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Evidence for Several Cysteine Transport Mechanisms in the
Mitochondrial Membranes of Arabidopsis thaliana
Chun Pong Lee1,2, Markus Wirtz1 and Rüdiger Hell1,*
Regular Paper
Centre for Organismal Studies (COS) Heidelberg, Im Neuenheimer Feld 360, University of Heidelberg, D-69120 Heidelberg, Germany
Present address: Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK.
*Corresponding author: E-mail, [email protected]; Fax, +49-6221-54-5859.
(Received July 11, 2013; Accepted October 22, 2013)
Cysteine is essential for many mitochondrial processes in
plants, including translation, iron–sulfur cluster biogenesis
and cyanide detoxification. Its biosynthesis is carried out by
serine acetyltransferase (SAT) and O-acetylserine (thiol)
lyase (OAS-TL) which can be found in the cytosol, plastids
and mitochondria. Mutants lacking one compartmentspecific OAS-TL isoform show viable phenotypes, leading
to the hypothesis that the organellar membranes are permeable to substrates and products of the cysteine biosynthetic pathway. In this report, we show that exogenouslly
supplied [35S]cysteine accumulates in the mitochondrial
fraction and is taken up into isolated mitochondria for in
organello protein synthesis. Analysis of cysteine uptake by
isolated mitochondria and mitoplasts indicates that cysteine
is transported by multiple facilitated mechanisms that operate in a concentration gradient-dependent manner. In
addition, cysteine uptake is dependent mainly on the pH
across the inner membrane. The rates of mitochondrial cysteine transport can be mildly altered by specific metabolites
in the cyanide detoxification-linked sulfide oxidation, but
not by most substrates and products of the cysteine biosynthetic pathway. Based on these results, we propose that the
transport of cysteine plays a pivotal role in regulating
cellular cysteine biosynthesis as well as modulating the
availability of sulfur for mitochondrial metabolism.
Keywords: Arabidopsis thaliana Cysteine biosynthesis and
catabolism Cysteine transport Mitochondria Proton
gradient Sulfur assimilation.
Editor-in-Chief’s choice
Abbreviations: BSA, bovine serum albumin; CCCP, carbonyl
cyanide m-chlorophenylhydrazone; CSC, cysteine synthase
complex; c, membrane potential; pH, proton gradient;
ETHE1, sulfur dioxygenase; IMM, inner mitochondrial membrane; MCF, mitochondrial carrier family; OAS, O-acetylserine; OMM, outer mitochondrial membrane; OAS-TL,
O-acetylserine (thiol) lyase; OAS-TL-C, mitochondrial isoform
of O-acetylserine (thiol) lyase; SAT, serine acetyltransferase;
SHAM, salicylhydroxamic acid; VDAC, voltage-dependent
anion channel.
Cysteine fulfills a number of essential cellular roles in both
multicellular and unicellular organisms, including protein synthesis, oxidative stress response, iron–sulfur (Fe–S) cluster biogenesis, and regulatory and structural changes in proteins. In
plants, cysteine is synthesized from two sequential enzymatic
reactions: serine acetyltransferase (SAT) facilitates the production of O-acetylserine (OAS) from serine and acetyl-CoA, while
O-acetylserine (thiol) lyase (OAS-TL) incorporates OAS with
sulfide to synthesize cysteine. These enzymes have multiple
isoforms with distinct kinetic, biochemical and regulatory characteristics, and can be found in plastids, mitochondria and
cytosol (Smith 1972, Rolland et al. 1992, Noji et al. 1998).
OAS-TL can interact with SAT to form the cysteine synthase
complex (CSC) which regulates the activity of each enzyme in
the cysteine biosynthetic pathway but does not facilitate substrate channeling (Droux et al. 1998, Wirtz and Hell 2007). It has
been hypothesized that the presence of SAT and OAS-TL in
each compartment eliminates the necessity for any cysteine
transport systems in mitochondria and plastids (Lunn et al.
1990, Rolland et al. 1992). However, numerous biochemical
and reverse genetic analyses have shown that cysteine biosynthesis is regulated in a compartment-specific manner: the mitochondrion is responsible for providing most of the OAS in the
cell, while cytosol is the major site of cysteine production (Wirtz
and Hell 2007, Haas et al. 2008, Heeg et al. 2008, Watanabe et al.
2008a, Watanabe et al. 2008b). These findings raise the possibility that the metabolites in sulfur assimilation and cysteine
biosynthesis can move across the organellar membranes.
The low OAS-TL to SAT activity ratio in mitochondria compared with those in the cytosol and plastids suggests that the
majority of the OAS produced in the mitochondrion is more
likely to leave the organelle (Droux et al. 1998, Droux 2004, Heeg
et al. 2008). Nuclear magnetic resonance analysis of [13C]serine
uptake by isolated mitochondria revealed that the rate of OAS
formation from serine in the mutant lacking mitochondrial
OAS-TL (OAS-TL-C) is lower than that in the wild type
(Wirtz et al. 2012). Knock-out of OAS-TL-C does not lead to
Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155, available FREE online at
! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013.
Plant mitochondrial cysteine transport
a defect in mitochondrial translation, a drop in total cellular
cysteine content or a reduction in the abundance of mitochondrial proteins containing an iron–sulfur cluster, although it is
important for the detoxification of H2S to protect the respiratory chain (Heeg et al., 2008, Álvarez et al. 2012, Birke et al.
