Uncoupling proteins 1 and 2 (UCP1 and UCP2) from Arabidopsis thaliana are mitochondrial transporters of aspartate, glutamate and dicarboxylates

Abstract

The Arabidopsis thaliana genome contains 58 members of the solute carrier family SLC25, also called the mitochondrial carrier family, many of which have been shown to transport specific metabolites, nucleotides and cofactors across the mitochondrial membrane. Here two Arabidopsis members of this family, AtUCP1 and AtUCP2, which were previously thought to be uncoupling proteins and hence named UCP1/PUMP1 and UCP2/PUMP2, respectively, are assigned with a novel function. They were expressed in bacteria, purified and reconstituted in phospholipid vesicles. Their transport properties demonstrate that they transport amino acids (aspartate, glutamate, cysteinesulfinate and cysteate), dicarboxylates (malate, oxaloacetate and 2-oxoglutarate), phosphate, sulfate and thiosulfate. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors at various degrees. AtUCP1 and AtUCP2 catalyzed a fast counter-exchange transport as well as a low uniport of substrates with transport rates of AtUCP1 being much higher than those of AtUCP2 in both cases. The aspartate/glutamate hetero-exchange mediated by AtUCP1 and AtUCP2 is electroneutral, in contrast to that mediated by the mammalian mitochondrial aspartate glutamate carrier. Furthermore, both carriers were found to be targeted to mitochondria. Metabolite profiling of single and double knockouts show changes in organic acid and amino acid levels. Notably, AtUCP1 and AtUCP2 are the first reported mitochondrial carriers in Arabidopsis to transport aspartate and glutamate. It is proposed that the primary function of AtUCP1 and AtUCP2 is to catalyze an aspartateout/glutamatein exchange across the mitochondrial membrane and thereby contribute to the export of reducing equivalents from the mitochondria in photorespiration.

Full text

Uncoupling proteins 1 and 2 (UCP1 and UCP2) from
Arabidopsis thaliana are mitochondrial transporters of
aspartate, glutamate, and dicarboxylates
Received for publication, November 9, 2017, and in revised form, January 15, 2018 Published, Papers in Press, January 25, 2018, DOI 10.1074/jbc.RA117.000771
Magnus Monne´
‡§
, Lucia Daddabbo
‡
, David Gagneul
¶1
, Toshihiro Obata
储
, Bjo¨rn Hielscher
¶2
, Luigi Palmieri
‡
**,
Daniela Valeria Miniero
‡
, Alisdair R. Fernie
储3
, Andreas P. M. Weber
¶4
, and Ferdinando Palmieri
‡
**
5
From the
‡
Department of Biosciences, Biotechnologies and Biopharmaceutics, Laboratory of Biochemistry and Molecular Biology,
University of Bari, via Orabona 4, 70125 Bari, Italy, the
§
Department of Sciences, University of Basilicata, Via Ateneo Lucano 10,
85100 Potenza, Italy, the
¶
Cluster of Excellence on Plant Science (CEPLAS), Institute of Plant Biochemistry, Heinrich-Heine-
Universita¨t, Universita¨tsstrasse 1, 40225 Du¨sseldorf, Germany, the
储
Department Willmitzer, Max-Planck-Institut fur Molekulare
Pflanzenphysiologie, Am Muhlenberg 1, 14476 Potsdam-Golm, Germany, and the **Center of Excellence in Comparative
Genomics, University of Bari, via Orabona 4, 70125 Bari, Italy
Edited by Joseph M. Jez
The Arabidopsis thaliana genome contains 58 members of
the solute carrier family SLC25, also called the mitochondrial
carrier family, many of which have been shown to transport spe-
cific metabolites, nucleotides, and cofactors across the mito-
chondrial membrane. Here, two Arabidopsis members of this
family, AtUCP1 and AtUCP2, which were previously thought to
be uncoupling proteins and hence named UCP1/PUMP1 and
UCP2/PUMP2, respectively, are assigned with a novel function.
They were expressed in bacteria, purified, and reconstituted in
phospholipid vesicles. Their transport properties demonstrate
that they transport amino acids (aspartate, glutamate, cysteine
sulfinate, and cysteate), dicarboxylates (malate, oxaloacetate,
and 2-oxoglutarate), phosphate, sulfate, and thiosulfate. Trans-
port was saturable and inhibited by mercurials and other mito-
chondrial carrier inhibitors to various degrees. AtUCP1 and
AtUCP2 catalyzed a fast counterexchange transport as well as a
low uniport of substrates, with transport rates of AtUCP1 being
much higher than those of AtUCP2 in both cases. The aspartate/
glutamate heteroexchange mediated by AtUCP1 and AtUCP2 is
electroneutral, in contrast to that mediated by the mammalian
mitochondrial aspartate glutamate carrier. Furthermore, both
carriers were found to be targeted to mitochondria. Metabolite
profiling of single and double knockouts shows changes in
organic acid and amino acid levels. Notably, AtUCP1 and
AtUCP2 are the first reported mitochondrial carriers in Arabi-
dopsis to transport aspartate and glutamate. It is proposed that
the primary function of AtUCP1 and AtUCP2 is to catalyze
an aspartate
out
/glutamate
in
exchange across the mitochondrial
membrane and thereby contribute to the export of reducing
equivalents from the mitochondria in photorespiration.
Mitochondrial carriers (MCs)
6
are a large family of mem-
brane proteins that transport nucleotides, amino acids, carbox-
ylic acids, inorganic ions, and cofactors across the mitochon-
drial inner membrane (1–3). Many metabolic pathways and
cellular processes with complete or partial localization in the
mitochondrial matrix are dependent on transport steps cata-
lyzed by MCs (e.g. oxidative phosphorylation, metabolism of
fatty acids and amino acids, gluconeogenesis, thermogenesis,
mitochondrial replication, transcription, and translation)
(3). The protein sequences of the MC family members have
a characteristic three times tandemly repeated 100-residue
domain (4), which contains two hydrophobic segments and a
signature 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) (5). In atomic resolution 3D
structures of the only MC family member determined to date
(the carboxyatractyloside-inhibited ADP/ATP carrier) (6, 7),
the six hydrophobic segments form a bundle of transmembrane
␣
-helices with a central substrate translocation pore, and the
three PX(D/E)XX(K/R) motifs form a gate toward the matrix
side. In most cases, the MC signature motif has been used to
identify family members in genomic sequences; Homo sapiens
has 53 members, Saccharomyces cerevisiae has 35, and Arabi-
dopsis thaliana has 58. About half of these carriers have been
identified and characterized in terms of substrate specificity,
transport proteins, and kinetic parameters by direct transport
assays (1, 8, 9).
This work was supported by grants from the Center of Excellence on Compar-
ative Genomics and Italian Human ProteomeNet Grant RBRN07BMCT_009
(MIUR). The authors declare that they have no conflicts of interest with the
contents of this article.
This article contains Tables S1–S4 and Figs. S1–S6.
1
Present address: Universite´ deLille, INRA, ISA, Universite´ d’ Artois, Universite´
du Littoral Coˆte d’Opale, EA 7394-ICV-Institut Charles Viollette, F-59000
Lille, France.
2
Supported by an iGRAD-Plant doctoral fellowship (IRTG 1525).
3
Work in the laboratory of this author was supported by the Max-Planck-Society.
4
Supported by the Cluster of Excellence on Plant Science CEPLAS (EXC 1028)
and CRC 1208.
5
To whom correspondence should be addressed: Dept. of Biosciences, Bio-
technologies, and Biopharmaceutics, Laboratory of Biochemistry and
Molecular Biology, University of Bari, via Orabona 4, 70125 Bari, Italy. Tel.:
39-080-5443323; Fax: 39-080-5442770; E-mail: ferdpalmieri@gmail.com.
