Homocysteine and cysteine - albumin binding in homocystinuria: assessment of cysteine status and implications for glutathione synthesis?

Summary. Measurement of plasma total cysteine rather than free dimeric cystine gives a better indication of cysteine status in homocystinuric patients. This is the result of displacement of cysteine from albumin by homocysteine and is related to the plasma homocysteine concentration. In control subjects the free/bound cyst(e)ine ratio was independent of albumin and total cysteine concentrations. In homocystinuric (HCU) patients both free and total cyst(e)ine values differed significantly from control values (P < 0.001) but whilst free cystine considerably overlapped control values the total cysteine concentrations were almost invariably lower. The possible consequences of this on glutathione synthesis was explored by assay of plasma total glutathione but no evidence for glutathione deficiency was found. Measurement of total cysteine, rather than free cystine, provides a better indication of cysteine status in HCU. Received February 1, 2001 Accepted November 13, 2001     

Analysis of cytosolic and plastidic serine acetyltransferase mutants and subcellular metabolite distributions suggests interplay of the cellular compartments for cysteine biosynthesis in Arabidopsis

In plants, the enzymes for cysteine synthesis serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL) are present in the cytosol, plastids and mitochondria. However, it is still not clearly resolved to what extent the different compartments are involved in cysteine biosynthesis and how compartmentation influences the regulation of this biosynthetic pathway. To address these questions, we analysed Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms. In addition, the subcellular distribution of enzyme activities and metabolite concentrations implicated in cysteine and glutathione biosynthesis were revealed by non-aqueous fractionation (NAF). We demonstrate that cytosolic SERAT1.1 and plastidic SERAT2.1 do not contribute to cysteine biosynthesis to a major extent, but may function to overcome transport limitations of O-acetylserine (OAS) from mitochondria. Substantiated by predominantly cytosolic cysteine pools, considerable amounts of sulphide and presence of OAS in the cytosol, our results suggest that the cytosol is the principal site for cysteine biosynthesis. Subcellular metabolite analysis further indicated efficient transport of cysteine, γ -glutamylcysteine and glutathione between the compartments. With respect to regulation of cysteine biosynthesis, estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for cysteine biosynthesis and that SAT is mainly present bound in the cysteine–synthase complex.     

Diallyl trisulfide induces apoptosis in Jurkat cells by the modification of cysteine residues in thioredoxin

We reported the regulation of protein function by oxidative modification of the specific cysteine residue(s) by diallyl trisulfide (DATS). In this study, we examined if DATS modifies the cysteine residue of thioredoxin (Trx) by urea-polyacryl amide gel electrophoresis. DATS modified two specific cysteine residues in Trx and this oxidative modification of cysteine residues would be sole causative of the apoptosis induced by DATS in leukemic cells.     

Taurine deficiency,synthesis and transport in the mdx mouse model for Duchenne Muscular Dystrophy

The amino acid taurine is essential for the function of skeletal muscle and administration is proposed as a treatment for Duchenne Muscular Dystrophy (DMD). Taurine homeostasis is dependent on multiple processes including absorption of taurine from food, endogenous synthesis from cysteine and reabsorption in the kidney. This study investigates the cause of reported taurine deficiency in the dystrophic mdx mouse model of DMD. Levels of metabolites (taurine, cysteine, cysteine sulfinate and hypotaurine) and proteins (taurine transporter [TauT], cysteine deoxygenase and cysteine sulfinate dehydrogenase) were quantified in juvenile control C57 and dystrophic mdx mice aged 18 days, 4 and 6 weeks. In C57 mice, taurine content was much higher in both liver and plasma at 18 days, and both cysteine and cysteine deoxygenase were increased. As taurine levels decreased in maturing C57 mice, there was increased transport (reabsorption) of taurine in the kidney and muscle. In mdx mice, taurine and cysteine levels were much lower in liver and plasma at 18 days, and in muscle cysteine was low at 18 days, whereas taurine was lower at 4: these changes were associated with perturbations in taurine transport in liver, kidney and muscle and altered metabolism in liver and kidney. These data suggest that the maintenance of adequate body taurine relies on sufficient dietary intake of taurine and cysteine availability and metabolism, as well as retention of taurine by the kidney. This research indicates dystrophin deficiency not only perturbs taurine metabolism in the muscle but also affects taurine metabolism in the liver and kidney, and supports targeting cysteine and taurine deficiency as a potential therapy for DMD.     

Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells

Cysteine levels are carefully regulated in mammals to balance metabolic needs against the potential for cytotoxicity. It has been postulated that one of the major regulators of intracellular cysteine levels in mammals is cysteine dioxygenase (CDO). Hepatic expression of this catabolic enzyme increases dramatically in response to increased cysteine availability and may therefore be part of a homeostatic response to shunt excess toxic cysteine to more benign metabolites such as sulfate or taurine. Direct experimental evidence, however, is lacking to support the hypothesis that CDO is capable of altering steady-state intracellular cysteine levels. In this study, we expressed either the wild-type (WT) or a catalytically inactivated mutant (H86A) isoform of CDO in HepG2/C3A cells (which do not express endogenous CDO protein) and cultured them in different concentrations of extracellular cysteine. WT CDO, but not H86A CDO, was capable of reducing intracellular cysteine levels in cells incubated in physiologically relevant concentrations of cysteine. WT CDO also decreased the glutathione pool and potentiated the toxicity of CdCl(2). These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.     

Potentiometric determination of cysteine with thiol sensitive silver-mercury electrode

A potentiometric procedure for cysteine thiol group concentration monitoring in media generating free radicals was developed using a thiol specific silver-mercury electrode. Electrolytic deposition of mercury on a silver wire and treatment with 20 mM cysteine in 0.5 M NaOH were used to produce the electrode. A silver-chloride electrode in saturated KCl was the reference. A glass capillary with 1 M KNO3 in 1% agarose gel was the liquid junction. The electrode responded to cysteine concentration in the range from 0.01 to 20 mM yielding a perfect linear relationship for the dependence of log [cysteine] versus electrode potential [mV], with b0 (constant) = -373.43 [mV], b1 (slope) = -53.82 and correlation coefficient r2 = 0.97. The electrode potential change per decade of cysteine concentration was 57 mV. The minimal measurable signal response was at a cysteine concentration of 0.01 mM. The signal CV amounted to 4-6% for cysteine concentrations of 0.01 to 0.05 mM and to less than 1% for cysteine concentrations of 0.5 to 20 mM. The response time ranged from about 100 s for cysteine concentrations of 0.01 to 0.1 mM to 30 s at higher cysteine concentrations. The standard curve reproducibility was the best at cysteine concentrations from 0.1 to 20 mM. In a reaction medium containing cysteine and copper(II)-histidine complex ([His-Cu]2+) solution in 55 mM phosphate buffer pH 7.4 the electrode adequately responded to changes in cysteine concentration. Beside cysteine, the silver-mercury electrode responded also to thiol groups of homocysteine and glutathione, however, the Nernst equation slope was about half of that for cysteine.     

Identification and characterization of bacterial cysteine dioxygenases: a new route of cysteine degradation for eubacteria

In metazoa and fungi, the catabolic dissimilation of cysteine begins with its sulfoxidation to cysteine sulfinic acid by the enzyme cysteine dioxygenase (CDO). In these organisms, CDO plays an important role in the homeostatic regulation of steady-state cysteine levels and provides important oxidized metabolites of cysteine such as sulfate and taurine. To date, there has been no experimental evidence for the presence of CDO in prokaryotes. Using PSI-BLAST searches and crystallographic information about the active-site geometry of mammalian CDOs, we identified a total of four proteins from Bacillus subtilis, Bacillus cereus, and Streptomyces coelicolor A3(2) that shared low overall identity to CDO (13 to 21%) but nevertheless conserved important active-site residues. These four proteins were heterologously expressed and purified to homogeneity by a single-step immobilized metal affinity chromatography procedure. The ability of these proteins to oxidize cysteine to cysteine sulfinic acid was then compared against recombinant rat CDO. The kinetic data strongly indicate that these proteins are indeed bona fide CDOs. Phylogenetic analyses of putative bacterial CDO homologs also indicate that CDO is distributed among species within the phyla of Actinobacteria, Firmicutes, and Proteobacteria. Collectively, these data suggest that a large subset of eubacteria is capable of cysteine sulfoxidation. Suggestions are made for how this novel pathway of cysteine metabolism may play a role in the life cycle of the eubacteria that have it.     