2012). In mammals, the sulfur dioxygenase ETHE1 detoxifies
sulfide generated by cysteine degradation (Hildebrandt and
Grieshaber 2008). The Arabidopsis ortholog AtETHE1 is localized in mitochondria and shares a number of conserved residues that are critical for regulating enzymatic activity (Holdorf
et al. 2012). The loss-of-function AtETHE1 mutant is lethal to
early seed development (Holdorf et al. 2012), demonstrating
the essential role of AtETHE1 in plant metabolism. It is apparent that the lack of cysteine degradation but not production
leads to a lethal phenotype, while OAS-TL-C plays a more
predominant role in regulating OAS formation through CSC
formation than cysteine production.
Despite the importance of cysteine in many plant mitochondrial functions, the biochemical characteristics/properties and
the molecular identities of the transporters for cysteine remain
elusive to date. While early mitochondrial transport analyses
using organelle swelling technique shows that L-cysteine can be
diffused/transported across the mitochondrial membranes in
rats (Cybulski and Fisher 1977), nothing is known about the
kinetics and properties of such a process. This is also unknown
in plants and highly questioned due to the compartmentspecific regulation of cysteine biosynthesis, since cysteine can
be significantly synthesized in plant mitochondria as demonstrated by the viability of the oastlAB mutant lacking cysteine
synthesis in cytosol and plastids (Heeg et al. 2008). In this
report, we analyzed the kinetic characteristics and specificities
of cysteine transport by isolated mitochondrial particles from
Arabidopsis using a silicone oil centrifugation approach.
According to our data, we could show that there are multiple
cysteine transport modes with distinct biochemical characteristics in isolated mitochondria, indicative of the existence of
multiple specific cysteine transporters in the mitochondrial
Import of cysteine into isolated mitochondria
The mitochondrial fraction incubated with radiolabeled
[35S]cysteine for 20 min at 25 C was layered on top of a modified 1.4 ml mini-Percoll step gradient (Millar et al. 2001, Howell
et al. 2006). After centrifugation, the mitochondrial fraction
appeared as a yellow-brownish band between the 25% and
40% Percoll interfaces (Fig. 1A). A total of fifteen 0.1 ml fractions were collected. About 80% of the total radioactivity
detected in the Percoll gradient was accumulated in fractions
11–14 (Fig. 1B) where the mitochondria were located.
Externally applied radiolabeled cysteine was incorporated into
the mitochondrial-encoded translation products within 60 min
(Fig. 1C), indicating that the outer (OMM) and inner (IMM)
mitochondrial membranes are permeable to cysteine. The
major protein bands detected have previously been identified
to be some of the key mitochondria-encoded components of
the electron transport chain (Kühn et al. 2011) that contain at
least four cysteine residues.
Kinetic characteristics of cysteine uptake
by mitochondria
Using the single silicone oil layer centrifugation procedure
(Heldt 1980), the accumulation of radioactivity in isolated
mitochondria in the presence of two external concentrations
of [35S]cysteine, namely 10 and 150 mM, over 3 min at 25 C was
examined (Fig. 2A, B). The uptake of 10 and 150 mM radiolabeled cysteine was very rapid at a rate of 8.2 and
59.2 pmol mg 1 protein s 1, respectively, in the first 30 s
(assuming the kinetics within the time interval strictly followed
linear characteristics), and reached equilibrium within 1 min
after the addition of substrate. Isolated mitochondria took up
different amounts of total cysteine after 3 min of incubation,
accumulating a maximum of 0.4 (Fig. 2A) and 4.0 (Fig. 2B)
nmol cysteine mg 1 protein for 10 and 150 mM of external
substrate, respectively.
We then measured the rate of [35S]cysteine uptake by isolated mitochondria in the first 20 s at 18 different concentrations of external cysteine (0–1,000 mM, Fig. 2C). Within the
concentration range tested, cysteine uptake by isolated plant
mitochondria displayed at least two phases of kinetics. The
uptake rate reached saturation at approximately 300 mM cysteine and was stable in the presence of up to 500 mM cysteine.
The uptake rate became non-saturating when a higher cysteine
amount was supplied, most probably due to the activity of less
specific general amino acid transporters that significantly transport cysteine only at these high concentrations (750 mM).
In contrast to mitochondria, mitoplasts appear to show nonsaturable uptake of cysteine from 0 to 1,000 mM cysteine. The
cysteine uptake of mitoplasts was almost indistinguishable
from mitochondrial cysteine uptake at low cysteine concentrations (200 mM, Fig. 2C). We conclude from these results that
(i) the saturable cysteine uptake system is localized in the
OMM, which is absent in mitoplasts; and that (ii) the saturable
uptake system of the OMM has similar or even higher affinities
for cysteine than the apparently non-saturable uptake system
of the IMM. The latter explains why uptake of cysteine into
mitoplasts, having only an IMM, was similar at low cysteine
concentrations (200 mM) when compared with mitochondria, having an IMM and OMM (Fig. 2C).
Based on the information obtained thus far, we examined
the kinetic parameters of the cysteine-specific saturable components of cysteine transport in mitochondria (0–500 mM)
using a Lineweaver–Burk plot (Supplementary Fig. S1). The
Km and Vmax values for cysteine uptake by mitochondria
derived from this plot were 0.39 ± 0.11 mM cysteine and
27.2 ± 4.4 nmol min 1 mg 1 protein, respectively. When the
uptake kinetics of mitoplasts by Michaelis–Menten kinetics
Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013.
