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The Plant Journal (2011) 66, 161–181
doi: 10.1111/j.1365-313X.2011.04516.x
THE PLANT GENOME: AN EVOLUTIONARY VIEW ON STRUCTURE AND FUNCTION
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,
2
European Institute of Oncology, IFOM-IEO Campus, Via Adamello 16, 20139 Milan, Italy,
3
Max-Planck Partner Group, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, 36570-000 Viçosa,
MG, Brasil, and
4
Max-Planck-Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany
1
Received 3 November 2010; accepted 25 January 2011.
*
For correspondence (fax +39 080 5442770; e-mail [email protected]).
SUMMARY
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.
INTRODUCTION
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
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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.
STRUCTURE AND TRANSPORT MECHANISM
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
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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
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841
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).
EXTENSION OF THE MCF
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
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Mitochondrial carriers 165
Table 1 MC subfamilies defined by substrate specificity and symmetry-related amino acid triplets
Subfamilies
Aliases
1. For nucleotides and dinucleotides
ADP/ATP
AAC
Coenzyme A/PAP
Coa/PAP
ATP-Mg/Pi
APC
Main substrates
References
Triplets
ADP, ATP
Klingenberg (2008) and
Fiore et al. (1998)
Coa, PAP,
dephospho-coa, AXP
ATP-Mg, Pi, 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),
88(KDS)
23(R[T/S]K), 34(IT[K/R]),
80 (L[A/T]K), 85(GAT)
Thiamine
pyrophosphate
TPC
Thpp, thmp;
(d)NDP, (d)NTP
Pyrimidine
nucleotides
PNC
Pyrimidine
(deoxy)nucleotides
FAD/folate
FAD
Folates, FAD
ANT
ANT
ATP, ADP, AMP
NAD+
NDT
NAD+, (d)AMP, (d)GMP
GTP/GDP
GGC
GTP, GDP, dgtp,
dgdp, ITP, IDP
2. For di-/tri-carboxylates and keto acids
Dicarboxylates
DIC
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,
thiosulfate
Palmieri et al. (1996),
Fiermonte et al. (1998b)
and Palmieri et al. (2008a)
Oxoglutarate
OGC
Oxoglutarate, malate
Indiveri et al. (1987) and
Fiermonte et al. (1993)
Di-/tri-carboxylates
DTC
Oxoglutarate, citrate
Picault et al. (2002)
Succinate/fumarate
SFC
Succinate, fumarate
Citrate
CTP
Oxodicarboxylates
ODC
Citrate, malate, isocitrate,
cis-aconitate, PEP
Oxoadipate, oxoglutarate
Palmieri et al. (1997b) and
Catoni et al. (2003)
Kaplan et al. (1993, 1995)
Oxaloacetate/sulfate
OAC
Oxaloacetate, sulfate,
thiosulfate,
a-isopropylmalate
Palmieri et al. (1999b) and
Marobbio et al. (2008)
GC
Glutamate
Fiermonte et al. (2002)
AGC
Aspartate, glutamate,
cysteinesulfinate
Palmieri et al. (2001c) and
Cavero et al. (2003)
3. For amino acids
Glutamate
Aspartate/glutamate
Palmieri et al. (2001a) and
Fiermonte et al. (2001)
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843
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)
Subfamilies
Aliases
Main substrates
References
Triplets
Ornithine
ORC
Ornithine, (lysine,
citrulline, arginine,
histidine)
Palmieri et al. (1997a),
Hoyos et al. (2003) and
Fiermonte et al. (2003)
Carnitine
CAC
Carnitine, acylcarnitines
S-adenosylmethionine
SAMC
S-adenosylmethionine,
S-adenosylhomocysteine
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)
Pic
Phosphate
4. For other substrates
Phosphate
UCP
UCP, KMCP
Unnamed
MRS3-4,
MFRN1-2
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
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
844
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).
SIZE OF THE MCF IN PLANT GENOMES
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.
FUNCTION PREDICTION OF MCS IN A. THALIANA,
B. DISTACHYON AND O. LUCIMARINUS
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
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
845
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.
EVOLUTION OF MCS DEDUCED BY PHYLOGENETIC TREES
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-
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
846
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-
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
847
170 Ferdinando Palmieri et al.
Table 2 MCs present in each chromosome of plant genomes
Dicots
Monocots
A. thaliana
Chr
M. truncatula
G. max
Algae
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
0
–
1
30
2
19
3
23
4
18
5
26
6
–
7
–
8
–
9
–
10
–
11
–
12
–
13
–
14
–
15
–
16
–
17
–
18
–
19
–
20
–
21
–
Unknown
–
Total
119
–
9
10
9
10
20
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
58
17
33
32
44
41
43
23
35
37
–
–
–
–
–
–
–
–
–
–
–
–
–
–
308
0
5
3
9
7
7
1
2
3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
37
29
55
51
47
49
41
50
44
46
46
50
39
40
44
49
50
37
41
62
50
46
–
–
979
0
6
10
6
9
8
8
9
15
4
3
3
2
5
6
3
7
5
4
6
6
–
–
125
–
74
59
59
48
28
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1
272
–
17
17
10
8
4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0
55
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.
