©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Detection and Characterization of a Transport System Mediating Cysteamine Entry into Human Fibroblast Lysosomes
SPECIFICITY FOR AMINOETHYLTHIOL AND AMINOETHYLSULFIDE DERIVATIVES (*)

(Received for publication, August 25, 1994; and in revised form, October 27, 1994)

Ronald L. Pisoni Grace Y. Park Vanessa Q. Velilla Jess G. Thoene

From the Department of Pediatrics and Communicable Diseases, The University of Michigan Medical School, Ann Arbor, Michigan 48109-2029

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The uptake of [^3H]cysteamine by Percoll-purified human fibroblast lysosomes was investigated to determine whether lysosomes contain a transport system recognizing cysteamine. Lysosomal cysteamine uptake is a Na-independent process which rapidly attains a steady state within 1 min at pH 7.0 and 37 °C. A biphasic Arrhenius plot is observed for cysteamine uptake, giving a Q of 2.2 from 17 to 26 °C and a Q of 1.2 from 27 to 35 °C. The rate of lysosomal cysteamine uptake is maximal at pH 8.2, half-maximal at pH 6.8, and declines 50-fold from the maximum to show very little transport at pH 5.0. Cysteamine uptake into fibroblast lysosomes displays complete saturability with a K of 0.88 mM and V(max) of 1410 pmol of beta-N-acetylhexosaminidase/min at pH 7.0 and 37 °C. Analog inhibition studies demonstrated that all analogs recognized thus far by the cysteamine carrier are either aminothiols or aminosulfides and contain an amino group and sulfur atom separated by a carbon chain, 2 carbon atoms in length. The K constants for these analogs as competitive inhibitors of lysosomal cysteamine uptake are 2-(ethylthio)ethylamine (0.64 mM), 1-amino-2-methyl-2-propanethiol (0.74 mM), 2-dimethylaminoethanethiol (0.87 mM), thiocholine (1.6 mM), and bis(2-aminoethyl)sulfide (4.9 mM). L-Cysteine, D-penicillamine, and analogs lacking either a sulfur atom or amino group are not recognized by the cysteamine carrier including ethanolamine, choline, taurine, beta-mercaptoethanol, ethylenediamine, cadaverine, spermine, spermidine, histamine, dopamine, and 3-hydroxytyramine. In a cystine-depletion assay, a 2-h exposure of cystinotic fibroblasts to 1 mM 1-amino-2-methyl-2-propanethiol lowers cell cystine levels to the same low level obtained with cysteamine. Thus, all four aminothiols, known to deplete cystinotic fibroblasts of their accumulated cystine, are recognized as substrates by the lysosomal cysteamine carrier, suggesting the importance of this transporter in the delivery of aminothiols to the lysosomal compartment.


INTRODUCTION

Lysosomes are a major intracellular site for the degradation of a wide variety of macromolecules including proteins, nucleic acids, complex carbohydrates, and lipids. The metabolites that form in lysosomes from macromolecule degradation are recognized by specific lysosomal transport systems which mediate metabolite exodus from the lysosomal compartment. At the present time 20 different lysosomal transport systems have been described including carriers for various amino acids(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) , monosaccharides(14, 15, 16, 17, 18, 19, 20) , nucleosides(21) , cobalamin(22, 23) , phosphate(24) , sulfate(25) , calcium(26) , dipeptides(27) , and methotrexate polyglutamates(28) . However, not all lysosomal transport systems are believed to function solely for facilitating metabolite egress from lysosomes. Previously, we described a cysteine-specific lysosomal transport system serving as a major route for the delivery of thiol into human fibroblast lysosomes(13) . In addition, Rome and colleagues have characterized a lysosomal membrane enzyme, acetyl-CoA:alpha-glucosaminide N-acetyltransferase, which catalyzes the transmembrane transfer of acetyl groups from cytosolic acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate located within the lysosomal compartment (29, 30).