C. P. Lee et al.
B Incorporated radioactivity
Time (min)
10 30 60
(1000 x cpm fraction-1)
5 10 15 20 25 30 35
Fig. 1 Cysteine is incorporated into isolated mitochondria and required for in organello protein synthesis. (A) The mitochondrial fraction
incubated with [35S]cysteine was re-isolated by a short discontinuous Percoll gradient. (B) A graph showing the radioactivity in each of the 0.1 ml
fraction collected from the top (fraction 1) to the bottom (fraction 15) of the Percoll gradient. (C) In vitro protein synthesis by isolated
mitochondria in the presence of [35S]cysteine for 10, 30 and 60 min. Proteins were visualized after gel electrophoresis and protein transfer.
An equal amount of proteins (100 mg) was loaded onto each lane. The identity of major protein bands detected based on previous identification
is indicated on the right.
Cysteine uptake
(nmol mg-1 protein)
Time (s)
Cysteine uptake
(nmol min-1 mg-1 protein)
Cysteine uptake
(nmol min-1 mg-1 protein)
Time (s)
Cysteine (µM)
Cysteine (µM)
Fig. 2 Uptake of [35S]cysteine into isolated mitochondria or mitoplasts as a function of time or external concentration. (A and B) Isolated
mitochondria were incubated with either (A) 10 mM or (B) 150 mM cysteine for the time indicated. (C) The rate of [35S]cysteine accumulation
was measured from 0 to 1,000 mM cysteine in mitochondria (dark circle/line) or mitoplasts (gray square/line). For (C), mitochondrial/mitoplast
fractions were incubated with [35S]cysteine for 20 s. (D) Close-up of C showing specifically the rate of [35S]cysteine accumulation by mitoplasts in
the presence of 0–200 mM external cysteine. Each data point represents the average initial rate of uptake calculated from at least three
independent experiments, with the error bar showing the SEM.
Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013.
* *
Other amino acids
basic amino acids
sulfur-containing metabolites
The OMM houses a very limited number of membrane proteins
(Duncan et al. 2011). In contrast, the IMM, which hosts the
electron transport chain, has limited permeability to many metabolites that require specialized membrane transport proteins,
most of which remain unknown to date. To minimize the interference by the OMM with the rate of IMM cysteine uptake, we
examined the characteristics of cysteine transport in the presence of two physiologically relevant substrate concentrations in
isolated mitoplasts. We chose 60 and 200 mM cysteine for the
analysis of cysteine uptake by mitoplasts, because at >300 mM
cysteine the OMM would limit cysteine transport in intact
mitochondria (Fig. 2C).
Various unlabeled amino acids, cyanide or sulfur-containing
compounds were tested to determine if they could competitively reduce the initial uptake rate of [35S]cysteine into isolated
mitoplasts (Fig. 3). In the presence of excess cysteine (5 mM),
the rate of uptake of 60 and 200 mM radiolabeled cysteine by
isolated mitoplasts was reduced by 75% and 45%, respectively. This might suggest that cysteine transport by the IMM
could be facilitated specifically at 60 mM cysteine, but less specifically so at 200 mM cysteine, which indicates the presence of
several cysteine uptake systems with different affinities.
Interestingly, the key metabolites required for the cysteine biosynthetic pathway, namely serine and OAS, did not have a significant effect on the cysteine uptake rate. Among all other
Na2SO 3
Specificity of cysteine transport
Rate of cysteine uptake
(nmol/mg protein/min)
was explored statistically using Sigmaplot, we found that the
uptake was apparently non-saturable in this concentration
range under the experimental conditions applied here
(Supplementary Table S1). We conclude from these results
that in planta the uptake of cysteine into mitochondria would
be limited by the specific cysteine uptake system in the OMM
at cytosolic cysteine concentrations (300 mM; Krueger et al.
2009). Cysteine transport became non-saturable at higher cytosolic cysteine concentrations (750 mM), indicating the presence of non-specific cysteine transport systems in the OMM.
The apparent non-saturable uptake kinetics of the mitoplasts
between 0 and 1,000 mM cysteine was surprising and could be a
result of (i) non-specific uptake of cysteine via a pore in the
IMM; (ii) a single cysteine-specific transporter with a low affinity but high transport rate of cysteine; or (iii) the combined
action of several saturable cysteine uptake systems with different affinities in the IMM that mimic a non-saturable cysteine
uptake under the experimental conditions applied here. A very
careful inspection of cysteine uptake kinetics at selected cysteine concentrations was indicative of the existence of multiple
cysteine transport systems in the IMM (Fig. 2D, close-up of
Fig. 2C). In order to distinguish between these possibilities
and to characterize the unexpected apparent non-saturable
transport over the IMM further, we tested the impact of
amino acids, metabolites of the sulfur assimilation pathways
and inhibitors of the electron transport chain on cysteine
uptake of mitoplasts, having only an IMM.
