Characterization of the lysosomal cystine transport system in mouse L-929 fibroblasts.

We characterize here a lysosomal cystine transporter in mouse L-929 fibroblasts. Granular fractions from cells preloaded with cystine demonstrated countertransport that showed no dependence on Na+ or K+. The Michaelis constant for infinite-trans influx was 0.27 +/- 0.06 mM (n = 3), and a nonsaturable component of cystine entry was observed with Kd = 0.8-1.8 nmol of cystine.min-1.unit of hexosaminidase-1.mM-1. We found no evidence that cystine was also carried on any of the other known lysosomal amino acid transporters. Over 50 analogs were tested for their ability to inhibit countertransport. The inhibition constants are reported for selenocystine, cystathionine, selenomethionine, and leucine. Significant requirements for recognition by the transporter were the presence of amino groups, L configuration, and a chain length not greater than eight atoms. A net positive or negative charge was not required. Some di- as well as tetrapolar amino acids were recognized. We have surmised that the binding site has polar and apolar domains, the latter being large enough to accommodate branching on C-3 and the substitution of selenium or carbon in place of sulfur.

We found no evidence that cystine was also carried on any of the other known lysosomal amino acid transporters. Over 50 analogs were tested for their ability to inhibit countertransport.
The inhibition constants are reported for selenocystine, cystathionine, selenomethionine, and leucine.
Significant requirements for recognition by the transporter were the presence of amino groups, L configuration, and a chain length not greater than eight atoms. A net positive or negative charge was not required.
Some di-as well as tetrapolar amino acids were recognized.
We have surmised that the binding site has polar and apolar domains, the latter being large enough to accommodate branching on C-3 and the substitution of selenium or carbon in place of sulfur. Lysosomes are a major site for intracellular degradation of many types of macromolecules including proteins. The first realization that lysosomes have specific transport systems for amino acids derived from the demonstration that the accumulation of cystine found in the inherited metabolic disease, cystinosis, was the result of the absence of a functional lysosomal transport system for cystine (l-3).
Cystine transport in normal lysosomes can be studied experimentally by raising the internal cystine concentration by means of cystine dimethyl ester (CDME)' (3)(4)(5)(6)(7)(8). Studies in this laboratory showed that the exodus of cystine from granular fractions or Percoll-purified lysosomes of normal human lymphoblasts, leukocytes, and fibroblasts proceeded with a first-order dependence on the initial intralysosomal cystine load. At these subsaturating concentrations, exodus was stimulated by the addition of Mg/ATP, by an increase in the transmembrane pH gradient, and by a decrease in the transmembrane potential (8). Only in leukocytes was it possible to load with sufficient cystine to saturate the internal binding site, a concentration at which the response to Mg/ATP disappeared (9). Countertransport has been used by Gahl et al. (5) to characterize the lysosomal cystine transport system in the granular fraction of human leukocytes. These studies (5) established the presence of a carrier-mediated system, provided an estimate of 0.5 mM cystine for the Koa of the external binding site, showed a preference for L-cystine and competition by cystathionine, cystamine, and cysteamine-L-cysteine mixed disulfide.
Countertransport of cystine has also been demonstrated in FRTL-5 rat thyroid epithelial cells (10). In this study we have taken advantage of the properties of mouse L-929 fibroblasts for further characterization of the lysosomal cystine transport system.

Countertransport into Granular
Fractions-Mouse L-929 fibroblasts have a specific lysosomal transport system for cystine and, like human leukocytes, show countertransport that is demonstrable through the uptake of L-['%]cystine into cystine-loaded granular fractions. Conditions were established so that the rate of uptake over the first 3 min was linear and proportional to the amount of granular fraction taken ( Fig. 1).
Experiments shown in Table I establish that the countertransport requires intact vesicles, physiological temperatures, and that external 50 mM NaCl or KC1 has no effect. Although cystine uptake decreases as expected with increasing osmolarity of the suspending medium, small changes in osmolarity such as might arise from unavoidable differences in the incubation mixtures containing competitors have negligible effect (data not shown).
