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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,* 1 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) 2 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. Introduction 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 www.pcp.oxfordjournals.org ! 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] 64 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 membranes. Results 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. 65 C. P. Lee et al. B Incorporated radioactivity A 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 4% 25% 40% Time (min) 10 30 60 C (1000 x cpm fraction-1) 5 10 15 20 25 30 35 kD 100 70 F r a c t i o n ATP1 COB1 50 40 35 COXII 25 15 10 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) A B 0.5 6 5 0.4 4 0.3 3 0.2 2 0.1 1 0 0 0 30 60 90 120 150 0 180 30 60 Time (s) 120 150 180 D 50 Cysteine uptake (nmol min-1 mg-1 protein) C Cysteine uptake (nmol min-1 mg-1 protein) 90 Time (s) Mitoplasts Mitochondria 40 30 20 10 0 0 200 400 600 800 1000 Cysteine (µM) Mitoplasts 12 10 8 6 4 2 0 0 50 100 150 200 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. 66 Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013. 5 * 4 * * 3 2 * KCN Glycine Phenylalanine Leucine Aspartate Alanine Glutamine Lysine Arginine Cysteine OAS Serine control Methionine Na2S Na2SO4 Na2SO3 Na2S2O3 GSH 1 0 16 14 12 10 * 8 * * * * * * 6 4 Glycine Phenylalanine Leucine Other amino acids Aspartate Alanine Glutamine Lysine Arginine basic amino acids Cysteine OAS Serine control 0 KCN 2 cyanide B * 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 6 Methionine Na2S Na2SO4 Na2SO 3 Na2S2O3 GSH Specificity of cysteine transport A 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. 67 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 68 Table 1 Specificity of cysteine exchange with various external metabolites Addition Release of [35S]cysteine (nmol mg 1 protein) 0 ± 0.5 H2O control Photorespiration/cysteine biosynthesis and catabolism 27.0 ± 1.9** Cysteine Glycine 0.9 ± 1.0 Serine 2.4 ± 1.9 O-acetylserine 2.9 ± 2.2 b-Cyano-alanine 5.9 ± 2.9 Basic amino acids 3.1 ± 1.9 Lysine 17.0 ± 5.7 Arginine Sulfur-containing metabolites and cyanide-derived ions 5.8 ± 1.0 Methionine 31.7 ± 2.8** Na2S Na2SO4 3.4 ± 1.1 Na2SO3 21.7 ± 2.2** Na2S2O3 4.8 ± 0.6 Glutathione reduced (GSH) 8.7 ± 1.2* 9.2 ± 4.3 Glutathione oxidized (GSSG) 18.3 ± 1.6** KCN 1.4 ± 1.6 NH4SCN 35 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) 60 200 Addition nmol min 1 mg 1 protein nmol min 1 mg 1 protein 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 experiments. 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. Discussion 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. 69 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 transporter(s). 70 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. 71 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. Funding 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. Acknowledgments We would like to thank Professor Hans-Peter Braun and Professor Ulf-Ingo Flügge for critical reading of the manuscript, 72 and Dr. Stephen Krüger for introducing us to the silicone oil centrifugation technique. Disclosures The authors have no conflicts of interest to declare. References Álvarez, C., Garcı́a, I., Romero, L.C. and Gotor, C. (2012) Mitochondrial sulfide detoxification requires a functional isoform O-acetylserine(thiol)lyase C in Arabidopsis thaliana. Mol. Plant 5: 1217–1226. Baughman, J.M., Perocchi, F., Girgis, H.S., Plovanich, M., BelcherTimme, C.A. et al. (2011) Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476: 341–345. Birke, H., Haas, F.H., De Kok, L.J., Balk, J., Wirtz, M. and Hell, R. (2012) Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana. Biochem. J. 445: 275–283. Bricker, D.K., Taylor, E.B., Schell, J.C., Orsak, T., Boutron, A. et al. (2012) A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans. Science 337: 96–100. Colombini, M. (2004) VDAC: the channel at the interface between mitochondria and the cytosol. Mol. Cell. Biochem. 