–
–
1
4
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
–
–
14
38
–
9
9
7
3
3
7
6
4
4
6
2
9
6
4
3
6
6
–
–
–
–
10
112
–
2
3
3
3
0
9
3
0
4
3
0
0
0
0
1
3
0
–
–
–
–
3
37
The data were retrieved from Ensembl-plants (http://plants.ensembl.org/index.html) for A. thaliana and B. distachyon, NCBI and PGDD (http://
chibba.agtec.uga.edu/duplication/) for O. lucimarinus, and Phytozome (http://www.phytozome.net/) 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 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). 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.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
848
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
Genes
Function
Experimentally tested
A. thaliana
B. distachyon
O. lucimarinus
Predicted
In (partial)
agreement
In
disagreement
At3g08580
BD1G29260
OL10G01660
AAC1
AAC1
–
At5g13490
BD3G53520
–
AAC2
AAC2
–
At4g28390
At5g17400
At5g56450
At1g78180
At5g64970
At5g61810
At5g51050
At5g07320
At3g51870
At5g01500
At3g20240
–
–
At3g05290
–
–
BD2G18347
BD1G71800
–
BD1G36570
–
–
BD2G09790
–
BD3G40497
BD4G34100
BD5G26776
BD2G26480
–
–
OL03G05390
–
–
–
–
–
OL10G01620
–
OL02G00320
OL12G03180
–
–
AAC3
AAC4
Unknown1 (AAC?)
Unknown2 (AAC?)
Unknown3 (AAC?)
APC1
APC2
APC3
Unknown4 (CoA/PAP?)
Unknown5 (CoA/PAP?)
Unknown6 (Nt)
Unknown7 (Nt)
Unknown8 (Nt)
Unknown9 (Ant1?)
AAC3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
ER-ANT1
–
–
–
–
–
–
–
TAAC
–
–
–
PNC1
At5g27520
–
–
Unknown10 (Ant1?)
–
PNC2
At4g32400
–
–
At4g01100
At1g14560
At4g26180
–
At3g53940
At3g55640
At2g37890
At3g21390
At5g48970
At5g66380
–
At2g47490
At1g25380
At2g39970
BD2G34270
BD1G36670
BD3G07500
BD2G14840
BD2G47880
BD3G08557
BD2G41500
BD2G02880
BD1G66990
–
BD2G59930
–
BD1G09560
–
BD2G28120
–
BD1G69370
OL03G01320
OL12G00350
OL15G01450
OL03G04280
OL08G02620
–
–
OL07G00540
–
–
OL13G02340
OL21G00660
OL13G02140
OL21G00840
OL11G01860
–
OL20G02530
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?)
FAD2
NDT1
NDT2
Unknown23 (NT)
–
–
–
–
–
–
–
–
–
–
–
–
Folate (folt1)
–
NDT1
NDT2
–
ATBT1
–
–
ADNT1
–
–
–
–
–
–
–
–
–
–
–
–
PMP38
–
At2g22500
At4g24570
At5g09470
–
At5g19760
At4g03115
At5g01340
At4g11440
At5g42130
–
–
At2g33820
At1g79900
BD1G67180
BD3G38410
BD4G32510
–
–
BD2G06520
BD2G32600
BD1G65510
–
BD2G61677
–
–
BD1G15960
BD2G07420
–
–
–
–
OL14G02490
OL10G00520
–
OL07G03670
–
OL16G01220
OL03G02490
OL11G02620
OL01G00440
–
Unknown24 (NT)
DIC1
DIC2
DIC3
Unknown25 (DIC?)
DTC
Unknown26 (DTC?)
SFC1
Unknown27 (acids)
Unknown28 (acids)
Unknown29 (acids)
Unknown30 (acids)
ORNITHINE1
ORNITHINE2
–
DIC1
DIC2
DIC3
–
DTC
–
ATMSFC1
–
–
–
–
BAC1
BAC2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
849
References
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)
Genes
Function
Experimentally tested
A. thaliana
B. distachyon
O. lucimarinus
Predicted
In (partial)
agreement
In
disagreement
–
At5g46800
–
BD3G34077
OL02G00080
–
Unknown31 (ornithine)
Unknown32 (amino acid)
–
–
–
Carnitine (CAC)
At4g27940
At2g46320
–
At4g39460
BD3G34090
BD2G07390
BD3G34820
BD1G71410
OL12G01640
OL01G03790
–
OL01G03770
Unknown33 (amino acid)
Unknown34 (amino acid)
Unknown35 (amino acid)
SAMC1
–
–
–
SAMC1
–
–
–
–
At1g34065
BD1G01770
–
SAMC2
SAMC2
–
At1g74240
At2g26360
At2g35800
–
At3g54110
BD1G70017
BD4G45190
–
BD5G16330
BD4G09060
OL13G02410
OL21G00590
–
OL17G01690
OL07G04120
Unknown36 (samc?)
Unknown37 (samc?)
Unknown38 (samc?)
Unknown39 (amino acid)
UCP1
–
–
–
–
PUMP1
–
–
–
–
–
At5g58970
At1g14140
At2g30160
At1g07030
At2g17270
BD2G40390
BD5G11757
BD1G65170
–
BD4G37420
–
–
OL06G04180
–
OL17G01410
UCP2
UCP3
MRS3
Mrs4
Unknown40 (phosphate?)