The first detailed characterization of a lysosomal transport system was that of the lysosomal cystine carrier by Gahl et al.(1) and Jonas et al.(2) in 1982. These investigators demonstrated that the high levels of lysosomal cystine accumulation observed in the genetic disorder, nephropathic cystinosis, were due to a defect in lysosomal cystine transport. In the absence of treatment, nephropathic cystinosis leads to end stage renal failure by 10 years of age. In 1976, Thoene et al.(31) demonstrated that the addition of cysteamine to cultures of cystinotic fibroblasts produces rapid intralysosomal cystine depletion. Subsequent clinical studies have shown that cysteamine stabilizes renal function and improves linear growth in a large population of cystinotic patients(32) . Despite this knowledge of the therapeutic effects of cysteamine, however, the manner by which cysteamine enters lysosomes is not understood. In this report, we provide the first characterization of a lysosomal transport system which mediates the entry of cysteamine into human fibroblast lysosomes and raise questions regarding the role this transporter plays in lysosomal function.


EXPERIMENTAL PROCEDURES

Cell Culture and Preparation of Percoll-purified Lysosomes

The normal human fetal skin fibroblast cell line, GM0010, and cystinotic fibroblast cell line, GM0090A, were obtained from the Human Genetic and Mutant Cell Repository. Fibroblasts were grown and maintained in an atmosphere of 95% air, 5% CO(2) in 100-mm tissue culture dishes or 850-cm^2 roller bottles in Coon's modification of Ham's F-12 medium (Sigma) supplemented with 10% fetal bovine serum. Confluent human fibroblast monolayers were routinely split 1:4 when passed using a trypsin/chicken serum/collagenase mixture (24) and were not used beyond the 16th passage. Fibroblast lysosomes were purified on density gradients as described previously (6, 8) and were resuspended in 40 mM Mops(^1)/Tris, pH 7.0, buffer containing 0.25 M sucrose for use in most of the transport studies. Typically, three to four confluent roller bottles were required to provide a sufficient quantity of lysosomes for accurate measurement of lysosomal cysteamine transport activity.

Kinetic Studies of Lysosomal Cysteamine Uptake

For the analog inhibition and K(m) experiments, an 18-µl aliquot of an inhibitor or substrate solution in 40 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose and 60 mM DTT was mixed with 9 µl of 142 µM [^3H]cysteamine in 20 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose and 1 mM DTT; incubation mixtures were warmed to 37 °C, then 9-µl aliquots of ice-cold lysosomal suspension in 40 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose were added at time 0 and incubated for the indicated period of time at 37 °C; a 30-µl aliquot was removed from each incubation mixture, added to 12 ml of ice-cold PBS, and filtered through a GF/A filter (Whatman, 24 mm); the filter was washed twice with 12-ml portions of ice-cold PBS and mixed with 16 ml of Cytoscint scintillation fluid (ICN). Filters were allowed to equilibrate in scintillation fluid for 18-24 h prior to counting for radioactivity. Lysosome-independent radioactivity retained on the filters, determined by substituting 40 mM Mops/Tris, pH 7.0, buffer in 0.25 M sucrose for the lysosomes, was subtracted from radioactivity retained on filters of lysosome-containing samples.

beta-N-Acetylhexosaminidase and Protein Assay

The integrity of individual lysosomal preparations was generally found to be 80% intact as judged by the latency of beta-N-acetylhexosaminidase activity, determined as described previously(21) , and which was calculated as the difference in hexosaminidase activity in the presence or absence of 0.1% Triton X-100. Cell protein was quantified using bicinchoninic acid as described in the precipitation assay of Brown et al.(34) .

Analysis of Kinetic Data

The kinetic constants, K(m) and V(max), were calculated by applying the Gauss-Newton nonlinear least squares method to the kinetic data using Cleland's HYPERL program(35) . Inhibition constants, K(i) values, were calculated using Cleland's COMP program(35) . K(i) values and the competitive nature of inhibition were verified by linear regression analysis of slope replots generated from Lineweaver-Burk plots.

Measurement of Cell Cystine Content

Cystinotic fibroblasts were harvested by incubation for 5 min in 1 ml of a trypsin/collagenase/EDTA solution(24) , washed twice by centrifugation in 13-ml portions of ice-cold PBS, and the cell pellets were suspended in 0.3 ml of 10 mM sodium phosphate, pH 7.5, buffer containing 5 mMN-ethylmaleimide. Cell pellet suspensions were sonicated for 5 s with a KONTES Microsupersonic Cell Disruptor, and cell proteins were precipitated by the addition of 100 µl of 12% sulfosalicylic acid. Precipitated proteins were collected by centrifugation for 5 min in an Eppendorf model 5415 microcentrifuge. Supernatants were analyzed for free cystine using the cystine binding protein assay described by Oshima et al.(36) .