6
The abbreviations used are: MC, mitochondrial carrier; UCP, uncoupling pro-
tein; AtUCP1, A. thaliana UCP1; AtUCP2, A. thaliana UCP2; hUCP2, human
UCP2; DIC, dicarboxylate carrier; DTC, di- and tricarboxylate carrier; GDC,
glycine decarboxylase; GFP, green fluorescent protein; GS/GOGAT, gluta-
mine synthetase/glutamine oxoglutarate aminotransferase; MAS, malate
aspartate shuttle; IVD, isovaleryl-CoA-dehydrogenase; CTD, C-terminal
domain.
cro
ARTICLE
J. Biol. Chem. (2018) 293(11) 4213–4227 4213
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
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Studies aiming to biochemically characterize MCs from
A. thaliana were initiated by comparing selected Arabidopsis
genes with those of yeast and humans encoding MCs with pre-
viously identified substrates (9). Arabidopsis has been demon-
strated to express MCs for the four main types of substrates (1)
(i.e. nucleotide carriers for ADP/ATP (AAC1–4, PNC1 and -2,
AtBT1, PM-ANT1, and TAAC) (10–16), adenine nucleotides
(ADNT1) (17), ATP-Mg/P
i
(APC1–3) (18, 19), NAD
⫹
(NDT1
and -2) (20), NAD
⫹
, NADH, CoA, and adenosine 3⬘,5⬘-phos-
phate (PXN) (21, 22); carboxylate carriers for di- and tricar-
boxylates (DTC) (23) and dicarboxylates (DIC1–3) (24); amino
acid carriers for basic amino acids (BAC1 and -2) (25, 26) and
S-adenosylmethionine (SAMC1 and -2) (27, 28); and inorganic
ion carriers for phosphate and sulfate (29)). It is important to
note that some of the carriers characterized from Arabidopsis
have broader substrate specificities than their human and yeast
counterparts, and additionally some of them are localized in
compartments other than the mitochondria, such as peroxi-
somes, chloroplasts, the endoplasmic reticulum, and the plasma
membrane (1). It is also noteworthy that the molecular identity of
an Arabidopsis MC corresponding to the human aspartate/gluta-
mate exchangers (AGC1 and -2) (30) or glutamate uniporters of
any type (GC1 and -2) (31) has, to date, not been established.
The mammalian uncoupling protein 1 (UCP1) was demon-
strated to transport protons, thereby uncoupling oxidative
phosphorylation (32, 33). On the basis of homology with sub-
sequently sequenced MCs, a UCP subfamily was identified con-
taining six members in both humans (hUCP1–6) and Arabi-
dopsis (AtUCP1–6). However, AtUCP4 – 6 were subsequently
renamed dicarboxylate carriers (DIC1–3), following the dem-
onstration that they transport malate, oxaloacetate, succinate,
P
i
, sulfate, thiosulfate, and sulfite (24), and hUCP2 was demon-
strated to be a four-carbon metabolite/P
i
carrier transporting
aspartate, malate, malonate, oxaloacetate, P
i
, and sulfate (34).
In the current study, we investigated the potential transport
properties of the two closest homologs of hUCP2 in Arabidop-
sis: AtUCP1 and AtUCP2, also known as PUMP1 and PUMP2.
Previously, AtUCP1 was shown to be localized to mitochondria
and display an uncoupling activity similar to that of hUCP1
(35–37). By contrast, very little is known about AtUCP2; in a
proteomic study, it was detected in the Golgi (38), but in
another, it was detected at the plasma membrane (39). The
results presented here demonstrate that AtUCP1 and the less-
studied AtUCP2 are mitochondrially localized isoforms and
have a broad substrate specificity, transporting a variety of sub-
strates, including aspartate, glutamate, malate, oxaloacetate,
and other metabolites. Characterization of metabolite profiles
of T-DNA insertional knockout mutants, including a ucp1/
ucp2 double mutant, revealed clear changes in organic acid lev-
els, some of which were exacerbated by the application of salt
stress.
Results
Identification of the closest homologs of AtUCP1 and AtUCP2
in various species
The protein sequences of AtUCP1 and AtUCP2 homologs
were collected, aligned, and analyzed (Fig. S1). AtUCP1 and
AtUCP2 share 72% identical amino acids. Their sequences are
much more similar to each other than to any other Arabidopsis
protein; in Arabidopsis, the closest relative to AtUCP1 and
AtUCP2 is AtDIC2, which shares 41 and 42% sequence identity
with AtUCP1 and AtUCP2, respectively. In humans and
S. cerevisiae, the closest homologs are hUCP2 (34), having 51
and 44% identical amino acids, and yeast Dic1p (40), exhibiting
30 and 33% sequence identity with AtUCP1 and AtUCP2,
respectively. Putative orthologs with high sequence identity
with AtUCP1 and AtUCP2 above 75% were found in several
plant species. Moreover, from structural sequence alignments
using the X-ray structure of the bovine ADP/ATP carrier (6)as
atemplate, it can be deduced that 85 and 54% of the residues
lining the surface of the substrate translocation pore are iden-
tical between AtUCP1 and AtUCP2 and between AtUCP1,
AtUCP2, and hUCP2, respectively. These results suggest that
AtUCP1 and AtUCP2 are isoforms, and their closest homolog
with identified substrates is hUCP2.
Bacterial expression of AtUCP1 and AtUCP2
AtUCP1 and AtUCP2 were expressed in Escherichia coli
BL21(DE3) strains (Fig. 1,lanes 4 and 7). They accumulated as
inclusion bodies and were purified by centrifugation and wash-
ing (see “Experimental procedures”). The apparent molecular
masses of purified AtUCP1 and AtUCP2 (Fig. 1,lanes 5and 8)
were ⬃31 kDa, which is in good agreement with the calculated
value of 33 kDa for both AtUCP1 and AtUCP2. The identities of
the recombinant proteins were confirmed by MALDI-TOF
mass spectrometry, and the yield of the purified proteins was
about 10 and 2 mg/liter of culture for AtUCP1 and AtUCP2,
respectively. The proteins were detected neither in non-
Figure 1. Expression in Escherichia coli and purification of AtUCP1 and
AtUCP2. Proteins were separated by SDS-PAGE and stained with Coomassie
Blue. Lanes 1–5, AtUCP1; lanes 6 – 8, AtUCP2. Markers were Bio-Rad prestained
SDS-PAGE standards: bovine serum albumin, 84 kDa; ovalbumin, 50 kDa; car-
bonic anhydrase, 37 kDa; soybean trypsin inhibitor, 29 kDa; lysozyme, 21
kDa). Lanes 1– 4,E. coli BL21(DE3); lanes 6 and 7,E. coli BL21 CodonPlus(DE3)-
RIL containing the expression vector, without (lanes 1,3, and 6) and with the
coding sequence of AtUCP1 (lanes 2 and 4) and the coding sequence of
AtUCP2 (lane 7). Samples were taken immediately before induction (lanes 1
and 2) and 5 h later (lanes 3,4,6, and 7). The same number of bacteria were
analyzed in each sample. Lanes 5 and 8, purified AtUCP1 protein (5
␮
g) and
purified AtUCP2 (3
␮
g) derived from bacteria shown in lanes 4 and 7,
respectively.
Transport properties of AtUCP1 and AtUCP2
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induced cultures nor in cultures with an empty vector (Fig. 1,
lanes 1,2,3, and 6).
Functional characterization of recombinant AtUCP1 and
AtUCP2
Recombinant AtUCP1 and AtUCP2 were reconstituted into
liposomes, and their transport activities for various radioactive
substrates were tested in homo-exchange experiments (i.e.
with the same external (1 mM) and internal (10 mM) substrate).