Inactivation of parasite cysteine proteinases by the NO-donor 4-(phenylsulfonyl)-3-((2-(dimethylamino)ethyl)thio)-furoxan oxalate

NO-donors block Plasmodium, Trypanosoma, and Leishmania life cycle by inactivating parasite enzymes, e.g., cysteine proteinases. In this study, the inactivation of falcipain, cruzipain, and Leishmania infantum cysteine proteinase by the NO-donor 4-(phenylsulfonyl)-3-((2-(dimethylamino)ethyl)thio)-furoxan oxalate (SNO-102) is reported. SNO-102 inactivates dose- and time-dependently parasite cysteine proteinases; one equivalent of NO, released from SNO-102, inactivates one equivalent of L. infantum cysteine proteinase. With SNO-102 in excess over the parasite cysteine proteinase, the time course of enzyme inhibition corresponds to a pseudo-first-order reaction for more than 90% of its course. The concentration dependence of the pseudo-first-order rate constant is second-order at low SNO-102 concentration but tends to first-order at high NO-donor concentration. This behavior may be explained by a relatively fast pre-equilibrium followed by a limiting pseudo-first order process. Kinetic parameters of L. infantum cysteine proteinase inactivation by SNO-102 are affected by the acidic pK shift of one apparent ionizing group (from pK(unl)=5.8 to pK(lig)=4.7) upon enzyme inhibition. Falcipain, cruzipain and L. infantum cysteine proteinase inactivation is prevented and reversed by dithiothreitol and L-ascorbic acid. Furthermore, the fluorogenic substrate N-alpha-benzyloxycarbonyl-Phe-Arg-(7-amino-4-methylcoumarin) protects parasite cysteine proteinases from inactivation by SNO-102. The absorption spectrum of the inactive S-nitrosylated SNO-102-treated L. infantum cysteine proteinase displays a maximum at about 340 nm. These results indicate that the parasite cysteine proteinase inactivation by SNO-102 occurs via the NO-mediated S-nitrosylation of the Cys25 catalytic residue.     

Cloning, in silico characterization and interaction of cysteine protease and cystatin for establishing their role in early blight disease in tomato

Cysteine protease (CP) and Cysteine protease inhibitor (CPI) or cystatin constitute a critical point in programmed cell death (PCD), a basic biological phenomenon which takes place in the plants, when they are exposed to varying biotic and abiotic stresses. In the present study we isolated and cloned cDNAs encoding cysteine protease and cystatin from early blight infected tomato plants. Using computational biology tools the sequence-structure-function relationships for the tomato cystatin and cysteine protease were elucidated. Interaction between the cystatin and cysteine protease of host and pathogen is higher as compared to interaction shown by cystatin and cysteine protease within the host. The interaction energy of (a)tomato cystatin—tomato cysteine protease, (b)tomato cystatin—fungal cysteine protease and (c)tomato cysteine protease—fungal cystatin are ?319.33 Kcal/mol, ?504.71 Kcal/mol and ?373.731 Kcal/mol respectively. Comparative protein sequence analysis with different plant cystatins and cysteine protease were also done with the sequences of cystatin and cysteine protease isolated from tomato. Structures for all the cystatin and cysteine protease were modeled along with their interactions with fungal cystatin and cysteine protease in order to explore the structural variability and its manifestation at the functional level. This helped to relate the already known functions of these proteins with their sequences as well as the predicted structures. This also served to better understand the CP-CPI interaction operational in developing this protein family and its implication in plant defense during fungal pathogenesis in tomato plants.     

Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes

Rat hepatocytes cultured for 3 days in basal medium expressed low levels of cysteine dioxygenase (CDO) and high levels of gamma-glutamylcysteine synthetase (GCS). When the medium was supplemented with 2 mmol/l methionine or cysteine, CDO activity and CDO protein increased by >10-fold and CDO mRNA increased by 1.5- or 3.2-fold. In contrast, GCS activity decreased to 51 or 29% of basal, GCS heavy subunit (GCS-HS) protein decreased to 89 or 58% of basal, and GCS mRNA decreased to 79 or 37% of basal for methionine or cysteine supplementation, respectively. Supplementation with cysteine consistently yielded responses of greater magnitude than did supplementation with an equimolar amount of methionine. Addition of propargylglycine to inhibit cystathionine gamma-lyase activity and, hence, cysteine formation from methionine prevented the effects of methionine, but not those of cysteine, on CDO and GCS expression. Addition of buthionine sulfoximine to inhibit GCS, and thus block glutathione synthesis from cysteine, did not alter the ability of methionine or cysteine to increase CDO. GSH concentration was not correlated with changes in either CDO or GCS-HS expression. The effectiveness of cysteine was equivalent to or greater than that of its precursors (S-adenosylmethionine, cystathionine, homocysteine) or metabolites (taurine, sulfate). Taken together, these results suggest that cysteine itself is an important cellular signal for upregulation of CDO and downregulation of GCS.     

Regulation of arylsulfatase synthesis by sulfur compounds in Klebsiella aerogenes.

In Klebsiella aerogenes, arylsulfatase synthesis was repressed by inorganic sulfate, sulfite, sulfide, thiosulfate, and cysteine, but not by methionine under normal growth conditions. We isolated cysteine-requiring mutants (Cys minus), and mutants (AtsS minus, AtsR minus) in which the regulation of arylsulfatase synthesis was altered. In the cysteine auxotroph, enzyme synthesis was also repressed by inorganic sulfate or cysteine. Kinetic studies on mutants of the cysteine auxotroph showed that inorganic sulfate repressed arylsulfatase synthesis and that this was not due to cysteine formed by reduction of sulfate. Arylsulfatase synthesis in the AtsS minus mutant was not repressed by inorganic sulfate but was repressed by cysteine. This mutant strain had a normal level of inorganic sulfate transport. Another mutant strain, defective in the inorganic sulfate transport system, synthesized arylsulfatase in the presence of inorganic sulfate but not in the presence of cysteine. The AtsS minus mutant could synthesize the enzyme in the presence of inorganic sulfate but not cysteine. The AtsR minus mutant could synthesize the enzyme in the presence of either inorganic sulfate or cysteine. These results suggest that there are two independent functional corepressors of arylsulfatase synthesis in K. aerogenes.     

The K(m) for Radiosensitization of Human Tumor Cells by Oxygen is Much Greater than 3 mmHg and is Further Increased by Elevated Levels of Cysteine