The rate of trans-stimulated radioactive cystine uptake will be maximal when the lysosomal interior binding site is saturated by unlabeled cystine (16, 17). We have measured the relation between interior cystine load and rate of cystine countertransport using granular fractions from a preparation of cells loaded to different levels. The relation is shown in Fig. 2  load. Mouse L-929 tibroblasts were harvested, pooled, divided into portions, and incubated in 0, 0.1, 0.6, 1.5, and 3.2 mM CDME. The granular fraction from each portion was prepared separately, and the rate of countertransport was measured as described under "Experimental Procedures." Each point represents the mean of duplicate 1-min rates; the error bars represent the range. the data to a rectangular hyperbola of the Michaelis form using the explicit weighting dictated by the error bars. This curue is an approximation because the exact form of the equation (equation 6 of White and Christensen (17) or equation IX-56 of Segel (16)) which describes countertransport at partial saturation requires values for parameters unavailable to us. The same experiment performed on another day shows the same Ko.5 for internal saturation but a different maximum rate. Such day-to-day variation has been observed by others (1% Kinetics-Initial rates of countertransport uersw external substrate concentration were measured in a preparation loaded to saturation and analyzed as described under "Experimental Procedures." The data were test fitted both to the simple Michaelis equation and to the Michaelis equation plus a term for nonsaturable migration as defined under "Experimental Procedures." The resulting parameters for three experiments are shown in Table II. Although the sum of the squared residuals (the difference between the calculated and experimental values) is less when the nonsaturable term is included, it is not so much less that a choice in favor of the additional term can be made on this basis alone. The consistent value for V","../K: obtained when the analysis includes the nonsaturable component provides additional evidence for its inclusion. When only the simple Michaelis equation is used the experimental rate values could include some component of transport which would not be accounted for by a rectangular hyperbola, and VzJpO would therefore be too large. When the first-order component, Kd, is included in the descriptive equation to which the experimental rates are fitted, VGJK: becomes consistent, as would be expected for a saturated mediated component even when the contribution of the nonsaturable component varies among the experiments. The data for experiment '2 (Table II) and the calculated curves for the two hypothetical components are plotted in Fig. 3. The two different slopes found when the data are analyzed in an Eadie-Scatchard plot (Fig. 3, inset) also suggest two components, whether the second be saturable or not, within the experimental range.
We interpret these data to mean that in mouse L-929 lysosomes both a saturable and a nonsaturable pathway exist for passage of cystine. Because the influx for eystine is low in unloaded granules and presumably contains such a nonsaturable component, we have not been able to obtain data of sufficient precision to calculate reliable values for Kh and V,?,?, the parameters for the saturable component (zero-trans condition). The relationship between rate and external cystine concentration for these unloaded preparations is not significantly different from a straight line represented by a firstorder rate constant of 3.5 nmol.unit of hexosaminidase-' . min-' . mM-'. At 8 ELM cystine, this rate would account for 28 pmol of cystine. unit of hexosaminidase-' . min-' which approximates the highest rate for unloaded granules we have observed. Based on the Kd calculated from Table II for loaded granules, one could expect only 6-14 pmol of cystine . unit of hexosaminidase-' . min-' to penetrate via the nonsaturable route. Possibly the net nonsaturable flux may be larger in the absence of cystine on the opposite side (zero-trans condition). In addition, although NEM has very little effect on countertransport at 8 pM external cystine, the effect of NEM, if any, on the nonsaturable component revealed at high external cystine concentrations has not yet been evaluated.
We emphasize that our failure to saturate completely the external cystine-binding site and our subsequent assignment of the flux remainder to a "nonsaturable" component do not exclude the possibility that such a component could, in fact, also be a saturable mediated system with a high K,. We are limited experimentally to roughly 1.3 mM external cystine by its solubility, but we have attempted to circumvent this limitation by inundating the external binding site with a more soluble competitive inhibitor.
A Dixon plot (Fig. 4) shows that the effect of readily soluble L-selenomethionine is consistent with competitive inhibition because the family of curves crosses at a point above the 1: axis. The nonsaturable component contributes a significant portion of the uptake only at the highest external cystine concentration (see Fig. 3). By presentation of the data in the form of the Dixon plot shown in Fig. 4, the error in evaluation of the primary transporter is largely confined to those values in the plot for 408 PM external cystine. The value of z was derived from the slopes of the other curves and is represented in experiment 3 of Table II. The Kd for this experiment was derived from the difference between calculated and observed rates at 408 pM external cystine.