In a first set of homo-exchange experiments, time-dependent
uptake of several radioactive substrates (aspartate, malate, and
glutamate for reconstituted AtUCP1 and AtUCP2; malonate
and sulfate for AtUCP1; and 2-oxoglutarate for AtUCP2) dem-
onstrated typical curves for carrier-mediated transport (Fig. 2,
Aand B). Both AtUCP1- and AtUCP2-mediated homo-ex-
changes between external [
14
C]aspartate and internal aspartate
were temperature-dependent (Fig. 2, Cand D), as would be
expected for protein-catalyzed transport. Furthermore, no
[
14
C]aspartate/aspartate or [
14
C]malate/malate exchange activity
was detected if AtUCP1 and AtUCP2 had been boiled before
incorporation into liposomes or if proteoliposomes were
reconstituted with lauric acid/sarkosyl-solubilized material
from bacterial cells lacking the expression vector for AtUCP1
and AtUCP2 or harvested immediately before induction of
expression (data not shown). In all of these experiments, a mix-
ture of pyridoxal-5⬘-phosphate and bathophenanthroline was
used to block the AtUCP1- and AtUCP2-mediated transport
reactions at various time points. In addition, AtUCP1 and
AtUCP2 were found to catalyze homo-exchanges of glutamate,
malonate, malate, succinate, and P
i
, whereas no or very low
transport was observed with glutamine, arginine, phenylala-
nine, threonine, valine, proline,
␥
-aminobutyrate, citrate, ATP,
GTP, S-adenosylmethionine, or glutathione (Fig. 3).
The substrate specificities of AtUCP1 and AtUCP2 were
examined in detail by measuring the initial rate of [
14
C]aspar-
tate uptake into proteoliposomes that had been preloaded with
various potential substrates (Fig. 4). For both AtUCP1 and
AtUCP2, the highest activities were observed in the presence of
internal aspartate, glutamate, cysteine sulfinate, cysteate, mal-
onate, malate, oxaloacetate, maleate, and (for AtUCP2) 2-oxo-
glutarate. Both proteins also exchanged, albeit to a lower extent,
internal D-aspartate, cysteine, oxalate, succinate, 2-oxogl-
utarate,
␣
-aminoadipate, P
i
, sulfate, and thiosulfate. In addi-
tion, AtUCP2 exchanged [
14
C]aspartate with the internal
substrates (Fig. 4B) fumarate, glutarate, and nitrate, which were
not significantly transported by AtUCP1 (Fig. 4A). By contrast,
the uptake of labeled aspartate by AtUCP1 and AtUCP2 was
negligible with internal asparagine, D-glutamate, glutamine,
serine, glycine, homocysteate, adipate,
␣
-ketoadipate, pyro-
phosphate, citrate, pyruvate, lactate, phosphoenolpyruvate,
acetoacetate,
␤
-hydroxybutyrate, N-acetylaspartate, ATP, and
Figure 2. Substrate homo-exchanges in proteoliposomes reconstituted with AtUCP1 (Aand C) and AtUCP2 (Band D). Aand B, homo-exchanges of
aspartate (F), malate (‚), glutamate (f), malonate (), sulfate (䉬), and 2-oxoglutarate (ƒ)at25°C.Cand D, aspartate/aspartate homo-exchange at 4 °C (䉬),
8°C(Œ), 16 °C (f), and 25 °C (F). Transport was initiated by adding radioactive substrate (concentration, 1 mM) to proteoliposomes preloaded internally with
the same substrate (concentration, 10 mM). The reaction was terminated at the indicated times. Similar results were obtained in at least three independent
experiments.
Transport properties of AtUCP1 and AtUCP2
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glutathione (Fig. 4, Aand B). The activity in the presence of
these substrates was approximately the same as that observed
in the presence of NaCl and no substrate.
The effects of other mitochondrial carrier inhibitors on the
[
14
C]aspartate/aspartate exchange reaction catalyzed by recon-
stituted AtUCP1 and AtUCP2 were also examined. This trans-
port activity was inhibited strongly by bathophenanthroline,
pyridoxal-5⬘-phosphate, and tannic acid and markedly by mer-
salyl, HgCl
2
, and butylmalonate (Fig. 5). Phenylsuccinate and
p-hydroxymercuribenzoate strongly inhibited AtUCP1 and
partially inhibited AtUCP2, whereas bromcresol purple and
␣
-cyano-4-hydroxycinnamate caused partial inhibition of both
carriers. By contrast, carboxyatractyloside, bongkrekic acid,
and N-ethylmaleimide had little or no effect on either AtUCP1
or AtUCP2 activity.
Kinetic characteristics of recombinant AtUCP1 and AtUCP2
proteins
In Fig. 6, the kinetics of 1 mM[
14
C]aspartate (Aand B)or1
mM[
14
C]malate (Cand D) uptake into proteoliposomes cata-
lyzed by recombinant AtUCP1 (Aand C) or AtUCP2 (Band D)
and measured either as uniport (with internal NaCl) or as
exchange (in the presence of 10 mMsubstrates) are compared.
The [
14
C]aspartate/aspartate and [
14
C]malate/malate exchanges
followed first-order kinetics (rate constants 1.6 and 1.4 min
⫺1
(AtUCP1) or 0.27 and 0.23 min
⫺1
(AtUCP2); initial rates 14 and
11 mmol/min ⫻g of protein (AtUCP1) or 1.9 and 1.3 mmol/
min ⫻g of protein (AtUCP2), respectively), isotopic equilib-
rium being approached exponentially. By contrast, with inter-
nal NaCl and no substrate, very low uptake of [
14
C]aspartate or
[
14
C]malate was observed by liposomes reconstituted either
with AtUCP1 or AtUCP2, suggesting that the two proteins cat-
alyze a minor unidirectional transport (uniport) of substrates.
In addition, Fig. 6 (A–D) illustrates the time courses of several
AtUCP1-mediated and AtUCP2-mediated hetero-exchanges
between [
14
C]aspartate or [
14
C]malate and other transported
substrates. The data of Fig. 6 (Aand C) show that AtUCP1
transports cysteate much better than D-aspartate and dicar-
boxylates with the following order of efficiency: malate ⬎oxa-
loacetate ⬎malonate ⬎succinate (these substrates better than
P
i
). Similarly, the data of Fig. 6 (Band D) demonstrate that
cysteate is transported slightly better than D-aspartate and
malate more efficiently than oxaloacetate by AtUCP2. The
uniport mode of transport was further investigated by measur-
ing the efflux of [
14
C]aspartate or [
14
C]malate from preloaded
active proteoliposomes because it provides a more convenient
assay for unidirectional transport (41). In the absence of exter-
nal substrate, significant efflux of [
14
C]aspartate (Fig. 7, Aand
B)or[
14
C]malate (Fig. 7, Cand D) catalyzed by both AtUCP1
and AtUCP2 was observed. However, in the presence of exter-
nal substrates, the efflux transport rates were at least one order
of magnitude higher. These experiments demonstrate that
AtUCP1 and AtUCP2 are capable of catalyzing both a rapid
antiport of substrates and a slow uniport transport.
The kinetic constants of AtUCP1 and AtUCP2 were deter-
mined from the initial transport rates of homo-exchanges at
various external labeled substrate concentrations in the pres-
ence of a constant saturating internal substrate concentration.
The Michaelis constants (K
m
) of the two recombinant proteins
for aspartate were about 0.8 mM, and for glutamate and malate,
they were between 1.9 and 2.5 mM. The maximal activities
(V
max
) for aspartate, glutamate, and malate varied between 24
and 33 mmol/min ⫻g of protein for AtUCP1 and 4.2 and 4.5
mmol/min ⫻g of protein for AtUCP2 (Table 1). Glutamate,
malate, cysteine sulfinate, cysteate, oxaloacetate,
␣
-ketogl-
utarate, and sulfate were competitive inhibitors of the AtUCP1-
and AtUCP2-mediated [
14
C]aspartate/aspartate exchanges, as
Figure 3. AtUCP1- and AtUCP2-mediated homo-exchanges of various
substrates. Proteoliposomes reconstituted with AtUCP1 (A) and AtUCP2 (B)
were preloaded internally with the substrates indicated in the figure (concen-
tration, 10 mM). Transport was initiated by adding radioactive substrate (con-
centration, 1 mM) to proteoliposomes containing the same substrate. The
reaction was terminated after 30 min. The values are means ⫾S.E. (error bars)
of at least three independent experiments. SAM,S-adenosylmethionine.