UCP2
–
–
–
–
–
–
–
–
PIC
At5g14040
BD3G57890
–
PIC1
PIC1
–
At3g48850
BD1G67330
–
PIC2
PIC2
–
–
–
At5g26200
At1g72820
At5g15640
At4g15010
–
BD4G32020
BD5G11740
BD1G72940
BD1G08301
BD4G31560
BD2G12390
BD3G51060
OL12G02770
OL01G01320
OL09G02540
OL16G02350
–
–
–
Unknown41 (phosphate?)
Unknown42 (phosphate?)
Unknown43
Unknown44
Unknown45
Unknown46
Unknown47
–
–
–
–
–
–
–
–
–
–
–
–
–
–
References
–
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.
EVOLUTION OF MCS DEDUCED BY COMPARATIVE
GENOMICS
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
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
850
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 (http://bioinformatics.psb.ugent.be/plaza/; Proost et al., 2009) for O. lucimarinus and from PGDD (http://
chibba.agtec.uga.edu/duplication/) 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; http://www.bar.utoronto.ca; 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
evolution.
PHYSIOLOGICAL CHARACTERIZATION OF PLANT MCS
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
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854
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
family.
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 (http://suba.plantenergy.uwa.edu.au/)
and ARAMEMNON (http://aramemnon.botanik.uni-koeln.de/
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
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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).
CONCLUDING REMARKS AND OUTLOOK
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.
ACKNOWLEDGEMENTS
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.
SUPPORTING INFORMATION
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.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
REFERENCES
Agrimi, G., Di Noia, M.A., Marobbio, C.M.T., Fiermonte, G., Lasorsa, F.M. and
Palmieri, F. (2004) Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional
characterization and tissue distribution. Biochem. J. 379, 183–190.
Arai, Y., Hayashi, M. and Nishimura, M. (2008) Proteomic identification and
characterization of a novel peroxisomal adenine nucleotide transporter
supplying ATP for fatty acid beta-oxidation in soybean and Arabidopsis.
Plant Cell, 20, 3227–3240.
Bedhomme, M., Hoffmann, M., McCarthy, E.A., Gambonnet, B., Moran, R.G.,
Rebeille, F. and Ravanel, S. (2005) Folate metabolism in plants: an Arabidopsis homolog of the mammalian mitochondrial folate transporter
mediates folate import into chloroplasts. J. Biol. Chem. 280, 34823–34831.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
856
Mitochondrial carriers 179
Benz, R., Kottke, M. and Brdiczka, F.R.G. (1990) The cationically selective state
of the mitochondrial outer membrane pore: a study with intact mitochondria and reconstituted mitochondrial porin. Biochim. Biophys. Acta 1022,
311–318.
Borecký, J., Maia, I.G., Costa, A.D., Jezek, P., Chaimovich, H., de Andrade,
P.B., Vercesi, A.E. and Arruda, P. (2001) Functional reconstitution of
Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1)
expressed in Escherichia coli. FEBS Lett. 505, 240–244.
Bouvier, F., Linka, N., Isner, J.C., Mutterer, J., Weber, A.P.M. and Camara, B.
(2006) Arabidopsis SAMT1 defines a plastid transporter regulating plastid
biogenesis and plant development. Plant Cell, 18, 3088–3105.
Cappello, A.R., Curcio, R., Miniero, D.V., Stipani, I., Robinson, A.J., Kunji,
E.R.S. and Palmieri, F. (2006) Functional and structural role of amino acid
residues in the even-numbered transmembrane a-helices of the bovine
mitochondrial oxoglutarate carrier. J. Mol. Biol. 363, 51–62.
Cappello, A.R., Miniero, D.V., Curcio, R., Ludovico, A., Daddabbo, L., Stipani,
I., Robinson, A.J., Kunji, E.R.S. and Palmieri, F. (2007) Functional and
structural role of amino acid residues in the odd-numbered transmembrane a-helices of the bovine mitochondrial oxoglutarate carrier. J. Mol.
Biol. 369, 400–412.
Catoni, E., Schwab, R., Hilpert, M., Desimone, M., Schwacke, R., Flügge, U.I.,
Schumacher, K. and Frommer, W.B. (2003) Identification of an Arabidopsis
mitochondrial succinate-fumarate translocator. FEBS Lett. 53, 87–92.
Cavero, S., Vozza, A., del Arco, A. et al. (2003) Identification and metabolic
role of the mitochondrial aspartate-glutamate transporter in Saccharomyces cerevisiae. Mol. Microbiol. 50, 1257–1269.
Colombini, M. (1979) A candidate for the permeability pathway of the outher
mitochondrial membrane. Nature, 279, 643–645.
Dolce, V., Fiermonte, F., Runswick, M.J., Palmieri, F. and Walker, J.E. (2001)
The human mitochondrial deoxynucleotide carrier and its role in toxicity of
nucleoside antivirals. Proc. Natl Acad. Sci. USA, 98, 2284–2288.
Douce, R. (1985). Mitochondria in Higher Plants. Structure, Function and
Biogenesis. New York: Academic Press.
Eubel, H., Meyer, E.H., Taylor, N.L., Bussell, J.D., O’Toole, N., Heazlewood,
J.L., Castleden, I., Small, I.D., Smith, S.M. and Millar, A.H. (2008) Novel
proteins, putative membrane transporters, and an integrated metabolic
network are revealed by quantitative proteomic analysis of Arabidopsis cell
culture peroxisomes. Plant Physiol. 148, 1809–1829.