We studied the role of cysteine as an intracellular radiation protector under conditions in which both oxygen and thiols were monitored at 37 degrees C. In HCT-116 human colon cancer cells, the intracellular cysteine content affects the radiation survival dramatically at intermediate oxygen levels, but not at zero or high oxygen levels. Using a spin-through-oil method with a dual radioactive label detection system, we measured intracellular cysteine and glutathione (GSH) levels for cells in suspension culture. A comparison of the cysteine levels of monolayer cells lysed in situ and of trypsinized monolayer cells in suspension (Horan et al., Cytometry 29, 76-82, 1997) revealed that, upon trypsinization from monolayer culture and transfer to a spinner apparatus at 37 degrees C, HCT-116 cells lose most of their intracellular cysteine. Over the 60-min time course of control experiments, these cells do not recover intracellular cysteine despite the availability of cystine (the disulfide of cysteine) in the medium. When cells in spinner culture are provided with exogenous cysteine, they initially concentrate it to 10-fold the extracellular concentration, with the concentration factor decreasing to about 5-fold over the course of an hour. The intracellular GSH concentration changes little throughout this period, regardless of the changes in cysteine levels. The same apparatus was used to assess the survival of HCT-116 cells irradiated at 37 degrees C under conditions of constant pO(2) monitoring. For cells without added cysteine, the oxygen concentration for half-maximal radiation sensitivity was about 7.5 mmHg (intermediate hypoxia), more than twice the commonly accepted value (3 mmHg). At 7.5 mmHg, cells with added cysteine (intracellular concentration 3.5 mM) were almost as radioresistant as severely hypoxic cells (approximately 0.005% oxygen). Cells in parallel experiments in which the cells were grown in monolayers on glass Petri dishes had intermediate cysteine values and also intermediate radiosensitivity. We conclude that the radiation response of cells at intermediate oxygen levels is controlled predominantly by intracellular cysteine levels and that the cysteine levels commonly found in tumors may increase the K(m) for radiosensitivity to values much higher than suggested previously.     

Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp

Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases present in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investigation, several cDNA sequences coding for cysteine proteinases in Vasconcellea x heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. x heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I-VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkat x mg protein(-1)) were isolated. After further purification, three of them were N-terminally sequenced. The observed microheterogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp.     

Probes of the catalytic site of cysteine dioxygenase

The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the alpha-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ alpha-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by alpha-ketoglutarate.     

Reduction of Cr(VI) by cysteine: Significance in human lymphocytes and formation of DNA damage in reactions with variable reduction rates

The induction of genotoxicity by Cr (VI) is dependent on its reductive activation inside the cell. Our recent studies have found that reduction of Cr (VI) by cysteine resulted in the formation of mutagenic Cr (III)-DNA adducts in the absence of oxidative DNA damage. In this work, we examined the formation of oxidative and Cr (III)-dependent types of DNA damage under a broader range of Cr (VI) and cysteine concentrations and investigated a potential role of this reducer in intracellular metabolism of Cr (VI). Peripheral lymphocytes from unexposed humans had 7.8-fold excess of glutathione over cysteine, whereas lymphocytes from stainless steel welders contained only 3 times higher amount of glutathione (p = 0.0009) which was entirely caused by the decrease in the concentration of glutathione. A strong correlation (r = 0.72) between the levels of both thiols was found in lymphocytes from controls. The number of DNA-protein crosslinks in lymphocytes from welders was 4.1 times higher than among controls, indicating the presence of Cr (VI)-dependent DNA damage. The average rate of Cr (VI) reduction by cysteine was approximately 5 times faster than that by glutathione. Higher reduction rate combined with the decrease in the intracellular concentration of glutathione should make cysteine a predominant Cr (VI)-reducing thiol in lymphocytes of welders. Analysis of the initial rates of Cr (VI) reduction by different concentrations of cysteine suggested the presence of one- and two-electron pathways, with one-electron mechanism dominating in the physiological range of concentrations. There was no detectable formation of DNA breaks or abasic sites under a broad range of Cr (VI) and cysteine concentrations, resulting in up to 68-fold differences in the rates of reduction and the production of as many as 3 Cr (III)-DNA adducts per 10 bp. The reactions with slow reduction rates (low concentrations of cysteine) led to the most extensive formation of Cr (III)DNA adducts. In summary, these results further establish Cr (III)-DNA adducts as the major form of DNA damage resulting from Cr (VI) metabolism by cysteine. The role of cysteine in reduction of Cr (VI) becomes more significant under conditions of occupational exposure to Cr (VI)-containing welding fumes.     