Having found a soluble competitive inhibitor, we applied the strategy of overwhelming the transporter with large concentrations of L-selenomethionine in an attempt to reach saturation of the primary transport system and expose a second if it should exist. Such a second system would not readily be inhibited within the test range because it is presumed to have an even larger K,,,. The results for L-selenomethionine and other competitors are shown in Fig. 5. The use of high concentrations of competitors does not imply that we believe that these competitors have any physiological role. Their use is a strategy to saturate the binding site with a soluble compound when it is impossible to do so with cystine. The extrapolation of these curves to maximum inhibition is charted in Table III  Whether both chiral centers of tetrapolar analogs must be of the L configuration for recognition by the mouse system is uncertain. Several examples found in Tables IV and V considered together indicate that the presence of 1yamino groups is more important than carboxylate groups for binding at the recognition site. LL-Lanthionine is a strong inhibitor; however, S-3,3-dipropionate, structurally equivalent to lanthionine but without its amino groups, is less effective. Although lacking carboxylate groups, the diamines cystamine and selenocystamine are able to compete quite well. We have demonstrated that the inhibition of [14C]cystine uptake is not due to partial loss of radioactive half-molecules by donation in a sulfhydryl-disulfide exchange. Cystine dimethyl ester in which the carboxyls are esterified also inhibits uptake of label. CDME is known to enter the lysosomes of the granular fraction since this route has been used for loading (11). In this experiment, however, the interior binding site is already saturated, and no stimulation is observed. As with the diamines above, inhibition might result from the competition of cystine monomethyl ester, derived by disulfide exchange, which either is not transported or which is transported carrying a depleted specific activity of label. We have shown, however, that the disulfide exchange measured by high voltage electrophoresis on paper amounts to only 1.1% of the total label under the conditions of the experiment. Most of the inhibition, then, must be the result of competition between CDME per se and cystine for the cystine transporter.
Table V also shows that sulfur seems to be accepted at the binding site as an apolar atom. The substitution of a sulfur atom for a carbon had little effect in the following paired examples: S-carboxymethyl-L-cysteine and L-2-aminoadipate; thialysine and lysine; methionine and norleucine. A departure from this generalization is seen on comparing the inhibition of lanthionine and diaminopimelate, which also differ from each other structurally only in the presence of a single sulfur atom in place of carbon. Of this pair, the sulfurcontaining analog is the better competitor (Table IV). These may be positioned more effectively by their four polar groups than analogs bearing a single a-amino and a-carboxylate but lacking either of the distal charged groups.
When selenium is substituted for sulfur as in selenocystine, selenocystamine, and selenomethionine, the selenium-containing compound proves the better inhibitor.
Although selenium may interact with proteins forming selenosulfides, this atom may also be bound by hydrophobic interactions (21). In view of our collective experience with the binding of phenylglycine, methionine, leucine, isoleucine, and valine (Table  IV), we believe that hydrophobic interactions may dominate at that part of the recognition site opposite the sulfur atoms of cystine. In addition to its greater mass compared with sulfur, selenium has approximately 10% larger effective diameter and slightly less electronegativity.
The recognition site of the transporter accommodates selenium in place of sulfur so that the analog acts as a competitive inhibitor (Figs. 4 and 5).
Shape of the Apolar Domain-Examination of these molecular determinants for recognition of analogs by the binding site leads us to some rather more speculative deductions.
If cystine should take a right-or left-handed spiral conformation with dihedral angles ranging from 80 to 103 ', as do cystine residues found in crystalline proteins (22), or of 106 ", as it does in crystalline cystine (23), then the central apolar domain of the cystine-binding site must provide sufficient space in three dimensions to accommodate such a shape. As illustrated in Fig. 6, left, the S-S bond may be visualized as the hinge of a door, and the dihedral angle formed by the flanking carbons may then be considered the angle to which the door is opened. If the C-S-S-C "door-hinge" of cystine were stretched open to 105 ", the molecule would have the same angle as the C-S-C of lanthionine.
Since lanthionine is recognized by the binding site in spite of having only one sulfur atom and a shorter chain length, we infer that the apolar region does in fact accommodate a change of direction at the sulfur. Furthermore, the inhibition by a group of analogs that branch on carbon 3 (valine, 66% of control; isoleucine, 50%; phenylglycine, 45%) is also instructive.
Because these compounds are positioned by only one pair rather than two pairs of a-carbon polar groups, they must fit well in the apolar region, which is large enough to permit access for the carbon 3 branch (Fig. 6, right). A group of analogs that branch at carbon 2 (2-aminoisobutyrate, 85%; 1,4-diamino-dicarboxycyclohexane, 72%; BCH, 85%) does not significantly inhibit, suggesting that the rec-    be narrow in this region. The failure of homocystine (89%) or djenkolate (101%) to inhibit may indicate that the binding site is too short to accommodate these longer analogs of cystine. Relation to Other Amino Acid Transport Systems in Lysosomes-Beyond a molecular description of the transporter site, the second goal of these studies is to determine whether cystine might be utilizing, in part, one of the previously described lysosomal amino acid carriers. The amino acids and analogs surveyed in Tables IV and V were chosen to provide  a profile of inhibitors for this purpose. Those analogs shown in Table V demonstrate the effect of net charge and of substrates for lysosomal transport systems c (24) and d (19).