Fiermonte, G., Walker, J.E. and Palmieri, F. (1993) Abundant bacterial
expression and reconstitution of an intrinsic membrane transport protein
from bovine mitochondria. Biochem. J. 294, 293–299.
Fiermonte, G., Dolce, V. and Palmieri, F. (1998a) Expression in Escherichia
coli, functional characterization, and tissue distribution of isoforms A and B
of the phosphate carrier from bovine mitochondria. J. Biol. Chem. 273,
22782–22787.
Fiermonte, G., Palmieri, L., Dolce, V., Lasorsa, F.M., Palmieri, F., Runswick,
M.J. and Walker, J.E. (1998b) The sequence, bacterial expression and
functional reconstitution of the rat mitochondrial dicarboxylate transporter
cloned via distant homologs in yeast and Caenorhabditis elegans. J. Biol.
Chem. 273, 24754–24759.
Fiermonte, G., Dolce, V., Arrigoni, R., Runswick, M.J., Walker, J.E. and
Palmieri, F. (1999) Organization and sequence of the gene for the human
mitochondrial dicarboxylate carrier: evolution of the carrier family.
Biochem. J. 344, 953–960.
Fiermonte, G., Dolce, V., Palmieri, L., Ventura, M., Runswick, M.J., Palmieri, F.
and Walker, J.E. (2001) Identification of the human mitochondrial oxodicarboxylate carrier: bacterial expression, reconstitution, functional
characterization, tissue distribution, and chromosomal location. J. Biol.
Chem. 276, 8225–8230.
Fiermonte, G., Palmieri, L., Todisco, S., Agrimi, G., Palmieri, F. and Walker, J.E.
(2002) Identification of the mitochondrial glutamate transporter: bacterial
expression, reconstitution, functional characterization, and tissue distribution of two human isoforms. J. Biol. Chem. 277, 19289–19294.
Fiermonte, G., Dolce, V., David, L., Santorelli, F.M., Dionisi-Vici, C., Palmieri, F.
and Walker, J.E. (2003) The mitochondrial ornithine transporter: bacterial
expression, reconstitution, functional characterization, and tissue distribution of two human isoforms. J. Biol. Chem. 278, 32778–32783.
Fiermonte, G., De Leonardis, F., Todisco, S., Palmieri, L., Lasorsa, F.M. and
Palmieri, F. (2004) Identification of the mitochondrial ATP-Mg/Pi transporter: bacterial expression, reconstitution, functional characterization and
tissue distribution. J. Biol. Chem. 279, 30722–30730.
Fiermonte, G., Paradies, E., Todisco, S., Marobbio, C.M.T. and Palmieri, F.
(2009) A novel member of solute carrier family 25 (SLC25A42) is a transporter of coenzyme a and adenosine 3¢,5¢-diphosphate in human mitochondria. J. Biol. Chem. 284, 18152–18159.
Fiore, C., Trezeguet, V., Le Saux, A., Roux, P., Schwimmer, C., Dianoux, A.C.,
Noel, F., Lauquin, G.J.-M., Brandolin, G. and Vignais, P.V. (1998) The
mitochondrial ADP/ATP carrier: structural, physiological and pathological
aspects. Biochemie 80, 137–150.
Floyd, S., Favre, C., Lasorsa, F.M. et al. (2007) The IGF-I–mTOR signaling
pathway induces the mitochondrial pyrimidine nucleotide carrier to promote cell growth. Mol. Biol. Cell, 18, 3545–3555.
Freeling, M. (2009) Bias in plant gene content following different sorts of
duplication: tandem, whole-genome, segmental, or by transposition.
Annu. Rev. Plant Biol. 60, 433–453.
Fukao, Y., Hayashi, Y., Mano, S., Hayashi, M. and Nishimura, M. (2001)
Developmental analysis of a putative ATP/ADP carrier protein localized on
glyoxysomal membranes during the peroxisome transition in pumpkin
cotyledons. Plant Cell Physiol. 42, 835–841.
Gallardo, K., Job, C., Groot, S.P., Puype, M., Demol, H., Vandekerckhove, J.
and Job, D. (2002) Importance of methionine biosynthesis for Arabidopsis
seed germination and seedling growth. Physiol. Plant. 116, 238–247.
Giangregorio, N., Tonazzi, A., Console, L., Indiveri, C. and Palmieri, F. (2010)
Site-directed mutagenesis of charged amino acids of the human mitochondrial carnitine/acylcarnitine carrier: insight into the molecular mechanism of transport. Biochim. Biophys. Acta, 1797, 839–845.
Giege, P., Heazlewood, J.L., Roessner-Tunali, U., Millar, A.H., Fernie, A.R.,
Leaver, C.J. and Sweetlove, L.J. (2003) Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells. Plant Cell,
15, 2140–2151.
Haferkamp, I. (2007) The diverse members of the mitochondrial carrier family
in plants. FEBS Lett. 581, 2375–2379.
Haferkamp, I., Hackstein, J.H., Voncken, F.G., Schmit, G. and Tjaden, J. (2002)
Functional integration of mitochondrial and hydrogenosomal ADP/ATP
carriers in the Escherichia coli membrane reveals different biochemical
characteristics for plants, mammals and anaerobic chytrids. Eur. J. Biochem. 269, 3172–3181.