Smectite Reduction by Shewanella Species as Facilitated by Cystine and Cysteine

Exogenous electron transfer mediators employed by Fe(III)-reducing bacteria are believed to govern the kinetics and equilibrium of bioreduction of Fe(III) in solid phase. In contrast to a large number of studies on humic substances and analog anthraquinone-2,6-disulfonate (AQDS), our knowledge of other potential electron shuttles involved in Fe(III) reduction is limited. The purpose of the present study was to understand the role of cystine and cysteine in reduction of iron-rich smectite (nontronite, NAu-2) by Shewanella species. A series of abiotic and biotic experiments were conducted in nongrowth media (bicarbonate buffered, pH = 7.0). Fe(II) and cysteine concentrations were monitored over the course of the bioreduction experiments with wet chemistry, and the unreduced and reduced nontronites were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results indicated that all Shewanella species tested here were capable of reducing cystine to cysteine. Either cystine or cysteine amendments significantly stimulated the Fe(III) bioreduction rate and extent. The initial reduction rate was linearly correlated with cystine or cysteine concentration. The reduction extent (18.7–22.3%) calculated from bioreactor with cystine or cysteine was slightly lower than those with AQDS (26.3%). Mineralogical analysis demonstrated that cystine or cysteine facilitated the reaction of smectite to illite as a result of Fe(III) bioreduction. Thus, we concluded that, in our experiments, cystine and cysteine functioned as electron carrier in the smectite reduction systems, and were favorable factors influencing smectite illitization.     

Degradation of L-djenkolate catalyzed by S-alkylcysteine alpha,beta-lyase from Pseudomonas putida

S-Alkylcysteine alpha, beta-lyase [EC 4.4.1.6] of Pseudomonas putida catalyzes alpha,beta-elimination of L-djenkolate [3,3'-methylenedithiobis(2-aminopropionic acid)] to produce pyruvate, ammonia, and S-(mercaptomethyl)cysteine initially. Secondly, S-(mercaptomethyl)-cysteine, which was identified in the form of S-(mercaptomethyl)cysteine thiolactone and S-(2-thia-3-carboxypropyl)cysteine in the absence and presence of iodoacetic acid, respectively, is decomposed enzymatically to pyruvate, ammonia, and bis(mercapto)methane, or spontaneously to cysteine, formaldehyde, and hydrogen sulfide. Balance studies showed that 1.3 mol each of pyruvate and ammonia and 0.2 mol each of formaldehyde and cysteine were produced with consumption of 1 mol of L-djenkolate. 1,2,4,5-Tetrathiane, 1,2,4-trithiolane, 1,2,4,6-tetrathiepane, and 1,2,3,5,6-pentathiepane, which are derivatives of bis(mercapto)methane, were also produced during the alpha,beta-elimination of L-djenkolate. In addition, a polymer with the general formula of -(CH2S)n- was produced as a white precipitate. When the alpha,beta-elimination of L-djenkolate was carried out in the presence of 20 mM iodoacetic acid, neither formaldehyde, cysteine, hydrogen sulfide, or the polymer were formed. Instead, the S-carboxymethyl derivatives of bis(mercapto)methane and S-(mercaptomethyl)cysteine were produced in addition to pyruvate and ammonia.     

Cysteine transport in melanosomes from murine melanocytes

The synthesis of pheomelanin requires the incorporation of thiol-containing compound(s) during the process of mammalian melanogenesis. Since melanins are produced only in specialized, membrane-bound organelles, known as melanosomes, such thiol donor(s) must cross the membrane barrier from the cytosol to the melanosome interior. Cysteine and/or glutathione (GSH) were proposed as suitable thiol donors, although uptake of these compounds into melanosomes was not previously characterized. In this study, we show that cysteine is transported, in a temperature- and concentration-dependent manner, across membranes of melanosomes derived from murine melanocytes. Additional proof that cysteine uptake results from a carrier-mediated process and is not due to simple diffusion or to a membrane channel, was obtained in countertransport experiments, in which melanosomes preloaded with cysteine methyl ester took up significantly more [35S]cysteine than did unloaded controls. In contrast, we were unable to detect any significant uptake of [35S]GSH over a wide concentration range, in the presence or in the absence of reducing agent. This study is the first demonstration of melanosomal membrane transport of cysteine, and it strongly suggests that free cysteine is the thiol source utilized for pheomelanin synthesis in mammalian melanocytes.