The presence or absence of a positive charge does not seem to increase the rather weak and variable inhibition by cationic amino acids. The cationic amino acid, p-methyl-L-lysine, which is accepted by the lysosomal transport system c, here shows no more competition than do citrulline and Ns-acetyl-L-ornithine, which lack a net charge. We conclude that cystine does not utilize transport system c, an observation confirmed by the reports that lysine does not exit the lysosome any faster when cystine is placed on the outside (25) and that cystinotic lysosomes ordinarily lose cystine only very slowly (8,25) in spite of having a normal transport system c. Our observations also eliminate any significant contribution by the system of the epithelial membrane type for cystine which is characteristically shared with lysine and a&nine (26). The absence of significant effect by aspartate or glutamate excludes system d as a route for cystine flow in lysosomes. This is a conclusion that confirms the observation of Gahl  System e has been shown to carry alanine, serine, and threonine but not methionine; and systems f and p have been shown to carry proline (28). We see no indication that these pathways are utilized by cystine since methionine does inhibit, and the other four analogs have only minimal effect on cystine countertransport (Table IV). Tryptophan and phenylalanine, substrates for system t in human fibroblast lysosomes (29) or h in rat thyroid lysosomes (lo), have little effect on the mouse lysosomal cystine transporter.
Although methionine, together with branched chain amino acids, may be recognized both by the rat thyroid system h and the cystine transporter, cystine is not in turn recognized by system h (10). The specific cysteine transporter described by Pisoni et al. (30) will not carry even homocysteine, and therefore it would be implausible for it to carry cystine. Since our incubation mixtures contain NEM, cysteine that might egress from the lysosome would not exchange with the labeled external cystine and thus carry label into the lysosome as cysteine.
These observations exclude the identity of the cystine transporter with e, f, p, t, h, or the cysteine transport systems. The effects of leucine, norleucine, and phenylglycine match rather well, with the exception of the ineffective BCH, the substrate pattern for lysosomal system 1 as described by Stewart et al. (29) in human fibroblasts.
We are not able to show, however, significant trans-stimulation of leucine uptake by cystine loading (data not shown). This result would seem to eliminate system 1 for cystine recognition although the existence of a species difference must be considered. In the mouse experiments, for example, we found no large nonsaturable component for leucine transport when excess leucine was present, a characteristic that differs from the observations of Stewart et al. (29).
Other Known Cystine Transport Systems--In Escherichia coli there are two cystine transport systems that differ in sensitivity to inhibitors. The specific system recognizes and is therefore inhibited only by selenocystine and cystathionine in addition to cystine. The general system recognizes and is inhibited not only by cystine, selenocystine, and cystathionine, but also by lanthionine, diaminopimelate, 3-hydroxyand 4-methyldiaminopimelate (31). A structural homology may conceivably be discovered between the recognition site of the general E. coli system and the mouse lysosomal cystine transporter.
Lanthionine, which is a considerably more effective inhibitor of the general compared with the specific system in E. coli, also reduces both mouse and human fibroblast lysosomal cystine countertransport to only 28% of control for 10 mM LL-lanthionine (data for human not shown). Furthermore, the mouse lysosomal transporter, like the general but not the specific E. coli system, shows a tolerance for branching at C-3 and C-4 (valine, leucine, isoleucine) and a modest inhibition by 10 mM L-(3R)-hydroxydiaminopimelate (62% of control). This similarity in the shape of the apolar domain between the primary mouse cystine transporter and the E. coli general system is another indication of possible homology. Significance-The work in this report shows that lysosomal cystine probably cannot make significant use of the transport systems described previously for other amino acids. It may, however, follow to a small degree an alternative transmembrane route that we have designated provisionally as nonsaturable and which should not automatically be assigned to diffusion. Since cystine is generated from protein degradation within the lysosome and is reduced to cysteine on the cytoplasmic side of the lysosome immediately after exodus, it is evident that these pathways that we have studied by countertransport for convenience of manipulation must operate in the living cell primarily in the direction of exodus. The inability of lysosomes to utilize an adequate alternative route has led to the accumulation of cystine in persons lacking the primary cystine transporter, the defect of the inherited human disease, cystinosis. If the presence of a nonsaturable component, especially a variable one, is also demonstrated for human lysosomal cystine transport, it would provide an explanation for the observation that although cystinosis patients lack a functional cystine transporter, their cells do not accumulate cystine beyond a level that is typical for each individual and each cell type. It might, in addition, provide an explanation for the partial cystine transport ability of lysosomes observed in benign cystinosis (32). The characterization of this alternative route would improve the understanding of cystinosis and its variants.
The nature of the chemical and spatial determinants for binding to the recognition site of the recognized lysosomal cystine transporter may provide an understanding for the design of effective irreversible inhibitors which will mark the transport protein for eventual isolation and purification.