Miscellaneous Methods

[^3H]Cystamine (0.5 mCi/ml in 0.01 N HCl, 3 Ci/mmol) was prepared as a custom synthesis by Moravek Biochemicals (Brea, CA). The radiolabeled cystamine was reduced to cysteamine on the day of each experiment by the addition of dithiothreitol as described in individual experiments. Percoll was purchased from Pharmacia Biotech Inc., and fetal bovine serum was obtained from Sigma or BioWhittaker. Thiocholine iodide was obtained from ICN and all other chemicals were obtained either from Sigma or Aldrich.


RESULTS

We initially defined the effects of time, temperature, substrate concentration, and pH on the rate of [^3H]cysteamine uptake by Percoll-purified human fibroblast lysosomes. Lysosomal cysteamine uptake at 37 °C and pH 7.0 occurs rapidly, attaining a steady state within 1 min following the initiation of uptake (Fig. 1). Similar to all other lysosomal transport systems, cysteamine transport was not affected by the presence of 77 mM NaCl in the incubation mixtures (data not shown), indicating that lysosomal cysteamine uptake is not a sodium dependent process. In order for further kinetic analysis to reflect initial rate kinetics, uptakes generally were performed for 30-45 s so as to occur during the linear portion of the uptake curve. The effect of temperature on lysosomal cysteamine uptake revealed a biphasic Arrhenius plot over the tested temperature range from 17 to 35 °C (Fig. 2). For the temperature range from 17 to 26 °C, a rate-limiting step with a Q of 2.26 is observed which corresponds to an activation energy of 14 kcal/mol. At temperatures above 26 °C, however, the activation energy for the rate-limiting transport step decreases to 3.7 kcal/mol, corresponding to a Q of 1.2. These two different activation energies may be a reflection of two completely different rate-limiting steps involved in the process of lysosomal cysteamine translocation or could correspond to essentially the same rate-limiting step occurring more easily at temperatures above 26 °C due to substantial lowering of a translocation barrier. Although we are not able to discriminate among these possibilities, changes in membrane fluidity and structure with changes in temperature could have a significant effect on the energetics of transport. This is the first report of a biphasic Arrhenius plot observed for a lysosomal transport system. All other lysosomal carriers characterized until the present time have displayed linear Arrhenius plots with Q values ranging from 1.8 to 3.2, with a mean of 2.15 and a median of 2.0(37) .


Figure 1: Time course of 0.071 mM [^3H]cysteamine uptake by human fibroblast lysosomes at pH 7.0. Time courses at 37 °C were initiated by mixing a prewarmed 70-µl aliquot of 142 µM [^3H]cysteamine in 20 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose and 1 mM DTT^1 with a 70-µl aliquot of Percoll-purified lysosomes suspended in 40 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose. At the indicated times, 15-µl aliquots were removed from the incubation mixture, added to 12 ml of ice-cold phosphate-buffered saline, and filtered through a GF/A filter. Filters were immediately washed twice with 12-ml portions of ice-cold phosphate-buffered saline, mixed with 16 ml of Cytoscint scintillation fluid, and counted for radioactivity. hex, beta-N-acetylhexosaminidase.




Figure 2: Arrhenius plot of 0.035 mM [^3H]cysteamine uptake by human fibroblast lysosomes. Prewarmed lysosomes (8 µl) in 40 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose were mixed with 24-µl aliquots of prewarmed 0.047 mM [^3H]cysteamine in 33 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose and 4.2 mM DTT. Uptakes were performed in triplicate for 30 s at a temperature of either 17, 20, 23, 26, 29, 32, or 35 °C. At the completion of each uptake, lysosomes were collected on glass fiber filters and washed with ice-cold phosphate-buffered saline, and filters were counted for radioactivity. hex, beta-N-acetylhexosaminidase.



