Characterization of lysosomal monoiodotyrosine transport in rat thyroid cells. Evidence for transport by system h.

Lysosomal transport of monoiodotyrosine was characterized in countertransport experiments using rat FRTL-5 thyroid cell lysosomes. Monoiodotyrosine carrier activity was temperature-dependent (Ea = 11.65 kcal/mol) and had a pH optimum of 7.5. Carrier activity was minimally inhibited by KCl and NaCl, but unaffected by the presence of other ions or ATP. Monoiodotyrosine transport was unaffected by the presence of carbonyl cyanide m-chlorophenylhydrazone, nigericin, or ammonium chloride, indicating that a proton or K+ gradient is not necessary for monoiodotyrosine transport across the lysosomal membrane. Monoiodotyrosine countertransport showed a 6-fold increase in lysosomes from FRTL-5 cells grown in medium containing thyrotropin by comparison to cells grown without this hormone. Thyrotropin responsiveness raised the possibility that monoiodotyrosine was transported by system h, the only known lysosomal carrier whose activity is enhanced by thyrotropin. Consistent with this, monoiodotyrosine-loaded lysosomes exhibited countertransport of [3H]tyrosine, [3H]phenylalanine, and [3H]leucine, three system h ligands, but not [3H]cystine, a nonsystem h ligand. Unlabeled tyrosine, phenylalanine, and leucine, but not cystine or proline, inhibited [125I]monoiodotyrosine countertransport, and leucine inhibition of [3H]tyrosine countertransport and [125I]monoiodotyrosine countertransport yielded virtually identical KI values, 3.5 and 3.2 microM, respectively. Competition studies with monoiodotyrosine analogues showed that system h recognizes a broad range of ligands with an alpha-amino acid configuration at one end and a hydrophobic region at the other. Ring-substituted halogens, regardless of mass or ring position, but not amino, nitro, hydroxy, or methoxy groups, enhanced carrier recognition of system h analogues. It appears that a single system effects the transport of iodinated (e.g. monoiodotyrosine) and noniodinated (e.g. tyrosine) thyroglobulin catabolites into the cytosol for salvage and reutilization by FRTL-5 thyroid cells.

Competition studies with monoiodotyrosine analogues showed that system h recognizes a broad range of ligands with an a-amino acid configuration at one end and a hydrophobic region at the other. Ring-substituted halogens, regardless of mass or ring position, but not amino, nitro, hydroxy, or methoxy groups, enhanced carrier recognition of system h analogues. It appears that a single system effects the transport of iodinated (e.g. monoiodotyrosine) and noniodinated (e.g. tyrosine) thyroglobulin catabolites into the cytosol for salvage and reutilization by FRTL-5 thyroid cells. The proteolysis of thyroglobulin in the lysosome also releases significant amounts of iodine, 11 atoms iodine/m01 thyroglobulin (l), in the form of monoiodotyrosine (MIT)' and diiodotyrosine (DIT). The mono-and diiodotyrosine residues formed after lysosomal degradation of the thyroglobulin are transported to the cytoplasm and deiodinized, and the iodine is reincorporated into newly synthesized thyroglobulin (2). The transport of iodotyrosines into the cytosol has recently been shown to occur by means of a lysosomal carrier for MIT, discovered in rat FRTL-5 thyroid cells (3).
In the present report, we characterize the lysosomal transport of MIT in FRTL-5 cells, demonstrate that it is stimulated by thyrotropin, and provide evidence that MIT is transported by system h, a lysosomal membrane carrier of neutral, hydrophobic amino acids (4,5). We also detail the system's structural requirements for ligand binding, in particular, the influence of a ring halogen upon ligand recognition and transport. TSH, thyrotropin. ml), glycyl-L-histidyl-L-lysine acetate (10 rig/ml), and somatostatin (2 rig/ml) (6,11). Cell growth conditions were 37 "C in 5% CO*, 95% air atmosphere; cells were passaged every 7-10 days with a maximal passage number of 28. Lysosomal Preparations-Lysosomal preparations were obtained as described previously (3). Summarized briefly, FRTL-5 cells were scraped from 75-cm* dishes after incubation in Hanks' balanced salt solution containing 4 mM EGTA for 30 min at 37 "C. After the cells were briefly sonicated (8 s) in 0.25 M sucrose, 10 mM Hepes, pH 7.0, a lysosome-rich granular fraction was produced by a lo-min, 17,000 x g centrifugation of the post-nuclear supernatant. This granular fraction was layered onto 22% Percoll in 0.25 M sucrose, 10 mM Hepes and centrifuged at 40,000 x g for 40 min in a Ti-50 vertical rotor. The lower third of the gradient, containing "heavy lysosomes," was washed twice by centrifugation in 300 and 50 ml of sucrose/Hepes for 10 min at 17,000 x g to remove the Percoll. The final pellet was routinely enriched 3-4-fold in lysosomes, as gauged by /3-hexosaminidase activity, and contained 7-20% of the total cell's hexosaminidase activity.
Small amounts of mitochondria and plasma membranes have been shown to contaminate this "heavy" lysosomal fraction (3). Contaminating organelles or membranes could not affect the outcome of any experiments since all transport results were based on the differential uptake of MIT into loaded and unloaded lysosomes (see "MIT Loading" and "MIT Countertransport" below).
Since only lysosomes contain the hydrolases necessary to achieve MIT loading when exposed to MIT methyl ester, any countertransport observed would be attributable to the lysosome.
MIT Loading-Lysosomes were loaded with MIT by incubating the lysosomal suspension in a tube into which MIT methyl ester in methanol had been added and the methanol evaporated, the amount of MIT methyl ester added was calculated to give a final concentration of 2 mM MIT methyl ester. After incubation at 37 "C for 15 min, the lysosomes were pelleted in a 9,000 x g centrifugation, 4 min, and washed by resuspending the pellet in 3 ml of sucrose/Hepes and centrifugation at 17,000 X g, 10 min. Under these conditions, MIT methyl ester is effectively taken up by the lysosomes and hydrolyzed to MIT in these FRTL-5 lysosomes ( Assay of He~osaminidase Activity-Each incubation mixture was corrected for lysosomal content by assay of hexosaminidase activity, a uniquely lysosomal enzyme, as described previously (12).