Transport properties of AtUCP1 and AtUCP2
4216 J. Biol. Chem. (2018) 293(11) 4213–4227
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they increased the apparent K
m
without changing the V
max
(not
shown). The inhibition constants (K
i
) of these compounds are
listed in Table 2.
Influence of membrane potential and pH gradient on the
AtUCP1- and AtUCP2-mediated exchange reactions
Given that the mammalian aspartate glutamate carriers
AGC1 and -2 have been shown to catalyze an electrophoretic
exchange between aspartate
⫺
and glutamate
⫺
⫹H
⫹
(30), we
investigated the influence of the membrane potential on the
[
14
C]aspartate/glutamate exchange catalyzed by recombinant
AtUCP1 and AtUCP2. A K
⫹
-diffusion potential was generated
across the proteoliposomal membranes with valinomycin in
the presence of a K
⫹
gradient of 1:50 (mM/mM, in/out), corre-
sponding to a calculated value of about 100 mV positive inside
(Table 3). The rate of the [
14
C]aspartate
out
/glutamate
in
hetero-
exchange was unaffected by valinomycin in the presence of the
K
⫹
gradient. By contrast, the aspartate
out
/glutamate
in
exchange,
mediated by recombinant AGC2 C-terminal domain (AGC2-
CTD) (30), was stimulated under the same experimental
conditions. These results indicate that the AtUCP1- and
AtUCP2-mediated aspartate/glutamate hetero-exchange is not
electrophoretic but electroneutral, suggesting that AtUCP1
and AtUCP2 transport either aspartate
⫺
for glutamate
⫺
or
both together with a H
⫹
. Also, the AtUCP1- and AtUCP2-me-
diated aspartate/aspartate, malate/malate, and malate
out
/as-
partate
in
(Table 3) and malate
out
/glutamate
in
(data not shown)
were unaffected by valinomycin in the presence of a K
⫹
gradi-
ent of 1:50. In view of the different charges carried by the amino
Figure 4. Substrate specificity of AtUCP1 and AtUCP2. Proteoliposomes were preloaded internally with various substrates (concentration, 10 mM). Trans-
port was started by adding 0.8 mM[
14
C]aspartate and stopped after 7 and 20 s for AtUCP1 (A) and AtUCP2 (B), respectively. The values are means ⫾S.E. (error
bars) of at least three independent experiments.
␣
-OG,
␣
-ketoglutarate; CSA, cysteinesulfinic acid; PEP, phosphoenolpyruvate.
Figure 5. Effect of mitochondrial carrier inhibitors on the rate of AtUCP1-
and AtUCP2-mediated [
14
C]aspartate/aspartate exchange. Proteolipo-
somes were preloaded internally with 10 mMaspartate, and transport was
initiated by adding 1 mM[
14
C]aspartate. The incubation time was 7 and 20 s
for AtUCP1 and AtUCP2, respectively. Thiol reagents and
␣
-cyanocinnamate
were added 2 min before the labeled substrate; the other inhibitors were
added together with [
14
C]aspartate. The final concentrations of the inhibitors
were as follows: 10
␮
Mfor mercuric chloride (HgCl
2
), carboxyatractyloside
(CAT), and bongkrekic acid (BKA); 0.1 mMfor mersalyl (MER) and p-hydroxy-
mercuribenzoate (pHMB); 0.2 mMfor bromcresol purple (BrCP);1mMfor
N-ethylmaleimide (NEM) and
␣
-cyanocinnamate (CCN);5mMfor butylma-
lonate (BMA) and phenylsuccinate (PHS); 25 mMfor bathophenanthroline
(BAT); 30 mMfor pyridoxal 5⬘-phosphate (PLP); and 0.2% for tannic acid (TAN).
The extents of inhibition (percentages) for AtUCP1 (black bars) and AtUCP2
(gray bars) from a representative experiment are given. Similar results were
obtained in at least three independent experiments.
Transport properties of AtUCP1 and AtUCP2
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acids aspartate and glutamate and dicarboxylates at physiolog-
ical pH levels, we explored whether the charge imbalance of the
malate/aspartate and malate/glutamate hetero-exchanges cat-
alyzed by AtUCP1 and AtUCP2 is compensated by proton
movement. A pH difference across the liposomal membranes
(basic inside the vesicles) was created by the addition of the
K
⫹
/H
⫹
exchanger nigericin to proteoliposomes in the presence
ofaK
⫹
gradient of 1:50 (mM/mM, in/out). Under these condi-
tions, the uptake of [
l4
C]malate in exchange for internal aspar-
tate or glutamate increased (Table 3), whereas the uptake of
[
l4
C]malate in exchange for internal malate or 2-oxoglutarate
was unaffected (data not shown). Therefore, the charge imbal-
ance of the substrates exchanged by AtUCP1 and AtUCP2 is
compensated by the movement of protons.
Subcellular localization of AtUCP1 and AtUCP2 proteins in
transiently transformed Nicotiana benthamiana leaf cells
C-terminal fusion proteins of AtUCP1 and AtUCP2 with the
green fluorescent protein (GFP) under the control of an Arabi-
dopsis ubiquitin-10 promoter were transiently expressed in
N. benthamiana to investigate the subcellular localization via
confocal laser-scanning microscopy. Simultaneously, N. ben-
thamiana was co-infiltrated with the mitochondrion-located
Arabidopsis isovaleryl-CoA-dehydrogenase tagged with a C-
terminal eqFP611 (IVD-eqFP611) under the control of the cau-
liflower mosaic virus 35S promoter. Two days after infiltration,
protoplasts were isolated from leaf tissue and directly used for
microscopy.
The C-terminal fusion proteins AtUCP1-GFP (Fig. 8A) and
AtUCP2-GFP (Fig. 8B) (shown in green)clearly overlap with
almost all mitochondrial IVD-eqFP611 fluorescent signals
(shown in red) in all observed protoplasts, indicating the mito-
chondrial localization of both proteins. 48 h after infiltration,
the mitochondrial marker was generally higher-expressed than
the GFP fusion proteins and was also found in the cytosol with
more prominent fluorescent signals detected in mitochondria.
Isolation, generation, and metabolic characterization of
AtUCP1–2 knockout mutants
After biochemically characterizing the properties of recom-
binant AtUCP1 and AtUCP2 proteins, we turned our attention
to evaluating their physiological role in Arabidopsis. For this
purpose, we acquired the individual T-DNA insertion mutants
and crossed them to obtain the ucp1/ucp2 double mutant (Fig.
S2). The ucp1 line used here was extensively characterized by
Sweetlove et al. (37), including functional complementation by
the UCP1 genomic sequence. This mutant harbors a T-DNA
insertion in the first intron and shows low residual UPC protein
amounts in the mitochondria (5% of the wildtype line (37)).
Congruent with this previous work, we also detected residual
expression of the UCP1 gene, which was much lower than that
of the wildtype (Fig. S3). The expression of the UCP2 gene was
Figure 6. Kinetics of [
14
C]aspartate or [
14
C]malate uptake by AtUCP1- and AtUCP2-reconstituted liposomes containing no substrate or various
substrates. Proteoliposomes containing AtUCP1 (Aand C) or AtUCP2 (Band D) were preloaded internally with 10 mMaspartate (F), cysteate (Œ), D-aspartate
(䉬), or 10 mMNaCl and no substrate ()(Aand B) and with malate (‚), oxaloacetate (䉬), malonate (F), succinate (Œ), phosphate (f), aspartate (䡺), or 10 mM
NaCl and no substrate ()(Cand D). Transport was initiated by adding 1 mM[
14
C]aspartate (Aand B)or1mM[
14
C]malate (Cand D) and terminated at the
indicated times. Similar results were obtained in at least three independent experiments.
Transport properties of AtUCP1 and AtUCP2
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virtually absent in the newly isolated ucp2 mutant (Fig. S3).