A Michaelis-Menten plot of the initial rate of cysteamine uptake as a function of the concentration of cysteamine demonstrates that lysosomal uptake of cysteamine is a saturable process at pH 7.0 and 37 °C (Fig. 3). The Lineweaver-Burk plot is linear suggesting that lysosomal cysteamine uptake is mediated by one transport route. The kinetic parameters of K(m) and V(max) were calculated using Cleland's HYPERL program yielding values of K(m) = 0.88 mM ± 0.03 and V(max) = 1410 ± 30 pmol of beta-N-acetylhexosaminidase/min. Although our data for these short uptake time intervals conform well to a right rectangular hyperbola characteristic of processes obeying Michaelis-Menten kinetics, our analysis does not exclude the possibility that a small nonsaturable component also may be associated with cysteamine uptake. The observed V(max) for cysteamine uptake is one of the largest rates of solute flux quantified thus far for a lysosomal transport process.


Figure 3: Kinetics of the initial rate of cysteamine uptake into human fibroblast lysosomes as a function of the cysteamine concentration. Fibroblast lysosomes were incubated for 30 s at 37 °C with [^3H]cysteamine of the indicated concentration, in 35 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose and 30 mM DTT. At the completion of the incubation period, lysosomes were collected on glass fiber filters and washed with ice-cold phosphate-buffered saline, and filters were counted for radioactivity. Determinations were performed in triplicate at each concentration. The results are displayed in a Michaelis-Menten plot of the initial rate of cysteamine uptake as a function of the cysteamine concentration. The graph provided in the inset is a double-reciprocal plot of the same data. hex, beta-N-acetylhexosaminidase.



Lysosomal cysteamine uptake shows a dramatic response to pH over the range from pH 5 to 8.5. An 50-fold greater rate of cysteamine uptake is observed at the optimal pH of 8.2 compared to the acidic pH of 5.0 (Fig. 4). If this pH dependence exists on both sides of the lysosomal membrane, then once cysteamine is transported into lysosomes from the cytosol, the intralysosomal pH of 5.3 (38) would not be favorable for cysteamine exodus from lysosomes by this transport route. Thus the pH dependence of the lysosomal cysteamine carrier appears to greatly favor net cysteamine influx compared to efflux and may allow lysosomes to concentrate cysteamine into this compartment when cystinotic cells are exposed to therapeutic levels of cysteamine.


Figure 4: pH dependence of 0.035 mM [^3H]cysteamine uptake by human fibroblast lysosomes. Fibroblast lysosomes (8 µl) suspended in 0.25 M sucrose were incubated with 24 µl of 0.047 mM [^3H]cysteamine in either 20 mM MES or 20 mM MOPS buffers containing 0.25 M sucrose and 1.67 mM DTT and which had been titrated to the indicated pH with Tris-free base. Incubations were performed at 37 °C for 0.75 min at which time 26.5-µl aliquots were removed from incubation mixtures, lysosomes were collected and washed on GF/A filters, and the filters were mixed with 16 ml of Cytoscint for measurement of radioactivity. hex, beta-N-acetylhexosaminidase.



To measure cysteamine efflux from lysosomes, lysosomes were incubated with 157 µM [^3H]cysteamine for 10 min at 37 °C and washed of unaccumulated radioactivity by centrifugation at 4 °C, and the rate of lysosomal cysteamine egress was then measured at 37 °C. The results, shown in Fig. 5, indicate that 40% of the radiolabeled cysteamine is rapidly released within the first 3 min of incubation at 37 °C. Thereafter, only a slow rate of release is observed for the remaining radioactivity accumulated within human fibroblast lysosomes. Cysteamine is known to readily form mixed disulfides by participating in sulfhydryl/disulfide exchange reactions (39, 40) . It is likely that much of the slowly released radioactivity could represent mixed disulfides of cysteamine, such as protein-bound cysteamine-cysteinyl-mixed disulfides, which would be expected to form within lysosomes and require peptide bond hydrolysis to be released as a free mixed disulfide, then transported out of lysosomes by lysosomal system c(5, 6) .