Hamel, P., Saint-Georges, Y., de Pinto, B., Lachacinski, N., Altamura, N. and
Dujardin, G. (2004) Redundancy in the function of mitochondrial phosphate
transport in Saccharomyces cerevisiae and Arabidopsis thaliana. Mol.
Microbiol. 51, 307–317.
Hanak, P. and Jezek, P. (2001) Mitochondrial uncoupling proteins and
phylogenesis – UCP4 as the ancestral uncoupling protein. FEBS Lett. 495,
137–141.
Hoyos, M.E., Palmieri, L., Wertin, T., Arrigoni, R., Polacco, J.C. and Palmieri, F.
(2003) Identification of a mitochondrial transporter for basic amino acids in
Arabidopsis thaliana by functional reconstitution into liposomes and
complementation in yeast. Plant J. 33, 1027–1035.
Iacobazzi, V., Lauria, G. and Palmieri, F. (1997) Organization and sequence of
the human gene for the mitochondrial citrate transport protein. DNA Seq. 7,
127–139.
Iacobazzi, V., Naglieri, M.A., Stanley, C.A., Wanders, R.J.A. and Palmieri, F.
(1998) The structure and organization of the human carnitine/acylcarnitine
translocase (CACT) gene. Biochem. Biophys. Res. Commun. 252, 770–
774.
Indiveri, C., Palmieri, F., Bisaccia, F. and Kramer, R. (1987) Kinetics of the
reconstituted 2-oxoglutarate carrier from bovine heart mitochondria. Biochim. Biophys. Acta, 890, 310–318.
Indiveri, C., Tonazzi, A. and Palmieri, F. (1990) Identification and purification of
the carnitine carrier from rat liver mitochondria. Biochim. Biophys. Acta,
1020, 81–86.
Indiveri, C., Iacobazzi, V., Giangregorio, N. and Palmieri, F. (1997) The mitochondrial carnitine carrier protein: cDNA cloning, primary structure, and
comparison with other mitochondrial transport proteins. Biochem. J. 321,
713–719.
International Brachypodium Initiative. (2010) Genome sequencing and
analysis of the model grass Brachypodium distachyon. Nature, 463, 763–
768.
Kaplan, R.S., Mayor, J.A. and Wood, D.O. (1993) The mitochondrial
tricarboxylate transport protein. cDNA cloning, primary structure, and
comparison with other mitochondrial transport proteins. J. Biol. Chem.
268, 13682–13690.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
857
180 Ferdinando Palmieri et al.
Kaplan, R.S., Mayor, J.A., Gremse, D.A. and Wood, D.O. (1995) High level
expression and characterization of the mitochondrial citrate transport
protein from the yeast Saccharomyces cerevisiae. J. Biol. Chem. 270, 4108–
4114.
Kirchberger, S., Tjaden, J. and Neuhaus, H.E. (2008) Characterization of the
Arabidopsis Brittle1 transport protein and impact of reduced activity on
plant metabolism. Plant J. 56, 51–63.
Klaus, S.M., Kunji, E.R., Bozzo, G.G., Noiriel, A., de la Garza, R.D., Basset, G.J.,
Ravanel, S., Rébeillé, F., Gregory, J.F. 3rd and Hanson, A.D. (2005) Higher
plant plastids and cyanobacteria have folate carriers related to those of
trypanosomatids. J. Biol. Chem. 280, 38457–38463.
Klingenberg, M. (1976) The ADP/ATP carrier in mitochondrial membranes. In
The Enzymes of Biological Membranes: Membrane Transport Vol. 3
(Martonosi, A.N., ed). New York/London: Plenum Publishing Corp, pp. 383–
438.
Klingenberg, M. (2005) Ligand–protein interaction in biomembrane carriers.
The induced transition fit of transport catalysis. Biochemistry, 44, 8563–
8570.
Klingenberg, M. (2008) The ADP and ATP transport in mitochondria and its
carrier. Biochim. Biophys. Acta, 1778, 1978–2021.
Krzywinski, M., Schein, J., Birol, I., Connors, J., Gascoyne, R., Horsman, D.,
Jones, S.J. and Marra, M.A. (2009) Circos: an information aesthetic for
comparative genomics. Genome Res. 19, 1639–1645.
Kuan, J. and Saier, M.H. Jr (1993) The mitochondrial carrier family of transport
proteins: structural, functional, and evolutionary relationships. Crit. Rev.
Biochem. Mol. Biol. 28, 209–233.
Kunji, E.R.S. and Harding, M. (2003) Projection structure of the atractylosideinhibited mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae.
J. Biol. Chem. 278, 36985–36988.
Kunji, E.R.S. and Robinson, A.J. (2010) Coupling of proton and substrate
translocation in the transport cycle of mitochondrial carriers. Curr. Opin.
Struct. Biol. 20, 440–447.
Lawand, S., Dorne, A.J., Long, D., Coupland, G., Mache, R. and Carol, P. (2002)
Arabidopsis À BOUT DE SOUFFLE, which is homologous with mammalian
carnitine acyl carrier, is required for postembryonic growth in the light.
Plant Cell, 14, 2161–2173.
Leroch, M., Neuhaus, H.E., Kirchberger, S., Zimmermann, S., Melzer, M.,
Gerhold, J. and Tjaden, J. (2008) Identification of a novel adenine nucleotide transporter in the endoplasmic reticulum of Arabidopsis. Plant Cell, 20,
438–451.