Having these genotypes in hand, we next assessed their meta-
bolic phenotypes via GC-MS–based metabolic profiling both
in plants grown on normal MS agar and in plants exposed to salt
stress. The effects of salt stress were investigated because UCPs
have been previously proposed to contribute to the abiotic
stress response (36). The clearest metabolic phenotype was that
observed in the organic acids; however, changes in phenylala-
nine, serine, the branched chain amino acids, ornithine, myo-
inositol, putrescine, and AMP were apparent in one or more of
the genotypes (Fig. 9 and Tables S1–S3). These metabolite pro-
files are thus consistent with the transport assay data suggesting
that AtUCP1 and AtUCP2 are important in organic and amino
acid metabolism in plants. The metabolic phenotypes of the
ucp1/ucp2 double mutant tend to be similar to those of ucp1,
suggesting a predominant role of UCP1 in Arabidopsis, at least
in the leaf tissue assayed in the work reported here. Regarding
the observed changes, interestingly, for the levels of some of the
metabolites, such as malate and fumarate, the imposition of salt
stress exacerbated intergenotypic differences. For others, such
Figure 7. Efflux of [
14
C]aspartate and [
14
C]malate from AtUCP1- and AtUCP2-reconstituted liposomes. Proteoliposomes containing AtUCP1 (Aand C)
and AtUCP2 (Band D)with5mMaspartate and 5 mMmalate internally were loaded with [
14
C]aspartate and [
14
C]malate, respectively, by carrier-mediated
exchange equilibrium, and external substrate was removed by Sephadex G-75. Efflux of [
14
C]aspartate (Aand B) and [
14
C]malate (Cand D) was started by
adding 5 mMaspartate (F), malate (‚), glutamate (E), 5 mMNaCl and no substrate (), and 5 mMaspartate (Aand B)or5mMmalate (Cand D) together with 20
mMpyridoxal 5⬘-phosphate and 20 mMbathophenanthroline (䉫). The transport was terminated at the indicated times. Similar results were obtained in at least
three independent experiments.
Table 1
Kinetic constants of recombinant AtUCP1 and AtUCP2
The values were calculated from linear regression of double reciprocal plots of the
initial rates of the indicated homo-exchanges versus the external substrate concen-
tration. The exchanges were started by adding appropriate concentrations of labeled
substrate to proteoliposomes preloaded internally with the same substrate (10 mM).
The reaction time was 7 and 20 s for AtUCP1 and AtUCP2, respectively. The values
are means ⫾S.E. of at least three independent experiments carried out in duplicate.
Carrier and substrate K
m
V
max
mMmmol/min ⫻g protein
AtUCP1
关
14
C兴Aspartate/aspartate 0.8 ⫾0.1 30 ⫾6
关
14
C兴Glutamate/glutamate 1.9 ⫾0.2 24 ⫾6
关
14
C兴Malate/malate 2.0 ⫾0.2 33 ⫾6
AtUCP2
关
14
C兴Aspartate/aspartate 0.8 ⫾0.1 4.5 ⫾0.5
关
14
C兴Glutamate/glutamate 2.5 ⫾0.2 4.2 ⫾0.4
关
14
C兴Malate/malate 2.4 ⫾0.1 4.3 ⫾0.4
Table 2
Competitive inhibition by various substrates of 关
14
C兴aspartate uptake
into proteoliposomes reconstituted with AtUCP1 or AtUCP2
The inhibition constants (K
i
) were calculated from Dixon plots of the inverse rate of
关
14
C兴aspartate transport versus the competing substrate concentration. The com-
peting substrates at appropriate concentrations were added together with labeled
asparate to proteoliposomes containing 10 mMaspartate. The values are means ⫾
S.E. of at least three independent experiments carried out in duplicate.
Inhibitor
K
i
AtUCP1 AtUCP2
mM
␣
-Ketoglutarate 3.3 ⫾0.2 2.6 ⫾0.2
Cysteate 2.2 ⫾0.3 2.4 ⫾0.2
Cysteinesulfinate 2.7 ⫾0.3 2.3 ⫾0.2
Glutamate 2.2 ⫾0.2 2.3 ⫾0.2
Malate 1.7 ⫾0.2 2.2 ⫾0.2
Oxaloacetate 2.6 ⫾0.3 3.2 ⫾0.2
Sulfate 3.6 ⫾0.3 3.3 ⫾0.3
Transport properties of AtUCP1 and AtUCP2
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as citrate, these differences were ameliorated (Fig. 9). The com-
plexity of these results suggests that further research is war-
ranted into the precise physiological role(s) of these proteins
under both optimal and suboptimal conditions and in different
plant tissues and developmental stages.
Discussion
A recent report has shown that the MC family member
hUCP2, which was thought to have a UCP1-like uncoupling
activity (42, 43), transports aspartate, 4-carbon dicarboxylates,
phosphate, and sulfate (34). The percentages of identical amino
acids between hUCP2 and AtUCP1 (51%) and AtUCP2 (44%)
suggest that these proteins are highly related to one another.
However, it is not possible to make reliable assumptions about
the substrate specificity or about the transport modes on the
basis of the amino acid similarity, given that even close MC
homologs, such as isoforms 1 and 2 of the human ornithine
carrier having 87% identical sequences, exhibit considerable
differences in substrate specificity and transport kinetics (44,
45). Therefore, we decided to investigate the transport proper-
ties of AtUCP1 and AtUCP2 by recombinant expression, puri-
fication, and reconstitution into liposomes (the EPRA method
(9)).
The results presented in this study demonstrate that AtUCP1
and AtUCP2 both transport aspartate, glutamate, cysteine sul-
finate, cysteate, malonate, malate, oxaloacetate, and 2-oxogl-
utarate and, to a lesser extent, D-aspartate, cysteine, oxalate,
succinate, P
i
, sulfate, and thiosulfate (Fig. 4). In addition,
AtUCP2 also transports fumarate, glutarate, and nitrate to
some extent (Fig. 4B). The substrate specificities of AtUCP1
and AtUCP2 are, therefore, (i) similar as expected in light of
their high sequence identity (72%) and (ii) broader than those of
previously characterized mitochondrial carriers (9), given that
their substrates overlap those of hUCP2 and those of the aspar-
Table 3
Influence of membrane potential and pH gradient on the activity of reconstituted AtUCP1 and AtUCP2
The exchanges were started by adding 0.8 mM[
14
C]aspartate or 0.8 mM[
14
C]malate to AtUCP1- and AtUCP2-reconstituted proteoliposomes. For the measurements of the
aspartate/glutamate carrier activity, 50
␮
M[
14
C]aspartate was added to proteoliposomes reconstituted with AGC2-CTD. K
in
⫹
was in included as KCl in the reconstitution
mixture, whereas K
out
⫹
was added as KCl together with the labeled substrate. Valinomycin or nigericin was added in 10
␮
l ethanol/ml of proteoliposomes, whereas the control
samples contained the solvent alone. 20 mMor2mMPIPES (pH 7.0) was present inside and outside the proteoliposomes in the experiments with valinomycin or nigericin,
respectively. The exchange reactions were stopped after 7, 20, and 30 s for AtUCP1, AtUCP2, and AGC2-CTD, respectively. The values are means ⫾S.E. of four independent
experiments carried out in duplicate.
Transport properties of AtUCP1 and AtUCP2
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tate/glutamate and dicarboxylate carriers (24, 30, 40, 46, 47).
AtUCP1 and AtUCP2 share a number of similar transport
properties; for example, both proteins catalyze a highly efficient
counterexchange of substrates; do not transport mono- and
tricarboxylates, nucleotides, and other amino acids; respond
similarly to the inhibitors tested; and have similar transport
affinities (K
i
) for aspartate, glutamate, and malate. However,
they greatly differ for their specific activities (V
max
), AtUCP1
being much more active than AtUCP2, although both activities
are similar or higher than those exhibited by most mitochon-
drial carriers characterized to date (1, 9). Furthermore, some
substrates (e.g. D-aspartate and 2-oxoglutarate) are transported
at higher rates by AtUCP2 than AtUCP1 compared with the
respective [
14
C]aspartate/aspartate exchanges.