Figure 5: Release of radioactivity accumulated within lysosomes following [^3H]cysteamine uptake. Percoll-purified lysosomes were loaded with radioactivity by incubation with 0.079 mM [^3H]cysteamine in 29 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose and 1.1 mM DTT, for 10 min at 37 °C. The lysosomal incubation (0.1 ml) was then diluted to 1.5 ml with ice-cold 40 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose (wash buffer), centrifuged at 15,600 times g in an Eppendorf microcentrifuge at 4 °C for 15 min. The supernatant was discarded, and the lysosomes were washed once more by resuspension in 1.5 ml of ice-cold wash buffer followed by centrifugation. The final lysosomal pellet was resuspended to 200 µl with ice-cold wash buffer. To assay for exodus of radioactivity from the ^3H-loaded lysosomes, the lysosomal suspension was then incubated at 37 °C. At the indicated time points, 15-µl aliquots were removed from the incubation mixture, lysosomes were collected and washed on GF/A filters, and filters were counted for radioactivity. hex, beta-N-acetylhexosaminidase.



Several years ago, we used L-[S]cysteine uptake studies to describe a cysteine-specific lysosomal transport system displaying an extraordinary specificity for the amino acid, cysteine(13) . During that investigation, we observed that cysteamine could strongly inhibit the lysosomal uptake of L-[S]cysteine, indicating that cysteamine could be recognized by the cysteine-specific lysosomal transport system. Whether cysteamine actually could be transported by the cysteinespecific carrier, however, was not determined at that time because of the unavailability of radiolabeled cysteamine. In the present investigation, we now have applied analog inhibition analysis to determine the specificity of the lysosomal transport system mediating [^3H]cysteamine uptake. For this analysis, 30 different analogs, each at a concentration of 10 mM, were tested for their ability to inhibit 0.035 mM [^3H]cysteamine uptake (Table 1). Notably, L-cysteine, which is a model amino acid substrate for the cysteine-specific carrier, has very little effect on lysosomal cysteamine uptake, indicating that cysteamine is not transported into fibroblast lysosomes by the cysteinespecific lysosomal transport system. Six other analogs, however, were found to be well recognized by the cysteamine carrier. These include cysteamine, 2-(ethylthio)ethylamine, 1amino-2-methyl-2-propanethiol, 2-dimethylaminoethanethiol, thiocholine, and bis(2-aminoethyl)sulfide. All of these compounds share the general structure,



where R = -H or -CH(3) and X = -H, -CH(2)-CH(3), or -CH(2)-CH(2)-NH(2). The amino group and sulfur atom are both required for substrate recognition by the carrier. Replacing the SH group of cysteamine or thiocholine with an OH group to give ethanolamine or choline results in poor recognition by the cysteamine transporter. Likewise, replacing the SH group of cysteamine with an SO(3) group to give taurine, or an NH(3) group to give ethylenediamine, also yields structures that are poorly recognized by the cysteamine transport system. A free thiol is not required for recognition as indicated by the strong inhibitory action of 2-(ethylthio)ethylamine and bis(2-aminoethyl)sulfide, which contain a sulfur atom in the form of a sulfide. The requirement for an amino group as part of the substrate recognition unit is indicated by the lack of inhibitory effect of beta-mercaptoethanol which contains the thiol of cysteamine but lacks the amino group. Methylation of cysteamine's amino group is well tolerated as indicated by the strong recognition of thiocholine and 2-dimethylaminoethanethiol. Acetylation of the amino group, however, is not permitted as demonstrated by the complete inability of N-acetylcysteamine to inhibit [^3H]cysteamine uptake. As part of our analog inhibition analysis, we determined if lysosomal cysteamine uptake could be inhibited by various amines which are known substrates for amine transporters in other biological membranes. These analogs included tyramine, dopamine, spermine, spermidine, hydroxytryptamine, acetylcholine, histamine, and cadaverine. Tyramine demonstrated a modest inhibitory effect producing 50% inhibition. None of the other amines, however, had any significant effect on lysosomal cysteamine uptake including 3-hydroxytyramine which failed to show any inhibitory effect on lysosomal cysteamine transport. Finally, the presence of an alpha-carboxyl group prevents recognition by the cysteamine transport system as indicated by the failure of L-cysteine and D-penicillamine to inhibit cysteamine uptake, whereas their decarboxylated analogs, cysteamine and 1-amino-2-methyl-2-propanethiol, each strongly inhibit lysosomal transport of [^3H]cysteamine.