Lindhurst, M.J., Fiermonte, G., Song, S. et al. (2006) Knockout of Slc25a19
causes mitochondrial thiamine pyrophosphate depletion, embryonic
lethality, CNS malformations, and anemia. Proc. Natl Acad. Sci. USA, 103,
15927–15932.
Linka, N. and Weber, A.P.M. (2010) Intracellular metabolite transporters in
plants. Mol. Plant, 3, 21–53.
Linka, N., Theodoulou, F.L., Haslam, R.P., Linka, M., Napier, J.A., Neuhaus,
H.E. and Weber, A.P.M. (2008) Peroxisomal ATP import is essential
for seedling development in Arabidopsis thaliana. Plant Cell, 20, 3241–
3257.
Ludwig, O., De Pinto, V., Palmieri, F. and Benz, R. (1986) Pore formation by the
mitochondrial porin of rat brain in lipid bilayer membranes. Biochim.
Biophys. Acta, 860, 268–276.
Marobbio, C.M.T., Vozza, A., Harding, M., Bisaccia, F., Palmieri, F. and
Walker, J.E. (2002) Identification and reconstitution of the yeast
mitochondrial transporter for thiamine pyrophosphate. EMBO J. 21,
5653–5661.
Marobbio, C.M.T., Agrimi, G., Lasorsa, F.M. and Palmieri, F. (2003) Identification and functional reconstitution of yeast mitochondrial carrier for
S-adenosylmethionine. EMBO J. 22, 5975–5982.
Marobbio, C.M., Di Noia, M.A. and Palmieri, F. (2006) Identification of a
mitochondrial transporter for pyrimidine nucleotides in Saccharomyces
cerevisiae: bacterial expression, reconstitution and functional characterization. Biochem. J. 393, 441–446.
Marobbio, C.M.T., Giannuzzi, G., Paradies, E., Pierri, C.L. and Palmieri, F.
(2008) a-Isopropylmalate, a leucine biosynthesis intermediate in yeast, is
transported by the mitochondrial oxaloacetate carrier. J. Biol. Chem. 283,
28445–28453.
Millar, A.H. and Heazlewood, J.L. (2003) Genomic and proteomic analysis
of mitochondrial carrier proteins in Arabidopsis. Plant Physiol. 131, 443–
453.
Millar, A.H., Carrie, C., Pogson, B. and Whelan, J. (2009) Exploring the function–location nexus: using multiple lines of evidence in defining the subcellular location of plant proteins. Plant Cell, 21, 1625–1631.
Morozzo della Rocca, B., Miniero, D.V., Tasco, G. et al. (2005) Substrateinduced conformational changes of the mitochondrial oxoglutarate carrier:
a spectroscopic and molecular modelling study. Mol. Membr. Biol. 22, 443–
452.
Nelson, D.R., Felix, C.M. and Swanson, J.M. (1998) Highly conserved
charge-pair networks in the mitochondrial carrier family. J. Mol. Biol. 277,
285–308.
Palenik, B., Grimwood, J., Aerts, A. et al. (2007) The tiny eukaryote Ostreococcus provides genomic insights into the paradox of plankton speciation.
Proc. Natl Acad. Sci. USA, 104, 7705–7710.
Palmieri, F. (1994) Mitochondrial carrier proteins. FEBS Lett. 346, 48–54.
Palmieri, F. (2004) The mitochondrial transporter family (SLC25): physiological
and pathological implications. Pflugers Arch. Eur. J. Physiol. 447, 689–709.
Palmieri, F. (2008) Diseases caused by defects of mitochondrial carriers: a
review. Biochim. Biophys. Acta, 1777, 564–578.
Palmieri, F. and Pierri, C.L. (2010a) Structure and function of mitochondrial
carriers: role of the transmembrane helix P and G residues in the gating and
transport mechanism. FEBS Lett. 584, 1931–1939.
Palmieri, F. and Pierri, C.L. (2010b) Mitochondrial metabolite transport. Essays
Biochem. 47, 37–52.
Palmieri, F., Agrimi, G., Blanco, E. et al. (2006) Identification of mitochondrial
carriers in Saccharomyces cerevisiae by transport assay of reconstituted
recombinant proteins. Biochim. Biophys. Acta, 1757, 1249–1262.
Palmieri, F., Rieder, B., Ventrella, A. et al. (2009) Molecular identification and
functional characterization of Arabidopsis thaliana mitochondrial and
chloroplastic NAD+ carrier proteins. J. Biol. Chem. 284, 31249–31259.
Palmieri, L., Palmieri, F., Runswick, M.J. and Walker, J.E. (1996) Identification
by bacterial expression and functional reconstitution of the yeast genomic
sequence encoding the mitochondrial dicarboxylate carrier protein. FEBS
Lett. 399, 299–302.
Palmieri, L., De Marco, V., Iacobazzi, V., Palmieri, F., Runswick, M.J. and
Walker, J.E. (1997a) Identification of the yeast ARG-11 gene as a mitochondrial ornithine carrier involved in arginine biosynthesis. FEBS Lett.
410, 447–451.
Palmieri, L., Lasorsa, F.M., De Palma, A., Palmieri, F., Runswick, M.J. and
Walker, J.E. (1997b) Identification of the yeast ACR1 gene product as a
succinate-fumarate transporter essential for growth on ethanol or acetate.
