computer-based
predictions (Millar et al., 2009).
In planta function
The above sections have detailed impressive advances in
identifying the evolution of the MCF in plants as well as
its biochemical properties, characteristics of expression and
subcellular location of its members. Whilst our level of
knowledge lags behind that apparent in the microbial and
mammalian fields, the recent adoption of genomic and
comparative-genomic approaches has ensured immense
progress in furthering understanding of the plant MCF in
recent years. The availability of knock-out mutants in Arabidopsis and rice has additionally facilited studies to ascertain the in planta role of the various MCF proteins. To date,
the reverse genetic analysis of five MCF proteins has been
reported as summarized below.
In the knockout mutant of AT3G54110_UCP1, which
dissipates the proton gradient across the inner mitochondrial membrane, a restriction in photorespiration and a
subsequent reduction in the rate of photosynthetic carbon
assimilation were detected (Sweetlove et al., 2006). The
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
178 Ferdinando Palmieri et al.
knockout mutant of AT4G01100_ADNT1, which catalyzes
the exchange of AMP for ATP (Palmieri et al., 2008b),
displayed no clear photosynthetic phenotype, but it was
characterized by a remarkable reduction in root growth
suggesting an important role for this protein in supporting
growth in plant heterotrophic tissues (Palmieri et al.,
2008b). The knockout mutant of AT5G66380_FOLT1, which
catalyzes the transport of folate into chloroplasts, displayed no difference in terms of germination efficiency,
growth rate, morphology, seed production and fertility or
rates of photosynthesis and respiration (Bedhomme et al.,
2005), which seemingly indicates that its function can be
compensated for by another folate chloroplast transport
protein, for example by the AT2G32040 protein which does
not belong to the MCF and is structurally similar to
cyanobacterial plasma membrane folate transporters
(Klaus et al., 2005). The characterization of a knockout
mutant for AT1G79900_BAC2 (Toka et al., 2010) showed
that AT1G79900_BAC2 can work as a hyperosmotic stressinducible transporter of basic amino acids and that it
contributes to proline accumulation in response to hyperosmotic stress in Arabidopsis. The phenotype of the ‘à
bout de souffle’ Arabidopsis mutant is similar to that of
mutants defective in peroxisomal fatty acid b-oxidation
suggesting that it is an acyl-carnitine carrier (Lawand et al.,
2002). However, the transported substrate for this protein
has not yet been identified. Given that AT5G46800_CAC is
structurally highly similar to BAC carriers of plants it
remains possible that this carrier is also involved in amino
acid transport (Hoyos et al., 2003; Palmieri et al., 2006b;
Linka and Weber, 2010).
Understanding the evolution, structure and function of the
MCF members in plants, in general, is strongly tied not only
to understanding their role in the model plant A. thaliana
but also in microbial and mammalian systems. Impressive
progress has recently been made on substrate specificity,
modes of transport, expression and localization of these
carriers in Arabidopsis (Picault et al., 2004; Linka and Weber,
2010). However, increased utilization of advanced genomic
tools applied to a wider range of green lineages will likely
allow us to address numerous functional genomic issues.
What is clear from the information presented in this study is
that there is a high level of conservation in gene function
across species and even kingdoms with many of the MCF
members clearly having been in existence in the ancestral
eukaryote from which all kingdoms derive. This important
finding suggests that it is often relatively easy to predict oneto-one orthologs of MCs from different species in the green
lineage. However, due to different rates of genome-wide
duplication and/or gene loss this is not always the case.
Despite the great progress made in recent years, a number
of important questions need to be answered in order to
understand the function of the plant MC subfamilies of
various divergent plant species. The most pressing challenge is probably to gain more information concerning the
substrate specificities of each and every transporter. In order
to achieve this, heterologous expression of transporters and
detailed biochemical characterization will be required as
well as experiments involving isolated plant mitochondria.
An additional and equally important question is to elucidate
the in vivo function of all MCs which will require the isolation
and characterization of knockout mutants of all the family
proteins. Due to the possibility of functional redundancy, the
generation and characterization of multiple mutants will also
be necessary. The type of characterization will be largely
dependent on their location of expression; a survey of the
data presented here will be an useful resource to obtain
information about which tissues and/or cellular conditions
are the focus of future studies. Once this essential information is achieved we will be able to clarify the functional
importance of the events underlying the evolution of this
gene family.
This work was supported by grants from MIUR, the Center of
Excellence in Genomics (CEGBA), the Fondazione Cassa di Risparmio di Puglia, the Apulia Region, and the Italian Human ProteomeNet No. RBRN07BMCT_009.
This paper is dedicated to the memory of Professor Gian
Tommaso Scarascia Mugnozza.
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Co-expression analysis of genes encoding characterized
MCs of A. thaliana.
Table S1. Subcellular localization of functionally characterized
mitochondrial carriers from plants.
Table S2. A. thaliana_58 sequences.
Table S3. B. distachyon_55 sequences.
Table S4. O. lucimarinus_38 sequences.
Table S5. H. sapiens_53 sequences.
Table S6. S. cerevisiae_35 sequences.
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ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181