For the six analogs in Table 1that were found to be the strongest inhibitors of lysosomal cysteamine uptake, further inhibition analysis revealed that all six analogs act as competitive inhibitors of cysteamine transport. In this analysis, five different concentrations of inhibitor were chosen for their ability to inhibit either 0.025, 0.05, 0.1, 0.2, or 0.5 mM [^3H]cysteamine uptake. Results, when displayed in a double-reciprocal plot, yielded inhibition lines intersecting on the y axis. Fig. 6A demonstrates such a plot for the inhibition of [H]cysteamine uptake by several different concentrations of thiocholine. When the slopes of individual inhibition curves from the double-reciprocal analyses were plotted as a function of the inhibitor concentration (Fig. 6B), a direct linear proportionality indicative of pure competitive inhibition (41) was obtained for each analog. The nearly identical K values for cysteamine and 2-(ethylthio)ethylamine, obtained from this kinetic analysis (Table 2), indicate that the cysteamine transport system does not discriminate whether the sulfur atom exists as a free thiol or in the form of a sulfide. Moreover, the transport system easily accommodates groups as long as an ethyl group bonded onto the non-amino side of the sulfur atom without any loss in recognition. The addition of an ethylamino group onto both sides of the sulfur atom, however, results in an 8-fold decrease in recognition by the cysteamine carrier as shown for bis-(2-aminoethyl)sulfide. Because of the unavailability of suitable analogs, we have not been able to examine if this large decrease in affinity of bis-(2-aminoethyl)sulfide compared to 2-(ethylthio)ethylamine is due to the carrier having difficulty in accommodating the overall length of bis-(2-aminoethyl)sulfide, is due to the effect of the additional amino group, or is the result of both structural changes significantly affecting substrate recognition. Another possibility for the higher K of bis-(2-aminoethyl)sulfide is that, at pH 7.0, the carrier may only recognize a minor ionic species of bis-(2-aminoethyl)sulfide such as the species in which one amino group is positively charged and the other amino group is uncharged. Branching at the beta-carbon of cysteamine has only a slight effect upon recognition by the cysteamine transport system as demonstrated by the similar K values for cysteamine and 1-amino-2-methyl-2-propanethiol. However, di- and trimethylation of the substrate amino group modestly diminishes recognition by the carrier as indicated by the larger K values of 0.87 mM and 1.63 mM for 2-dimethylaminoethanethiol and thiocholine, respectively, as compared to 0.58 mM for cysteamine.


Figure 6: Inhibition of lysosomal cysteamine uptake by thiocholine. Fibroblast lysosomes were incubated with either 0, 0.6, 1.2, 2.4, or 4.8 mM thiocholine (TC) for 30 s at 37 °C with [^3H]cysteamine of the indicated concentration in 35 mM Mops/Tris, pH 7.0, buffer containing 0.25 M sucrose and 26 mM DTT. At the completion of the incubation period, lysosomes were collected on GF/A filters, washed with ice-cold phosphate-buffered saline, and the filters were washed to determine the amount of radioactive cysteamine taken up. A, double-reciprocal plot of the initial rate of [^3H]cysteamine uptake as a function of the cysteamine concentration for the different concentrations of thiocholine that were used. B, slope replot, the slope of each curve from the above double-reciprocal plot is graphed as a function of the thiocholine concentration. The negative of the x axis intercept yields a K of 1.6 mM for thiocholine. hex, beta-N-acetylhexosaminidase.





Our analog inhibition and K(i) analyses in Table 1and Table 2indicate that the aminothiol, 1-amino-2-methyl-2-propanethiol (AMPT), also is recognized by the lysosomal cysteamine transport system. Upon entering lysosomes, AMPT would be expected to react with free cystine to form a cationic mixed disulfide. Lysosomal system c, which has been shown to accept a broad range of cationic substrates(6) , would likely recognize this mixed disulfide and facilitate its exodus from the lysosomal compartment. Thus, the aminothiol, AMPT, would appear to have the necessary characteristics to deplete cystinotic cells of their accumulated cystine. To test this possibility, cystinotic fibroblasts were incubated in medium containing either 1 mM AMPT, 0.5 mM cysteamine or medium without any further additions. The results shown in Fig. 7demonstrate that during a 2-h exposure period, 1 mM AMPT is able to deplete cystinotic fibroblasts of their accumulated cystine to the same low level as occurs when cells are exposed to 0.5 mM cysteamine.