The results of recombinant protein studies are largely con-
sistent with the in vivo evaluation of the function of the proteins
that was possible via the isolation and crossing of the respective
knockout mutants. Thus, the metabolic phenotype of the
mutants was characterized by changes in organic acid and
amino acid levels, which are probably due to altered exchanges
of these metabolites between the mitochondria and cytosol.
However, the differences in metabolite content of the knock-
outs, which were dependent on salt stress, are difficult to dis-
entangle, and this will probably require considerable further
research effort.
Two additional remarks regarding the transport properties
of AtUCP1 and AtUCP2 should be made. First, the close bio-
chemical similarities between AtUCP1 and AtUCP2 are under-
standable, given the commonality of their gene structures; both
genes (At3g54110 and At5g58970) share an identical exon/in-
tron structure. We therefore assume that they derive from a
common molecular ancestor, accounting for their similarities
in biochemical properties. After gene duplication, independent
evolution took place allowing the development of individual
properties, such as the different specific activity and slightly
different substrate preference. Second, our transport measure-
ments, in agreement with the previous data on AtUCP3–6 (24)
and hUCP2 (34), are in contrast with the idea that AtUCP1–6
as well as the human UCP1–6 are all “uncoupling proteins”
transporting protons and dissipating the proton motive force
generated by the respiratory chain. In particular, our findings
show that AtUCP1 and AtUCP2 greatly differ from AtUCP4 –6
(previously demonstrated to be dicarboxylate carriers) and sug-
gest that they also differ from AtUCP3, which displays only 35
and 37% identical amino acids with AtUCP1 and AtUCP2,
respectively. Several protein sequences available in databases
Figure 8. Subcellular localization of AtUCP1-/AtUCP2-GFP fusion proteins in N. benthamiana protoplasts. Fluorescent signals of AtUCP1-/AtUCP2-GFP
(green), mitochondrial marker IVD-eqFP611 (red), chlorophyll A/chloroplasts (blue), and merge showing the overlap of the fluorescent signals (yellow) detected
via confocal laser-scanning microscopy. A, co-localization of AtUCP1-GFP with the mitochondrial marker. B, co-localization of AtUCP2-GFP with the mitochon-
drial marker. Scale bar,20
␮
M. Two independently transformed cells are shown in each panel.
Transport properties of AtUCP1 and AtUCP2
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are likely to be orthologs of AtUCP1 and AtUCP2 in mono-
cots, dicots, conifers, mosses, and green algae species. These
sequences include A9PAU0_POPTR and B9GIV8_POPTR
from Populus trichocarpa (86 and 79% identity with AtUCP1
and AtUCP2, respectively), A0A077DCK6_TOBAC from Nico-
tiana tabacum (84% identity with AtUCP1), C6T891_SOYBN
from Glycine max (84% identity), I3ST66_LOTJA from Lotus
japonicus (83% identity), A9RLI6_PHYP from Physcomitrella
patens (81% identity), A9P0D2_PICSI from Picea sitchensis
(79% identity), Q2QZ12_ORYSJ from Oryza sativa (77% iden-
tity), Q8S4C4_MAIZE from Zea mays (76% identity), and
A8J1X0_CHLRE from Chlamydomonas reinhardtii (76% iden-
tity) (Fig. S1). To our knowledge, none of these proteins have
been characterized biochemically.
Previous analyses of AtUCP1 knockout mutants revealed
impaired photorespiration due to a dramatic decrease in mito-
chondrial glycine oxidation rate, which led to the suggestion of
a physiological role of AtUCP1 in uncoupling the mitochon-
drial membrane potential for fine-tuning the cell redox state
concomitant with photorespiration (37). The substrate speci-
ficity and high transport activity of AtUCP1 revealed in this
study shed new light on its role in photorespiration. We suggest
that AtUCP1 is involved in the glycolate pathway by playing a
role in the transfer of reducing equivalents across the mito-
chondrial inner membrane (Fig. 10). In the glycolate pathway,
2-phosphoglycolate formed from O
2
usage by Rubisco in chlo-
roplasts needs to be transformed in a series of reactions local-
ized in peroxisomes and mitochondria in order to return to the
Calvin–Benson cycle as 3-phosphoglycerate. The mitochon-
drial reaction catalyzed by glycine decarboxylase (GDC), which
is the most abundant enzyme in plant mitochondria, reduces
NAD
⫹
to NADH, and peroxisomal hydroxypyruvate reductase
oxidizes NADH to NAD
⫹
. This implies that the reducing
equivalents of NADH are transported from the mitochondria
to the peroxisomes as malate (Fig. 10). In this respect, AtUCP1
(and AtUCP2) would mainly transport aspartate and glutamate
Figure 9. Levels of organic acids in the seedlings of AtUCP knockouts
exposed or not to salt stress. Plants were grown on the plates containing 0,
50, and 75 mMNaCl for 12 days, and the relative levels of malate (A), fumarate
(B), citrate (C), and pyruvate (D) in whole seedlings were determined. The
levels of the metabolites were normalized to the mean of those of wildtype
plants grown on the plate without salt. The means ⫾S.E. (error bars) from
plants grown on three individual plates are shown. Orange, wildtype; brown,
ucp1;green,ucp2;light green,ucp1/ucp2 double knockout.
Figure 10. Role of AtUCP1 and AtUCP2 in photorespiration. Blue and red
lines with arrows indicate the flow of the glycolate pathway and the transport
of reducing equivalents, respectively. Blue and red dashed lines with arrows
indicate several transformation steps and alternative paths, respectively. The
presence of GOT in the peroxisomes, which is uncertain, has been drawn.
Compounds are abbreviated in black as follows.
␣
-KG,
␣
-ketoglutarate; OAA,
oxaloacetate; OH-Pyr, hydroxypyruvate. Enzymes are abbreviated in green.
GDC, glycine decarboxylate; GOT, glutamate oxaloacetate transaminase; HPR,
hydroxypyruvate reductase; MDH, malate dehydrogenase. The figure is mod-
ified from Refs. 37 and 51.
Transport properties of AtUCP1 and AtUCP2
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as components of the mitochondrial malate/aspartate shuttle
(MAS), which has been suggested to exist in plants in connec-
tion to photorespiration (48, 49). More precisely, we propose
that the primary function of AtUCP1 (and AtUCP2) is to cata-
lyze an exchange of aspartate
out
for glutamate
in
across the inner
mitochondrial membrane, thus contributing to the export of
reducing equivalents of NADH from mitochondria in conjunc-
tion with the other enzymes of MAS. In mammals, MAS trans-
fers the reducing equivalents of NADH from the cytosol to the
mitochondria (i.e. in the opposite direction of that occurring
during photorespiration) because the aspartate glutamate car-
riers (AGC1 and -2) catalyze an electrophoretic exchange of
aspartate
⫺
for glutamate
⫺
⫹H
⫹
, and hence exit of aspartate
and entry of glutamate are greatly favored in active mitochon-
dria with a positive membrane potential outside, making the
aspartate/glutamate exchange and the entire MAS unidirec-
tional. By striking contrast, the aspartate/glutamate exchanges
mediated by AtUCP1 and AtUCP2 are electroneutral (Table 3)
and therefore independent from the proton motive force exist-
ing across the mitochondrial membrane. The hypothesis that
AtUCP1 and AtUCP2 are involved in the glycolate pathway by
catalyzing an aspartate
out
/glutamate
in
exchange and thereby
contributing to the export of reducing equivalents from the
mitochondrial matrix is supported by the following consider-
ations: (i) aspartate and glutamate, to the best of our knowledge,
are only transported by AtUCP1 (and AtUCP2) in Arabidopsis
mitochondria; (ii) these metabolites are present in the cytosol at
very high concentrations (about 20 mM)(50), which are much
higher than those of 2-oxoglutarate, malate, and oxaloacetate;
(iii) both mitochondrial glutamate-oxaloacetate transaminase
and malate dehydrogenase are involved in the regeneration of
NAD
⫹
by GDC in mitochondria (51), and both mitochondrial
and peroxisomal malate dehydrogenases are required for opti-
mal photorespiration rates (52, 53); and (iv) as shown by the
BAR Arabidopsis eFP Browser 2.0 (http:/bar.utoronto.ca),
7
AtUCP1 and AtUCP2 are expressed in many plant tissues,
being more highly expressed in photosynthetic ones (Figs. S4
and S5). Interestingly, the expression of AtUCP1 is co-regu-
lated with enzymes of the citric acid cycle, such as aconitase,
isocitrate dehydrogenase,
␣
-ketoglutarate dehydrogenase, and
succinyl-CoA ligase, as well as with the peroxisomal trans-
porter for NAD
⫹
(21, 22)(Fig. S6).