Rate of cysteine uptake
(nmol/mg protein/min)
Plant mitochondrial cysteine transport
Fig. 3 The effect of amino acids and sulfur-containing metabolites on
the rate of cysteine uptake by isolated mitoplasts. Each of these metabolites was added together with the specified amount of radiolabeled cysteine, and mitoplasts were collected by centrifugation
through silicone oil after a 20 s incubation period. Uptake of (A)
60 mM and (B) 200 mM cysteine was tested. The concentration of
amino acid added was 10 mM, with the exception of cysteine
(5 mM), OAS (4 mM), glutamine (3 mM), histidine (5 mM), phenylalanine (5 mM), leucine (2.3 M) and methionine (6 mM). For thiolcontaining metabolites, Na2S (500 mM), Na2SO4 (25 mM), Na2SO3
(25 mM), Na2S2O3 (25 mM) or GSH (3.2 mM) was tested. The concentration of KCN added was 50 mM. Asterisks (*) indicate P < 0.05. Each
value of uptake rate represents the average ± SE from at least five
independent experiments.
amino acids tested, only lysine, arginine and histidine have a
mild inhibitory effect on uptake only at 200 mM cysteine.
Overall, the unaffected transport of cysteine over the IMM in
the presence of these amino acids rules out the possibility of
cysteine transport by an unspecific pore. The presence of excess
sulfite (SO23 ) in the incubation medium also decreased the
level of cysteine uptake only at 200 mM cysteine when the
uptake of cysteine appeared to be less specific. Either sulfide
(S2 ) or cyanide caused a reduction in the cysteine uptake rate
Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013.
C. P. Lee et al.
at low and high cysteine concentrations. In comparison, the
addition of thiosulfate (S2 O23 ) or glutathione resulted in a
stimulation of the initial cysteine uptake rate only at 60 mM
cysteine. Taken together, our results demonstrate the specific
transport of cysteine over the IMM that is most probably facilitated by several cysteine transporters with different affinities.
The inhibition of cysteine import by selected metabolites
might indicate that these metabolites could be transported by
the same specific transporter as cysteine. Therefore, the release
of radioactivity into the medium by 60 mM [35S]cysteine-preloaded mitoplasts was measured in the presence of various
external compounds (Table 1; Supplementary Table S2).
A significant amount of radiolabeled cysteine was released
when excess unlabeled cysteine is externally applied, indicating
the presence of a cysteine or cysteine-like unknown metabolite
counter-exchange system in the IMM. Internal cysteine cannot
be or can be only marginally exchanged with most amino acids
including glycine, serine, OAS and basic amino acids (Table 1),
adenine nucleotides, phosphate, iron(III), pyruvate, malate and
succinate (Supplementary Table S2). Among cyanides and
sulfur-containing metabolites tested, sulfide, cysteine, sulfite
and cyanide (in descending order of exchange activity) can be
significantly exchanged at a higher rate with internal cysteine
compared with the control (Table 1, P < 0.01). In order to rule
out the possibility that detected 35S radioactivity in the supernatants of exchange experiments is due to release of [35S]sulfide
produced by breakdown of [35S]cysteine within the pre-loaded
mitochondria, we tested the stability of cysteine in mitochondria. Under the experimental conditions applied here, the
amount of cysteine remained unchanged even after 30 min
of incubation with mitochondria at room temperature
(Supplementary Fig. S3A–C). Cysteine also remained stable
in the presence of excess sulfide or sulfite and it did not
appear to be metabolized into sulfide (Supplementary
Fig. S3D, E). The cysteine level dropped and the sulfide level
increased substantially after cyanide was added to isolated
mitochondrial fractions (Supplementary Fig. S3F, L), possibly
due to the rapid action of b-cyanoalanine synthetase which
converts these metabolites into sulfide and b-cyanoalanine
(Yamaguchi et al. 2000). However, in this case, it is not clear
if the detected radioactivity is due to [35S]cysteine that was
exchanged with cyanide, or [35S]sulfide released from metabolized cysteine, or a combination of both. In contrast, the [35S]
sulfur detected in the supernatant in the presence of cysteine,
sulfide or sulfite was from [35S]cysteine, but not from
[35S]sulfide that was produced from cysteine.
The role of the electrochemical gradient across
the IMM and respiration on cysteine uptake
We also examined whether the electrochemical gradient generated across the IMM, mostly by the mitochondrial electron
transport chain, could have an influence on cysteine transport.
The absence of activation solution (to stimulate State III respiration) in the incubation medium had no overall effect on the
Table 1 Specificity of cysteine exchange with various external
Release of
(nmol mg 1
0 ± 0.5
H2O control
Photorespiration/cysteine biosynthesis and catabolism
27.0 ± 1.9**
0.9 ± 1.0
2.4 ± 1.9
2.9 ± 2.2
5.9 ± 2.9
Basic amino acids
3.1 ± 1.9
17.0 ± 5.7
Sulfur-containing metabolites and cyanide-derived ions
5.8 ± 1.0
31.7 ± 2.8**
3.4 ± 1.1
21.7 ± 2.2**
4.8 ± 0.6
Glutathione reduced (GSH)
8.7 ± 1.2*
9.2 ± 4.3
Glutathione oxidized (GSSG)
18.3 ± 1.6**
1.4 ± 1.6
Isolated mitoplasts were pre-loaded with 60 mM [ S]cysteine at 4 C for 30 min.
Back-exchange was initiated by adding 500 mM unlabeled substrate, and the
resulting mixture was incubated for 30 s at 25 C and was stopped by centrifugation through silicone oil. The radioactivity detected in the supernatant (the
layer above the silicone oil) is the amount of 35S released by the mitoplasts.