256–257: 107–115. Cybulski, R.L. and Fisher, R.R. (1977) Mitochondrial neutral amino acid transport: evidence for a carrier mediated mechanism. Biochemistry 16: 5116–5120. Day, D.A., Neuburger, M. and Douce, R. (1985) Biochemical characterization of chlorophyll-free mitochondria from pea leaves. Aust. J. Plant Physiol. 12: 219–228. Droux, M. (2004) Sulfur assimilation and the role of sulfur in plant metabolism: a survey. Photosynth. Res. 79: 331–348. Droux, M., Ruffet, M.L., Douce, R. and Job, D. (1998) Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plants. Structural and kinetic properties of the free and bound enzymes. Eur. J. Biochem. 255: 235–245. Duncan, O., Taylor, N.L., Carrie, C., Eubel, H., Kubiszewski-Jakubiak, S., Zhang, B. et al. (2011) Multiple lines of evidence localise signalling, morphology and lipid biosynthesis machinery to the mitochondrial outer membrane of Arabidopsis thaliana. Plant Physiol. 157: 1093–1113. Giegé, P., Sweetlove, L.J., Cognat, V. and Leaver, C.J. (2005) Coordination of nuclear and mitochondrial genome expression during mitochondrial biogenesis in Arabidopsis. Plant Cell 17: 1497–1512. Haas, F.H., Heeg, C., Queiroz, R., Bauer, A., Wirtz, M. and Hell, R. (2008) Mitochondrial serine acetyltransferase functions as a pacemaker of cysteine synthesis in plant cells. Plant Physiol. 148: 1055–1067. Halling, P.J., Brand, M.D. and Chappell, J.B. (1973) Permeability of mitochondria to neutral amino acids. FEBS Lett 34: 169–171. Heeg, C., Kruse, C., Jost, R., Gutensohn, M., Ruppert, T., Wirtz, M. et al. (2008) Analysis of the Arabidopsis O-acetylserine(thiol)lyase gene family demonstrates compartment-specific differences in the regulation of cysteine synthesis. Plant Cell 20: 168–185. Heldt, H.W. (1980) Measurement of metabolite movement across the envelope and of the pH in the stroma and the thylakoid space in intact chloroplasts. Methods Enzymol. 69: 604–613. Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013. Plant mitochondrial cysteine transport Hildebrandt, T.M. and Grieshaber, M.K. (2008) Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria. FEBS J. 275: 3352–3361. Holdorf, M.M., Owen, H.A., Rhee Lieber, S., Yuan, L., Adams, N., Dabney-Smith, C. et al. (2012) Arabidopsis ETHE1 encodes a sulfur dioxygenase that is essential for embryo and endosperm development. Plant Physiol. 160: 226–236. Howell, K.A., Millar, A.H. and Whelan, J. (2006) Ordered assembly of mitochondria during rice germination begins with promitochondrial structures rich in components of the protein import apparatus. Plant Mol. Biol. 60: 201–223. Krueger, S., Niehl, A., Martin, M.C., Steinhauser, D., Donath, A., Hildebrandt, T. et al. (2009) Analysis of cytosolic and plastidic serine acetyltransferase mutants and subcellular metabolite distributions suggests interplay of the cellular compartments for cysteine biosynthesis in Arabidopsis. Plant Cell Environ. 32: 349–367. Kühn, K., Carrie, C., Giraud, E., Wang, Y., Meyer, E.H., Narsai, R. et al. (2011) The RCC1 family protein RUG3 is required for splicing of nad2 and complex I biogenesis in mitochondria of Arabidopsis thaliana. Plant J. 67: 1067–1080. Lee, C.P., Eubel, H., O’Toole, N. and Millar, A.H. (2008) Heterogeneity of the mitochondrial proteome for photosynthetic and nonphotosynthetic Arabidopsis metabolism. Mol. Cell. Proteomics 7: 1297–1316. Lunn, J.E., Droux, M., Martin, J. and Douce, R. (1990) Localization of ATP sulfurylase and O-acetylserine(thiol)lyase in spinach leaves. Plant Physiol. 94: 1345–1352. Mannella, C.A. and Bonner, W.D. Jr. (1975) Biochemical characteristics of the outer membranes of plant mitochondria. Biochim. Biophys. Acta 413: 213–225. Millar, A.H. and Heazlewood, J.L. (2003) Genomic and proteomic analysis of mitochondrial carrier proteins in Arabidopsis. Plant Physiol. 