FEBS Lett. 417, 114–118.
Palmieri, L., Lasorsa, F.M., Iacobazzi, V., Runswick, M.J., Palmieri, F. and
Walker, J.E. (1999a) Identification of the mitochondrial carnitine carrier in
Saccharamyces cerevisiae. FEBS Lett. 462, 472–476.
Palmieri, L., Vozza, A., Agrimi, G., De Marco, V., Runswick, M.J., Palmieri,
F. and Walker, J.E. (1999b) Identification of the yeast mitochondrial
transporter for oxaloacetate and sulfate. J. Biol. Chem. 274, 22184–
22190.
Palmieri, L., Agrimi, G., Runswick, M.J., Fearnley, I.M., Palmieri, F. and
Walker, J.E. (2001a) Identification in Saccharomyces cerevisiae of two
isoforms of a novel mitochondrial transporter for 2-oxoadipate and
2-oxoglutarate. J. Biol. Chem. 276, 1916–1922.
Palmieri, L., Rottensteiner, H., Girzalsky, W., Scarcia, P., Palmieri, F. and
Erdmann, R. (2001b) Identification and functional reconstitution of the
yeast peroxisomal adenine nucleotide transporter. EMBO J. 20, 5049–
5059.
Palmieri, L., Pardo, B., Lasorsa, F.M. et al. (2001c) Citrin and aralar1 are Ca2+stimulated aspartate/glutamate transporters in mitochondria. EMBO J. 20,
5060–5069.
Palmieri, L., Arrigoni, R., Blanco, E., Carrari, F., Zanor, M.I., StudartGuimarães, C., Fernie, A.R. and Palmieri, F. (2006a) Molecular identification
of an Arabidopsis thaliana S-adenosylmethionine transporter: analysis of
organ distribution, bacterial expression, reconstitution into liposomes and
functional characterization. Plant Physiol. 142, 855–865.
Palmieri, L., Todd, C.D., Arrigoni, R., Hoyos, M.E., Santoro, A., Polacco, J.C.
and Palmieri, F. (2006b) Arabidopsis mitochondria have two basic amino
acid transporters with partially overlapping specificities and differential
expression in seedling development. Biochim. Biophys. Acta, 1757, 1277–
1283.
Palmieri, L., Picault, N., Arrigoni, R., Besin, E., Palmieri, F. and Hodges, M.
(2008a) Molecular identification of three Arabidopsis thaliana mitochon-
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
858
Mitochondrial carriers 181
drial dicarboxylate carrier isoforms: organ distribution, bacterial expression, reconstitution into liposomes and functional characterization. Biochem. J. 410, 621–629.
Palmieri, L., Santoro, A., Carrari, F., Blanco, E., Nunes-Nesi, A., Arrigoni, R.,
Genchi, F., Fernie, A.R. and Palmieri, F. (2008b) Identification and characterization of ADNT1, a novel mitochondrial adenine nucleotide transporter
from Arabidopsis. Plant Physiol. 148, 1797–1808.
Pebay-Peyroula, E., Dahout-Gonzalez, C., Kahn, R., Trézéguet, V., Lauquin,
G.J.-M. and Brandolin, G. (2003) Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature, 426, 39–44.
Pfaff, E., Klingenberg, M., Ritt, E. and Vogell, W. (1968) Correlation of the
unspecific permeable mitochondrial space with the ‘intermembrane
space.’ Eur. J. Biochem. 5, 222–232.
Picault, N., Palmieri, L., Pisano, I., Hodges, M. and Palmieri, F. (2002) Identification of a novel transporter for dicarboxylates and tricarboxylates in
plant mitochondria: bacterial expression, reconstitution, functional characterization, and tissue distribution. J. Biol. Chem. 277, 24204–24211.
Picault, N., Hodges, M., Palmieri, L. and Palmieri, F. (2004) The growing
family of mitochondrial carriers in Arabidopsis. Trends Plant Sci. 9, 138–
146.
Prohl, C., Pelzer, W., Diekert, K., Kmita, H., Bedekovics, T., Kispal, G. and Lill,
R. (2001) The yeast mitochondrial carrier Leu5p and its human homologue
Graves’ disease protein are required for accumulation of coenzyme A in the
matrix. Mol. Cell. Biol. 21, 1089–1097.
Proost, S., Van Bel, M., Sterck, L., Billiau, K., Van Parys, T., Van de Peer, Y. and
Vandepoele, K. (2009) PLAZA: a comparative genomics resource to study
gene and genome evolution in plants. Plant Cell, 21, 3718–3731.
Robinson, A. and Kunji, E. (2006) Mitochondrial carriers in the cytoplasmic
state have a common substrate binding site. Proc. Natl Acad. Sci. USA, 103,
2617–2622.
Robinson, A., Overy, C. and Kunji, E. (2008) The mechanism of transport by
mitochondrial carriers based on analysis of symmetry. Proc. Natl Acad. Sci.
USA, 105, 17766–17771.
Saeed, A.I., Sharov, V., White, J. et al. (2003) TM4: a free, opensource system
for microarray data management and analysis. BioTechniques, 34, 374–
3778.
Saraste, M. and Walker, J. (1982) Internal sequence repeats and the path of
polypeptide in mitochondrial ADP/ATP translocase. FEBS Lett. 144, 250–
254.