The average ± SE from at least four experiments is shown. *P < 0.05; **P < 0.01.
rate of cysteine uptake. In the presence of myxothiazol
(Complex III inhibitor), oligomycin (ATP synthase inhibitor)
or salicylhydroxamic acid (SHAM; alternative oxidase inhibitor),
there was no significant difference in the uptake rate of either
cysteine concentration in the mitoplasts (Table 2). Since the
application of various respiratory inhibitors has no effect on
cysteine uptake (Table 2), the reduction in uptake rate
caused by cyanide or sulfide inhibition of Complex IV activity
can be ruled out. Overall, these experiments indicate that ATP
synthesis and respiration driven by the electron transport chain
are not essential for cysteine transport over the IMM.
Upon treatment with the ionophore carbonyl cyanide
m-chlorophenylhydrazone (CCCP), the rate of cysteine uptake
by isolated mitoplasts increased by 1.9- to 2-fold in the presence
of 60 and 200 mM cysteine. Thus, it is likely that the electrochemical gradient primarily regulates the influx of cysteine into the
matrix. To examine further whether the membrane potential
(c) and/or pH have a role in cysteine transport, isolated
mitoplasts were incubated with phosphate (raises the c),
with nigericin (collapses the pH) or with valinomycin (collapses the c) in the presence of 10 mM K+ (Salvi et al. 2006).
Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013.
Plant mitochondrial cysteine transport
Table 2 The effect of uncouplers and respiratory inhibitors on the
rate of cysteine uptake by isolated mitoplasts
Cysteine (mM)
min 1
mg 1
min 1
mg 1
Control (+ activation solution)
4.3 ± 0.2
12.2 ± 0.6
H2O (– activation solution)
4.1 ± 0.3
12.0 ± 0.3
Myxothiazol (4 mM)
4.1 ± 0.2
11.2 ± 1
Oligomycin (50 mM)
5.5 ± 1.5
15.1 ± 3.6
Salicylhydroxamic acid (SHAM, 50 mM)
4.0 ± 1.5
10.2 ± 4.5
CCCP (50 mM)
8.2 ± 0.3*
23.3 ± 1.1**
NaCl (10 mM)
4.1 ± 0.3
12.9 ± 0.7
Na3PO4 (10 mM)
4.8 ± 0.4
KCl (10 mM)
4.5 ± 0.1
KCl (10 mM) + valinomycin (50 mM)
5.1 ± 0.3
16.0 ± 0.8***
KCl (10 mM) + nigericin (50 mM)
6.1 ± 0.3***
14.9 ± 0.5***
compartment without the need for organellar cysteine transporters (Lunn et al. 1990, Rolland et al. 1992). However, the
findings from our work unexpectedly demonstrated that externally applied cysteine can penetrate the lipid bilayer through
saturable transport mechanisms. We therefore conclude that
cysteine is neither non-permeable to the lipid bilayer nor
simply diffused across the membranes, but is more likely to be
transported by specific membrane proteins in the IMM.
Evidence for multiple cysteine transport
mechanisms in mitochondria
8.7 ± 0.9*
12.0 ± 0.7
Uncoupler or respiratory inhibitor (1 ml) was added together with the specified
amount of radiolabeled cysteine, and mitoplasts were collected by centrifugation
through silicone oil after a 20 s incubation period. Uptake of 60 and 200 mM
cysteine was tested.
*P < 0.05; **P < 0.01, compared with control (+activation solution); ***P < 0.01
compared with KCl.
Each value of the uptake rate represents the average ± SE from at least four
Nigericin, but not valinomycin or phosphate, caused an increase
in the uptake rate of 60 mM cysteine, although the extent of the
increase was not as high as observed in the CCCP treatment.
Both nigericin and valinomycin enhanced the uptake rate of
200 mM cysteine, whereas phosphate decreased it. From these
results, it appears that proton gradient dissipation across the
IMM may play a specific role in cysteine transport. This was
further confirmed by the analysis of the cysteine uptake rate at
different external pH values (Supplementary Fig. 2). The optimal external proton concentration in the presence of 60 or
200 mM cysteine is about 8.0, which is close to the physiological
pH in the mitochondrial matrix (matrix pH = 7.8, cytosol
pH = 7.4–7.5). At this pH, the side chain of cysteine remained
mostly uncharged (pKa 8.2–8.3; Tajc et al. 2004), ruling out the
possibility that the observed optimal uptake rate was due to
ionic modifications of the species.
In this study, we conducted a detailed investigation into the
characteristics of cysteine uptake by plant mitochondria. A few
early reports have postulated that neutral amino acids, including
cysteine, simply diffuse across the mitochondrial membranes
without mediation by any ion channels or carrier proteins
(Halling et al. 1973, Wiskich 1977). Also, the presence of different
SAT and OAS-TL isoforms in different compartments in plants
would indicate that cysteine biosynthesis is present in each
The equilibrium of cysteine between isolated mitochondria and
the surrounding in vitro uptake solution was achieved in almost
45 s, even when the mitochondrial transport system was challenged with a concentration of 150 mM cysteine (Fig. 2A, B).