131: 443–453. Millar, A.H., Liddell, A. and Leaver, C.J. (2001) Isolation and subfractionation of mitochondria from plants. Methods Cell Biol. 65: 53–74. Murcha, M.W., Lister, R., Ho, A.Y.Y. and Whelan, J. (2003) Identification, expression, and import of components 17 and 23 of the inner mitochondrial membrane translocase from Arabidopsis. Plant Physiol. 131: 1737–1747. Noji, M., Inoue, K., Kimura, N., Gouda, A. and Saito, K. (1998) Isoformdependent differences in feedback regulation and subcellular localization of serine acetyltransferase involved in cysteine biosynthesis from Arabidopsis thaliana. J. Biol. Chem. 273: 32739–32745. Palmieri, F., Pierri, C.L., De Grassi, A., Nunes-Nesi, A. and Fernie, A.R. (2011) Evolution, structure and function of mitochondrial carriers: a review with new insights. Plant J. 66: 161–181. Rolland, N., Droux, M. and Douce, R. (1992) Subcellular distribution of O-acetylserine(thiol)lyase in cauliflower (Brassica oleracea L.) inflorescence. Plant Physiol. 98: 927–935. Ruivo, R., Bellenchi, G.C., Chen, X., Zifarelli, G., Sagné, C., Debacker, C. et al. (2012) Mechanism of proton/substrate coupling in the heptahelical lysosomal transporter cystinosin. Proc. Natl Acad. Sci. USA 109: E210–E217. Salvi, M., Battaglia, V., Mancon, M., Colombatto, S., Cravanzola, C., Calheiros, R. et al. (2006) Agmatine is transported into liver mitochondria by a specific electrophoretic mechanism. Biochem. J. 396: 337–345. Smith, I.K. (1972) Studies of L-cysteine biosynthetic enzymes in Phaseolus vulgaris L. Plant Physiol. 50: 477–479. Sorgato, M.C., Moran, O. and Pedersen, P.L. (1993) Channels in mitochondrial membranes: knowns, unknowns, and prospects for the future. Crit. Rev. Biochem. Mol. Biol. 28: 127–171. Sweetlove, L.J., Lytovchenko, A., Morgan, M., Nunes-Nesi, A., Taylor, N.L., Baxter, C.J. et al. (2006) Mitochondrial uncoupling protein is required for efficient photosynthesis. Proc. Natl Acad. Sci. USA 103: 19587–19592. Tajc, S.G., Tolbert, B.S., Basavappa, R. and Miller, B.L. (2004) Direct determination of thiol pKa by isothermal titration microcalorimetry. J. Amer. Chem. Soc. 126: 10508–10509. Vander Heiden, M.G., Chandel, N.S., Li, X.X., Schumacker, P.T., Colombini, M. et al. (2000) Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival. Proc. Natl Acad. Sci. USA 97: 4666–4671. Watanabe, M., Kusano, M., Oikawa, A., Fukushima, A., Noji, M. and Saito, K. (2008a) Physiological roles of the beta-substituted alanine synthase gene family in Arabidopsis. Plant Physiol. 146: 310–320. Watanabe, M., Mochida, K., Kato, T., Tabata, S., Yoshimoto, N., Noji, M. et al. (2008b) Comparative genomics and reverse genetics analysis reveal indispensable functions of the serine acetyltransferase gene family in Arabidopsis. Plant Cell 20: 2484–2496. Wirtz, M., Beard, K.F.M., Lee, C.P., Boltz, A., Schwarzlaender, M., Fuchs, C. et al. (2012) Mitochondrial cysteine synthase complex regulates O-acetylserine biosynthesis in plants. J. Biol. Chem. 287: 27941–27947. Wirtz, M., Birke, H., Heeg, C., Müller, C., Hosp, F., Throm, C. et al. (2010) Structure and function of the hetero-oligomeric cysteine synthase complex in plants. J. Biol. Chem. 285: 32810–32817. Wirtz, M., Droux, M. and Hell, R. (2004) O-Acetylserine (thiol) lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited in Arabidopsis thaliana. J. Exp. Bot. 55: 1785–1798. Wirtz, M. and Hell, R. (2007) Dominant-negative modification reveals the regulatory function of the multimeric cysteine synthase protein complex in transgenic tobacco. Plant Cell 19: 625–639. Wiskich, J.T. (1977) Mitochondrial metabolite transport. Annu. Rev. Plant Physiol. 28: 45–69. Yamaguchi, Y., Nakamura, T., Kusano, T. and Sano, H. (2000) Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and beta-cyanoalanine synthase. Plant Cell Physiol. 41: 465–476. Yu, C., Claybrook, D.L. and Huang, A.H.C. (1983) Transport of glycine, serine, and proline into spinach leaf mitochondria. Arch. Biochem. Biophys. 227: 180–187. Zoglowek, C., Krömer, S. and Heldt, H.W. (1988) Oxaloacetate and malate transport by plant mitochondria. Plant Physiol. 87: 109–115. Plant Cell Physiol. 55(1): 64–73 (2014) doi:10.1093/pcp/pct155 ! The Author 2013. 73