Figure 7: Comparison of the ability of cysteamine and 1-amino-2-methyl-2-propanethiol to deplete cystinotic cells of their cystine content. Confluent monolayers of cystinotic fibroblasts, grown in 60-mm dishes in Coon's modified Hams' F-12 medium containing 10% fetal calf serum, were washed three times with PBS and placed in cystine-free culture medium with 10% dialyzed fetal calf serum containing either 0.5 mM cysteamine (MEA), 1 mM AMPT, or no further addition. After a 2-h incubation, plates were washed three times with PBS, cells were scraped off plates with a rubber policeman, and cell protein and cystine content was quantified as described under ``Experimental Procedures.'' The experiment was performed in triplicate for each incubation condition. Results are expressed as a percent of the initial cystine content of the cystinotic cells prior to the 2-h incubation which was 9.97 ± 0.15 nmol of cystine/mg of protein.




DISCUSSION

In this report, we provide the first characterization of a unique transport system that mediates cysteamine entry into human fibroblast lysosomes. The substrate specificity of this lysosomal cysteamine carrier is highly unusual among known transport systems in that all analogs recognized thus far are either aminothiols or aminosulfides, and contain a sulfur atom and amino group separated by a carbon chain, 2 carbon atoms in length. The observation that thiocholine, which contains a quaternary amine, is well recognized by the cysteamine carrier provides strong evidence that the substrate amino group is recognized in its positively charged form by the carrier. Throughout the physiological pH range, the amino group of cysteamine, with a pK of 10.5(42, 43) , also would be expected to exist largely in its protonated form. Therefore, the pH profile of lysosomal cysteamine uptake, showing 50-fold greater cysteamine uptake at pH 8.2 than at pH 5.0, does not reflect titration of the substrate amino group. Instead, this pH dependence likely corresponds to either titration of an amino acid residue on the carrier protein important for cysteamine transport, or reflects a dependence of lysosomal cysteamine uptake on a transmembrane proton gradient.

Cysteamine is therapeutically important in the treatment of nephropathic cystinosis by being able to enter lysosomes, react with free cystine to form the mixed disulfide of cysteamine and cysteine. These two products are then transported out of the lysosomal compartment by other functional lysosomal carriers(6, 13, 31, 32) . In addition to cysteamine, thiocholine and 2dimethylaminoethanethiol also have been shown to deplete cystinotic cells of their accumulated lysosomal cystine(6, 31) . How these aminothiols are able to enter lysosomes, however, has not been delineated. The results of the present investigation indicate that these three aminothiols and 1-amino-2-methyl-2-propanethiol are each recognized by the lysosomal cysteamine transport system and thus, carrier mediation by the cysteamine porter is an important step in the overall mechanism of cysteamine treatment of nephropathic cystinosis.

The biological importance of the lysosomal cysteamine transport system remains to be established. Cysteamine is the only naturally occurring aminothiol in man currently known to be recognized by the lysosomal cysteamine carrier. However, the levels of cysteamine generally detected in human fibroblasts are low(44) . It is noteworthy, however, that cysteamine is generated in animals as part of the coenzyme A degradative pathway(33, 44) . Some of the catabolic enzymes that comprise this pathway are lysosomal enzymes, and the cysteaminegenerating enzyme, pantethinase, appears to be located exclusively in the microsomal-lysosomal fraction in rat liver(33) . Although confusion remains as to the subcellular location of the complete coenzyme A degradative pathway, the lysosomal cysteamine transporter may serve for egress of cysteamine produced in lysosomes through the action of a lysosomal pantethinase.


FOOTNOTES

*
This work has been supported by National Institutes of Health, United States Public Health Service Grant DK25548. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: Mops, 3-(N-morpholino)propanesulfonic acid; DTT, dithiothreitol; PBS, phosphate-buffered saline; MES, 2-(N-morpholino)ethanesulfonic acid; AMPT, 1-amino-2-methyl-2propanethiol.


ACKNOWLEDGEMENTS

We thank Dr. Halvor N. Christensen for his helpful comments during the preparation of this manuscript.


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