This indicated a fast and efficient import of cysteine into the
mitochondria that was able to take up 20% or 12% of externally
applied cysteine at concentration of 10 or 150 mM within
60 s, respectively. The latter concentration is close to the determined cytosolic cysteine concentration (i.e. 300 mM; Krueger
et al. 2009), demonstrating that the mitochondrial cysteine
uptake systems evidenced here can import significant amounts
of cysteine under in vivo conditions. Cysteine uptake by plant
mitochondria displays two phases of kinetics (Fig. 2C). In the
presence of up to 500 mM cysteine in mitochondria, Michaelis–
Menten kinetics are apparent, with a Km value (380 mM) that
falls within the approximate in vitro cysteine inhibition constant for mitochondrial SAT in the CSC (IC50 = 250 mM) (Wirtz
et al. 2010) as well as within the steady-state cytosolic cysteine
concentration under normal conditions (Krueger et al. 2009).
During stress conditions in plant cells where higher cytosolic
cysteine concentrations (750 mM) can be reached, general
amino acid transport systems may contribute to cysteine
uptake of mitochondria.
Our initial analysis of the cysteine uptake rate by mitoplasts
(which only have an IMM) unexpectedly showed no clear
saturable transport characteristics over the physiologically
relevant cysteine concentrations (Fig. 2C; Supplementary
Table S1). Through our extensive analysis of mitoplast cysteine
uptake kinetics by various competitors and inhibitors and by
counter-exchange experiments, however, we have shown several
lines of evidence for the presence of multiple cysteine transport
modes with different substrate affinities in the IMM. Firstly, the
transport of 60 mM radiolabeled cysteine by mitoplasts can be
strongly inhibited by excess unlabeled cysteine (Fig. 3A, 75%
inhibition), suggesting the existence of a limited/saturable transport system in the IMM. Secondly, cysteine transport inhibition
by excess unlabeled cysteine became less pronounced in the
presence of 200 mM cysteine (Fig. 3B, 45% inhibition). This
might indicate: (i) the activation of a general non-specific transporter(s); and/or (ii) a switch in the specificity of the cysteine
transporter(s) from high-affinity into low-affinity mode. Thirdly,
the rates of cysteine uptake were largely unaffected in the presence of various amino acids (Fig. 3A, B) and cysteine can only be
Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013.
C. P. Lee et al.
exchanged at a very low rate with all the amino acids tested
(Table 1; Supplementary Table S2), ruling out the possibility
that cysteine can only be transported by non-specific/general
amino acid transporters in the IMM (Fig. 3A, B). Fourthly,
among the metabolites tested here, only sulfide and sulfite can
cause the release of a substantial amount of cysteine from the
mitoplasts (Table 1). This indicates the presence of the cysteinespecific transporter(s) which most probably facilitates the transport of specific metabolites responsible for mitochondrial cysteine catabolism, such as some of the substrates and products of
cyanide detoxification-associated sulfide oxidation (Yamaguchi
et al., 2000, Hildebrandt and Grieshaber 2008, Álvarez et al. 2012,
Birke et al. 2012)
Characteristics of cysteine transport by IMM
in Arabidopsis
The operation of a transport mode with higher specificity towards cysteine (at 60 mM) in the IMM appears to be induced
upon lowering the proton gradient across the inner membrane,
as evident by a mild increase in the initial uptake rate after the
treatment with CCCP or nigericin (Table 2). The acidification of
the matrix by the influx of protons into the matrix or the efflux
of hydroxyls into the cytosol should operate in conjunction
with the electron transport chain (through proton leakage)
but in a respiration-independent manner. Proton pumping by
ATP synthase does not appear to have a role in enhancing the
cysteine transport rate through matrix acidification, as demonstrated in the oligomycin treatment of mitoplasts (Table 2).
The only well-characterized mitochondrial carrier protein
which can pump protons into the matrix is the uncoupling
protein (Sweetlove et al. 2006). A reduction in proton gradient
across the IMM can also be achieved through a combined
action of internal NADH dehydrogenase and alternative oxidase. However, we cannot rule out the possibility that the cysteine transporter(s) itself may co-transport both the substrate
and proton(s). For example, it has recently been shown that
lysosomal cystine export by cystinosin in human is coupled to
the efflux of a proton (Ruivo et al. 2012).
While it is apparent that cysteine can be transported into
the matrix without any requirements for counter-exchange
metabolites (Fig. 2), an increase in the efflux of radiolabeled
cysteine from the matrix when unlabeled cysteine is externally
applied (Table 1) indicates the presence of an antiporter(s) in
the IMM. Unlike the translocators for glycine and malate where
their uptake activities are strongly inhibited by known counterexchange metabolites (Yu et al. 1983, Zoglowek et al. 1988), the
inhibition of cysteine transport by sulfide or sulfite is relatively
mild (Fig. 3). This further underlines the specificity of the transport mechanisms and might point to the existence of multiple
cysteine uniporter(s) and/or antiporter(s) in the IMM with different activities and specificities towards substrate and/or
counter-substrates. Alternatively, it is possible that we have
yet to uncover the true exchange substrate(s) for the cysteine
Possible molecular identity of the mitochondrial
cysteine transporters
Cysteine uptake by whole mitochondria showed Michaelis–
Menten saturation kinetics when slightly above cytosolic cysteine concentrations were supplied (up to 500 mM, Fig. 2C),
indicating that the OMM could act as a primary protective
barrier by limiting the rate of cysteine influx. Interestingly,
this protective ability was not apparent when a >2-fold
amount of cytosolic cysteine was supplied. In the OMM, voltage-dependent anion channels (VDACs) regulate the passage of
any metabolites of up to 10 kDa by adopting a specific metabolite conductance state in a manner that reflects the physiological state of mitochondria (Mannella et al. 1975, Sorgato and
Moran 1993, Vander Heiden et al. 2000, Colombini 2004).