Satrústegui, J., Pardo, B. and del Arco, A. (2007) Mitochondrial transporters as
novel targets for intracellular calcium signaling. Physiol. Rev. 87, 26–67.
Soltis, D.E., Bell, C.D., Kim, S. and Soltis, P.S. (2008) Origin and early evolution of angiosperms. Ann. NY Acad. Sci. 1133, 3–25.
Sweetlove, L.J., Lytovchenko, A., Morgan, M., Nunes-Nesi, A., Taylor, N.L.,
Baxter, C.J., Eickmeier, I. and Fernie, A.R. (2006) Mitochondrial uncoupling
protein is required for efficient photosynthesis. Proc. Natl Acad. Sci. USA,
103, 19587–19592.
Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular
Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol.
Evol. 24, 1596–1599.
Tang, H., Bowers, J., Wang, X., Ming, R., Alam, M. and Paterson, A. (2008)
Synteny and collinearity in plant genomes. Science, 320, 486–488.
Thomas, B.C., Pedersen, B. and Freeling, M. (2006) Following tetraploidy in an
Arabidopsis ancestor, genes were removed preferentially from one
homeolog leaving clusters enriched in dose-sensitive genes. Genome Res.
16, 934–946.
Thuswaldner, S., Lagerstedt, J.O., Rojas-Stutz, M. et al. (2007) Identification,
expression and functional analyses of a thylakoid ATP/ADP carrier from
Arabidopsis. J. Biol. Chem. 282, 8848–8859.
Titus, S.A. and Moran, R.G. (2000) Retrovirally mediated complementation of
the glyB phenotype: cloning of a human gene encoding the carrier for entry
of folates into mitochondria. J. Biol. Chem. 275, 36811–36817.
Todisco, S., Agrimi, G., Castegna, A. and Palmieri, F. (2006) Identification of
the mitochondrial NAD+ transporter in Saccharomyces cerevisiae. J. Biol.
Chem. 281, 1524–1531.
Toka, I., Planchais, S., Cabassa, C., Justin, A.M., De Vos, D., Richard, L.,
Savoure, A. and Carol, P. (2010) Mutations in the hyperosmotic stressresponsive mitochondrial BASIC AMINO ACID CARRIER2 enhance proline
accumulation in Arabidopsis. Plant Physiol. 152, 1851–1862.
Tonazzi, A., Giangregorio, N., Indiveri, C. and Palmieri, F. (2005) Identification
by site-directed mutagenesis and chemical modification of three vicinal
cysteine residues in rat mitochondrial carnitine/acylcarnitine transporter.
J. Biol. Chem. 280, 19607–19612.
Toufighi, K., Brady, S.M., Austin, R., Ly, E. and Provart, N.J. (2005) The botany
array resource: E-northerns, expression angling, and promoter analyses.
Plant J. 43, 153–163.
Traba, J., Froschauer, E., Wiesenberger, G., Satrustegui, J. and del Arco, A.
(2008) Yeast mitochondria import ATP through the calcium-dependent
ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobis growth in
glucose. Mol. Microbiol. 69, 570–585.
Traba, J., Satrustegui, J. and del Arco, A. (2009) Characterization of SCaMC3-like/slc25a41, a novel calcium-independent mitochondrial ATP-Mg/Pi
carrier. Biochem. J. 418, 125–133.
Tzagoloff, A., Jang, J., Glerum, D.M. and Wu, M. (1996) FLX1 codes for a
carrier protein involved in maintaining a proper balance of flavin nucleotides in yeast mitochondria. J. Biol. Chem. 271, 7392–7397.
Van Aken, O., Zhang, B., Carrie, C., Uggalla, V., Paynter, E., Giraud, E. and
Whelan, J. (2009) Defining the mitochondrial stress response in Arabidopsis thaliana. Mol. Plant, 2, 1310–1324.
Van de Peer, Y., Fawcett, J., Proost, S., Sterck, L. and Vandepoele, K. (2009)
The flowering world: a tale of duplications. Trends Plant Sci. 14, 680–688.
Vandepoele, K., Simillion, C. and Van de Peer, Y. (2002) Detecting the undetectable: uncovering duplicated segments in Arabidopsis by comparison
with rice. Trends Genet. 18, 606–608.
Vozza, A., Blanco, E., Palmieri, L. and Palmieri, F. (2004) Identification of the
mitochondrial GTP/GDP transporter in Saccharomyces cerevisiae. J. Biol.
Chem. 279, 20850–20857.
Walters, D.E. and Kaplan, R.S. (2004) Homology-modeled structure of the
yeast mitochondrialcitrate transport protein. Biophys. J. 87, 907–911.
Wibom, R., Lasorsa, F.M., Töhönen, V. et al. (2009) AGC1 deficiency associated with global cerebral hypomyelination. N. Engl. J. Med. 361, 489–495.
Wohlrab, H. (2004) Novel inter- and intrasubunit contacts between transportrelevant residues of the homodimeric mitochondrial phosphate transport
protein. Biochem. Biophys. Res. Commun. 320, 685–688.
Wohlrab, H. and Briggs, C. (1994) Yeast mitochondrial phosphate transport
protein expressed in Escherichia coli. Site-directed mutations at threonine43 and at a similar location in the second tandem repeat (isoleucine-141).
Biochemistry, 33, 9371–9375.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 161–181
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