It is noteworthy that AtUCP1 (and AtUCP2) may also cata-
lyze (i) the exchange between malate
in
and oxoglutarate
out
(i.e.
the other mitochondrial membrane reaction of MAS) (Fig. 10)
and (ii) an oxaloacetate
out
/malate
in
exchange, which per se
would result in export of the reducing equivalents of NADH
from the mitochondria. However, these exchanges are also cat-
alyzed by other Arabidopsis MCs, such as DTC, DIC1, DIC2,
and DIC3 (23, 24), and the affinities of AtUCP1 and AtUCP2 for
aspartate are much higher than those for the other substrates.
An additional hypothetical function of AtUCP1 and AtUCP2
in the photorespiratory glycolate pathway concerns the transfer
of nitrogen equivalents across the mitochondrial membrane.
During oxidative glycine decarboxylation by GDC in mitochon-
dria, ammonia is released and reassimilated by the plastidial
glutamine synthetase/glutamine oxoglutarate aminotransferase
(GS/GOGAT) reaction. How ammonia released by GDC in
mitochondria is shuttled to GS/GOGAT is still unknown, but it
has been suggested that shuttling of amino acids across the
mitochondrial membrane might be involved in this process
(54). One possible route that would involve AtUCP1 and/or
AtUCP2 would be incorporation of ammonia into 2-oxogl-
utarate by mitochondrial glutamate dehydrogenase, yielding
glutamate, which is exported to the cytoplasm in counterex-
change with external 2-oxoglutarate, thereby providing a new
acceptor molecule for the glutamate dehydrogenase reaction.
This hypothesis awaits experimental testing in future work.
Due to their broad substrate specificities, AtUCP1 (and
AtUCP2) may be multifunctional and play further important
physiological roles, depending on the metabolic conditions of
the organ/tissue and the light/dark phase. For example, they
might be involved in sulfur metabolism by exchanging cysteine
sulfinate, cysteate, and cysteine with sulfate. Furthermore, in
the dark, AtUCP1 (and AtUCP2) may catalyze the transport of
glutamate into the mitochondria and the exit of aspartate to the
cytosol, providing (together with the other enzymes of the
malate aspartate shuttle) reducing equivalents in the form of
NADH ⫹H
⫹
to the mitochondrial respiratory chain.
Experimental procedures
Sequence analysis
BLAST and reciprocal BLAST were used to search for homo-
logs of AtUCP1 and AtUCP2 (encoded by At3g54110 and
At5g58970, respectively) in e!Ensamble and UniProt. Sequences
were aligned with ClustalW.
Bacterial expression and purification of AtUCP1 and AtUCP2
PCR using complementary sequence-based primers was
used to amplify the coding sequences of AtUCP1 from A. thali-
ana leaf cDNA and AtUCP2 from a custom-made synthetic
gene (Invitrogen) with codons optimized for E. coli. The for-
ward and reverse oligonucleotide primers contained the restric-
tion sites NdeI and HindIII (AtUCP1) or XhoI and EcoRI
(AtUCP2). The amplified gene fragments were cloned into
pMW7 (AtUCP1) and pRUN (AtUCP2) vectors and trans-
formed into E. coli TG1 cells (Invitrogen). Transformants were
selected on LB (10 g/liter tryptone, 5 g/liter yeast extract, 5
g/liter NaCl, pH 7.4) plates containing 100
␮
g/ml ampicillin. All
constructs were verified by DNA sequencing.
AtUCP1 and AtUCP2 were overexpressed as inclusion bod-
ies in the cytosol of E. coli BL21(DE3) (AtUCP1) and BL21
CodonPlus(DE3)-RIL (AtUCP2) as described previously (55).
Control cultures with the empty vector were processed in par-
allel. Inclusion bodies were purified on a sucrose density gradi-
ent (56) and washed at 4 °C, first with TE buffer (10 mMTris-
HCl, 1 mMEDTA, pH 7.0); once with a buffer containing 3%
Triton X-114 (w/v), 1 mMEDTA, 10 mMPIPES (pH 7.0), and 10
mMNa
2
SO
4
; and finally three times with TE buffer (57). The
inclusion body proteins were solubilized in 2% lauric acid, 10
mMPIPES (pH 7.0), and 3% Triton X-114 (AtUCP1) or 1.6%
sarkosyl (w/v), 10 mMPIPES, pH 7.0, and 0.6% Triton X-114
7
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Transport properties of AtUCP1 and AtUCP2
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(AtUCP2). Unsolubilized material was removed by centrifuga-
tion (15,300 ⫻gfor 10 min).
Reconstitution of AtUCP1 and AtUCP2 into liposomes and
transport measurements
The solubilized recombinant proteins were reconstituted
into liposomes by cyclic removal of the detergent with a hydro-
phobic column of Amberlite beads (Bio-Rad), as described pre-
viously (41). The reconstitution mixture contained solubilized
proteins (about 6
␮
g), 1% Triton X-114, 1.4% egg yolk phospho-
lipids as sonicated liposomes, 10 mMsubstrate, 20 mMPIPES
(pH 7.0), 1 mg of cardiolipin, and water to a final volume of 700
␮
l. These components were mixed thoroughly, and the mixture
was recycled 13 times through a Bio-Beads SM-2 column pre-
equilibrated with a buffer containing 10 mMPIPES (pH 7.0) and
50 mMNaCl and the substrate at the same concentration used
in the starting mixture.
External substrate was removed from proteoliposomes on a
Sephadex G-75 column pre-equilibrated with 10 mMPIPES and
50 mMNaCl, pH 7.0. Transport at 25 °C was initiated by adding
the indicated radioactive substrates (American Radiolabeled
Chemicals Inc. or PerkinElmer Life Sciences) to substrate-
loaded (exchange) or empty (uniport) proteoliposomes. Trans-
port was terminated by adding 20 mMpyridoxal 5⬘-phosphate
and 20 mMbathophenanthroline, which in combination inhibit
the activity of several MCs completely and rapidly (58–60). In
controls, the inhibitors were added at the beginning together
with the radioactive substrate according to the “inhibitor-stop”
method (41). Finally, the external substrate was removed, and
the radioactivity in the proteoliposomes was measured. The
experimental values were corrected by subtracting control val-
ues. The initial transport rates were calculated from the radio-
activity incorporated into proteoliposomes in the initial linear
range of substrate transport. The kinetic constants K
m
,V
max
,
and K
i
were determined from Lineweaver–Burk and Dixon
plots. For efflux measurements, proteoliposomes containing 5
mMinternal aspartate or malate were loaded with 5
␮
M[
14
C]
aspartate and [
14
C]malate, respectively, by carrier-mediated
exchange equilibrium (61, 62). The external radioactivity was
removed by passing the proteoliposomes through Sephadex
G-75 columns pre-equilibrated with 50 mMNaCl. Efflux was
started by adding unlabeled external substrate or buffer alone
and terminated by adding the inhibitors indicated above.