However, the regulation of VDAC opening and closing has
been well studied with metabolites associated with respiration,
such as organic acids and adenine nucleotides, but not with
amino acids. Further study is necessary to address whether
cysteine transport is controlled by cysteine-specific opening/
closing of VDACs alone or together with several other uncharacterized general amino acid transporters in the OMM.
In the IMM, the carrier proteins for organic acids, basic
amino acids or adenine nucleotides have been well characterized (Palmieri et al. 2011). The substrate specificity of cysteine
transport, however, appeared to be very distinct: it could not be
inhibited by and/or exchanged with any of the organic
acids, basic amino acids or adenine nucleotides tested (Fig. 3,
Table 1). It is therefore very likely that these well-characterized
carrier proteins do not transport cysteine at all or do not translocate cysteine in a specific manner. The carriers/channels responsible for transporting neutral amino acids, such as cysteine,
glycine and tryptophan, across the IMM remain to be found in
plants and animals. In Arabidopsis, a proteomic survey of mitochondria revealed the presence of 45 putative members of the
mitochondrial carrier family (Millar and Heazlewood 2003), but
the number of metabolite transport proteins is likely to be
much larger due to the emergence of new classes of nonMCF (mitochondrial carrier family) transport proteins,
namely the calcium uniporter (Baughman et al. 2011) and
the pyruvate carrier (Bricker et al. 2012). The unexpected cysteine transport properties reported here and a large number of
potential mitochondrial membrane candidates make direct
identification of the cysteine transporter(s) through conventional cloning and kinetic analysis challenging. Nevertheless,
the biochemical and kinetic data obtained in this study will
be an indispensable prerequisite for designing a more targeted
screen of the plant cysteine transporter(s) in the near future.
Functional relevance of the mitochondrial
cysteine transport in cellular cysteine biosynthesis
The mitochondrion is the major producer of OAS in the cell, but
it has an insufficient capacity to convert all the OAS it produces
to cysteine due to the relatively low SAT:OAS-TL activity (Droux
et al. 1998, Heeg et al. 2008). As a result, OAS produced in the
Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013.
Plant mitochondrial cysteine transport
mitochondrion is mostly exported to the cytosol. The viability of
the oastlAB mutant demonstrates that reduced sulfur can be
transported into the mitochondria (Heeg et al. 2008). While
mitochondrial cysteine may also be derived from glutathione
breakdown or the degradation of proteins/peptides, our results
clearly show the presence of an efficient and specific cysteine
transport system that co-exists with the cysteine biosynthetic
pathway in mitochondria. The import of cytosolic cysteine into
mitochondria could act as part of the crucial feedback loop that
regulates cellular cysteine synthesis capacity, since the feedback
inhibition of mitochondrial SAT by cysteine contributes significantly to the control of the net cellular OAS formation (Haas
et al. 2008, Wirtz et al. 2010). Conversely, cysteine could not be
exchanged with eitherserine or OAS (Table 1). This is unexpected because the cysteine biosynthetic pathway is controlled
post-translationally by regulating the stability of the CSC, which
is dependent on the availability of substrates as well as the feedback inhibition by products (Noji et al. 1998, Wirtz et al. 2010). It
is likely that the activity of the mitochondrial cysteine transport
is strictly regulated by the cysteine concentration gradient across
the mitochondrial membranes and/or by the demand for sulfur
in the organelle, but is independent from the mitochondrial
OAS level.
Conclusion and perspectives
In summary, we have provided the first direct biochemical
evidence for the existence of transporter-mediated cysteine
uptake by mitochondria in plants. The kinetic properties and
substrate specificity of the cysteine transporters seem to be
unique compared with any mitochondrial carrier proteins identified to date. Hence, cysteine is most porbably imported via an
as yet uncharacterized inner membrane transporter(s) in the
mitochondrion. The knowledge gained from this study will
provide a platform for an integrated bioinformatics, proteomics and reverse genetics approach towards the molecular
identification of the mitochondrial cysteine transporters in
Arabidopsis as well as in other eukaryotes.
Materials and Methods
Preparation of mitochondria and mitoplasts from
Arabidopsis seedlings
Approximately 100–200 Arabidopsis seeds (ecotype Col-0)
were surface-sterilized according to Lee et al. (2008). Seeds
were carefully dispersed in a glass container containing
100 ml of growth medium [half-strength Murashige and
Skoog medium (Duchefa), 2 mM MES, 2% (w/v) sucrose, 0.1%
microagar, pH 5.8]. Arabidopsis seedlings were grown under a
16/8 h light/dark period with light intensity of 100–
125 mmol m 2 s 1 at 18 C (dark) or 23 C (light) over 16–20 d
under gentle agitation (60 r.p.m.).
Mitochondria were isolated from 20–40 g of 16- to 20day-old whole sterile Arabidopsis seedlings according to
Day et al. (1985) with slight modifications. After the Percoll
gradient centrifugation, the mitochondrial fraction was collected, diluted in sucrose wash buffer without bovine serum
albumin (BSA) (0.3 M sucrose, 10 mM TES pH 7.5) or in sorbitol
wash buffer (0.3 M sorbitol, 10 mM TES pH 7.5) and centrifuged
at 24,000g for 10 min. The wash was repeated once and the
resulting pellet was resuspended in residual wash buffer.
Osmotically shocked mitochondria (mitoplasts) were prepared
using a method modified from Murcha et al. (2003). The resulting mitoplast pellet was washed once and resuspended in
sorbitol wash buffer.
Validation of cysteine uptake by
isolated mitochondrion
About 0.1 mg of isolated mitochondrial protein fraction (in
1 mg ml 1) was incubated with 20 mCi of [35S]cysteine for
20 min at room temperature and centrifuged at 24,000g for
10 min at 4 C. The resulting pellet was resuspended in 0.1 ml of
sucrose wash buffer without BSA. The mitochondrial fraction
was carefully layered on top of a Percoll step gradient prepared
in a 2 ml Eppendorf tube consisting of, from top to bottom,
0.2 ml of 4% Percoll, 1 ml of 25% Percoll and 0.2 ml of 40%
Percoll in sucrose wash buffer without BSA (modified from
Howell et al. 2006). The tube was centrifuged at 20,000g for
30 min at 4 C in an Eppendorf centrifuge with the brake turned
off during deceleration. After centrifugation, 0.1 ml fractions
were collected and mixed with 3 ml of Ultima GoldTM scintillation fluid (PerkinElmer). Radioactivity was detected by either
a Beckman LS 6500 Scintillation Counter or a Tri-Carb 2910TR
Liquid Scintillation Analyzer controlled by the QuantaSmartÕ
software package (PerkinElmer).
In organello protein synthesis using [35S]cysteine was performed according to Giegé et al. (2005). The reaction mixture
was incubated at 25 C on an orbital shaker for 10, 30 and
60 min. The reaction was stopped by adding 1 ml of 10 mM
unlabeled cysteine. Sample clean-up was performed using PD
SpinTrapTM G-25 columns (GE Healthcare). Radiolabeled translation products were separated by SDS–PAGE and transferred
onto a polyvinylidene difluoride (PVDF) membrane. The membrane was exposed to an X-ray film and proteins were visualized
after the film was developed using the Optimax X-Ray Film
Processor (PROTEC Medizintechnik).
Cysteine uptake assay by silicone
oil centrifugation
Freshly prepared mitochondria/mitoplasts in sorbitol wash
buffer (50 mg per 200 ml of proteins) were first equilibrated at
25 C for 5–10 min. Uptake assays were initiated by adding an
appropriate concentration of freshly prepared cysteine and
radiolabeled [35S]cysteine (1.25 mCi, Hartmann Analytic) together with transport activation solution (20 mM KH2PO4 pH
7.4, 1.2 mM ATP, 1.2 mM ADP, 3.8 mM glutamate and 3.8 mM
malate) at 25 C. After the specified incubation time, the reaction was stopped by rapid centrifugation (at 14,000 r.p.m. for
Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013.
C. P. Lee et al.
1–2 min) through a 70 ml silicone oil layer (3 : 1 AR200 : AR20,
Sigma Aldrich) into the bottom sedimentation layer containing
20 ml of 10% (v/v) perchloric acid. Radioactivity in the aliquot of
sorbitol wash buffer or water-resuspended pellet was assayed as
described above, except that the samples were mixed with 2 ml
of scintillation fluid. Intramitochondrial cysteine concentrations were determined after correction for radiolabeled cysteine
in the extramatrix space (Heldt 1980).
For the analysis of counter-exchange of cysteine, freshly prepared mitoplasts were loaded with 60 mM unlabeled cysteine
and 40 mCi ml 1 [35S]cysteine by incubation at 4 C. After
30 min, mitoplasts were washed twice at 24,000g for 10 min
in sorbitol wash medium. The supernatant was removed and
the pellet was resuspended in sorbitol wash medium at a concentration of 0.25 mg ml 1. Unlabeled amino acid or substrate
was then added, followed by centrifugation through silicone oil
as specified above.
Measurement of cysteine and sulfide contents in
isolated mitochondria
The mitochondrial protein fraction (50 mg per 200 ml) was prewarmed to room temperature for 5 min before substrate incubation. Samples were boiled for 5 min to stop the reaction.
Thiol derivatives were derivatized by monobromobimane and
detected using reverse-phase HPLC (Waters 600E Multisolvent
Delivery system, Autosampler 717plus) connected to a NovaPak C18 4.6250 mm column (pore size 4 mm) as described
previously (Wirtz et al. 2004).
Data analysis
Unless stated otherwise, all data were expressed as the
mean ± SEM from at least three independent experiments.
Statistical significances were evaluated by unpaired Student’s
t-test. Lineweaver–Burk plots and kinetic parameters
(mean ± SE) for cysteine uptake were obtained from
SigmaPlot 12.5 (Systat Software).
Supplementary data
Supplementary data are available at PCP online.
This study was supported by the CellNetworks Excellence
Cluster (University of Heidelberg) [Post-Doc Program to
C.P.L.]; the German Research Foundation (DFG) [grant
He1848/14-1 to R.H.]; the Schmeil-Foundation Heidelberg.
We would like to thank Professor Hans-Peter Braun and
Professor Ulf-Ingo Flügge for critical reading of the manuscript,
and Dr. Stephen Krüger for introducing us to the silicone oil
centrifugation technique.
The authors have no conflicts of interest to declare.
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