Cloning and transient expression of GFP fusion constructs
For subcellular localization of AtUCP1 and AtUCP2, the
AtUCP1-GFP and the AtUCP2-GFP fusion constructs were
prepared. The AtUCP1 coding sequence was amplified via Phu-
sion High-Fidelity DNA Polymerase (New England Biolabs)
using primers BH254 and BH255 (Table S4) and cloned with
the Gibson Assembly Cloning Kit (New England Biolabs) into
the expression vector pTKan (63) using the restriction sites
ApaI and SacII. The GFP coding sequence for C-terminal GFP
fusion was inserted via SacII and SpeI into the pTKan vector.
The final vector contains AtUCP1 with a C-terminal GFP
(AtUCP1-GFP) under the control of an optimized Arabidopsis
ubiquitin-10 promoter (64) and the terminator of the nos
gene from Agrobacterium tumefaciens. The AtUCP2 coding
sequence was amplified via Phusion High-Fidelity DNA Polym-
erase (Thermo Fisher Scientific) using primers UCP2_BPF and
UCP2_BPR-s (Table S4) and cloned into pDONR207 vector by
aBP recombination reaction with BP Clonase II enzyme mix
(Invitrogen). The resulting entry clone was used for an LR
recombination reaction by LR Clonase II enzyme mix (Invitro-
gen) with pK7FWG2 destination vector (65) to construct
expression vector for the expression of AtUCP2-GFP under the
control of the 35S promoter.
A. tumefaciens strain GV3101 (pMP90) (66) was trans-
formed with the localization vectors (AtUCP1-GFP and AtUCP2-
GFP) and the mitochondrial marker IVD-eqFP611 expressing
the Arabidopsis IVD tagged at the C terminus with eqFP611
(67, 68). 5 ml of YPD medium (20 g/liter tryptone, 10 g/liter
yeast extract, 20 g/liter glucose) was inoculated with positively
transformed cells and grown overnight at 28 °C. Cells were har-
vested via centrifugation (10 min, 3000 ⫻g) and resuspended in
infiltration buffer (10 mMMgCl
2
,10mMMES, pH 5.7, 100
␮
M
acetosyringone) to an A
600
of 0.5. N. benthamiana leaves of the
same age were co-infiltrated with mitochondrial marker and
corresponding AtUCP1 and AtUCP2 fusion proteins (69). Pro-
toplasts were isolated 2 days after infiltration. Therefore, leaves
were cut into 0.5 ⫻0.5-cm pieces and incubated in Protoplast
Digestion Solution (1.5% (w/v) cellulase R-10, 0.4% (w/v)
macerozyme R-10, 0.4 Mmannitol, 20 mMKCl, 20 mMMES, pH
5.6, 10 mMCaCl
2
, 0.1% (w/v) BSA) for2hat30°C.Isolated
protoplasts were resuspended in W5 solution (154 mMNaCl,
125 mMCaCl
2
,5mMKCl, 2 mMMES, pH 5.6) and directly used
for microscopy. Protoplasts were observed using a Zeiss LSM
780 confocal microscope and Zeiss ZEN software. The follow-
ing excitation/emission wavelength settings were used: GFP
(488 nm/490–550 nm), IVD-eqFP611 (561 nm/580 – 625 nm),
and chlorophyll A (488 nm/640–710 nm). Pictures were pro-
cessed using Fiji software (75) and Adobe Photoshop CS6
(Adobe Systems).
Isolation, generation, and molecular characterization of
single- and double-knockout mutants of ucp1 and ucp2
T-DNA insertion lines for AtUCP1 (SAIL_536G01, referred
to as ucp1 (37)) and AtUCP2 (SALK_080188, referred to as
ucp2) were obtained from the ABRC. To identify homozygous
T-DNA insertion lines, genomic DNA was extracted and geno-
typed using gene-specific primer pairs (DG8/DG9 for ucp1
and DG6/DG5 for ucp2) and a primer pair for T-DNA/gene
junction (DG9/SAIL-LBa for ucp1 and DG6/SALK-LBa1 for
ucp2)(
Table S4). The position of the T-DNA was checked by
sequencing. Homozygous ucp1 and ucp2 were further propa-
gated. To generate double mutants (referred to as dKO),
homozygous ucp1 and ucp2 were crossed. Heterozygous plants
were selected by PCR in the T1 generation. After self crossing,
dKO were selected by PCR and further propagated.
Total RNA was extracted from wildtype, mutant, and trans-
genic Arabidopsis plants using the guanidinium thiocyanate-
phenol-chloroform method (70) and subjected after DNase
treatment (RQ1 RNase-Free DNase, Promega) to cDNA syn-
thesis (Superscript II Rnase H- reverse transcriptase, Invitro-
gen). Gene expression of AtUCP1 and AtUCP2 were analyzed
using gene-specific primer pairs. The following primer sets
Transport properties of AtUCP1 and AtUCP2
4224 J. Biol. Chem. (2018) 293(11) 4213–4227
at INRA Institut National de la Recherche Agronomique on April 25, 2019http://www.jbc.org/Downloaded from

were used: DG23/DG24 for AtUCP1 and DG25/DG26 for
AtUCP2 (Table S4). As a control for cDNA quality and quan-
tity, a cDNA fragment of an actin gene (ACT7, At5g09810) was
amplified using ML167 and ML168. PCR conditions were as
follows: 94 °C for 2 min, followed by cycles of 94 °C for 30 s,
58 °C for 45 s, 72 °C for 60 s, and a final extension for 2 min.
Products were visualized on an ethidium bromide–stained 1%
agarose gel.
Metabolite profiling
To obtain a broad overview of the major pathways of central
metabolism, an established GC-MS–based metabolite profil-
ing method was used to quantify the relative metabolite levels in
the Arabidopsis rosette (⬃50 mg fresh weight). The extraction,
derivatization, standard addition, and sample injection were
performed exactly as described previously (71). This analysis
allowed the determination of 46 different compounds, repre-
senting the main classes of primary metabolites (i.e. amino
acids, organic acids, and sugars).
Other methods
Proteins were analyzed by SDS-PAGE and stained with Coo-
massie Blue dye. The identity of the bacterially expressed, puri-
fied AtUCP1 and AtUCP2 was assessed by MALDI-TOF mass
spectrometry of trypsin digests of the corresponding band
excised from Coomassie-stained gels (25, 72). The amount of
purified AtUCP1 and AtUCP2 proteins was estimated by laser
densitometry of stained samples using carbonic anhydrase as a
protein standard (73). The amount of protein incorporated into
liposomes was measured as described (74) and was about 25%
of protein added to the reconstitution mixture. K
⫹
-diffusion
potentials were generated by adding valinomycin (1.5
␮
g/mg
phospholipid) to proteoliposomes in the presence of K
⫹
gradi-
ents. For the formation of an artificial ⌬pH (acidic outside),
nigericin (50 ng/mg phospholipid) was added to proteolipo-
somes in the presence of an inwardly directed K
⫹
gradient.
Author contributions—M.M., L.P., A.R.F., A.P.W., and F.P. concep-
tualization; M.M., L.D., D.G., T.O., B.H., L.P., and D.V.M. method-
ology; A.R.F., A.P.W., and F.P. supervision.
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Transport properties of AtUCP1 and AtUCP2
J. Biol. Chem. (2018) 293(11) 4213–4227 4227
at INRA Institut National de la Recherche Agronomique on April 25, 2019http://www.jbc.org/Downloaded from

Ferdinando Palmieri
Luigi Palmieri, Daniela Valeria Miniero, Alisdair R. Fernie, Andreas P. M. Weber and
Magnus Monné, Lucia Daddabbo, David Gagneul, Toshihiro Obata, Björn Hielscher,
mitochondrial transporters of aspartate, glutamate, and dicarboxylates areArabidopsis thalianaUncoupling proteins 1 and 2 (UCP1 and UCP2) from
doi: 10.1074/jbc.RA117.000771 originally published online January 25, 2018
2018, 293:4213-4227.J. Biol. Chem.
10.1074/jbc.RA117.000771Access the most updated version of this article at doi:
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