RESULTS
In CT experiments performed at 0 to 45 "C, the velocity of MIT CT was shown to increase with increasing temperature, with a Q10 of 1.95 and energy of activation of 11.7 kcal/mol. Velocity of MIT CT was maximal at pH 7.5 and fell off sharply above pH 7.5 ( Fig. 1). All countertransport experiments were performed at pH 7.0 to allow comparison of results with previously published studies performed at pH 7.0. There was, however, little difference in MIT CT between pH 7.5 and 7.0.
The presence in the incubation mixture of NaCl (100 mM) or KC1 (100 mM) inhibited MIT CT, 21 and 17%, respectively. NH&l (20 mM) had no effect on MIT CT, and MgC& (2 mM Linear regression analysis of a Lineweaver-Burk plot revealed an apparent K, for MIT of 1.8 PM and a V,,,,, of 8.5 pmol/unit of hexosaminidase/min (R = 0.99) ( Fig.   2A). The Km of tyrosine CT in tyrosine-loaded lysosomes was 20 pM and the V msX was 3.5 pmol/unit of hexosaminidase/ min (Fig. 2B).
Countertransport of MIT across lysosomal membranes was enhanced 4-g-fold by growth of FRTL-5 cells in thyrotropincontaining medium, compared with MIT CT in lysosomes from thyrotropin-depleted medium (Fig. 3) Therefore, the competing ligand concentrations used were 50 PM against MIT CT and 500 PM against tyrosine CT. The pattern of competition of the amino acids relative to each other was the same for MIT CT as for tyrosine CT (Table I). L-Tyrosine, L-leucine, and L-phenylalanine all competed strongly against both MIT and tyrosine CT, and D-tyrosine competed weakly against both. Valine and methionine each competed to an intermediate degree against both MIT and tyrosine countertransport.
Cystine and proline, which each have separate carriers (12,13), exhibited no competition against either MIT or tyrosine countertransport.
To further substantiate the identity of the system h and MIT carriers, MIT-loaded and nonloaded lysosomes were examined for countertransport of amino acids  (Fig. 4)  Percoll-purified lysosomes were loaded with MIT by incubation in 2 mM MIT methyl ester for 15 min at 37 "C. 0.1 pM ["'I]MIT CT was measured in the presence or absence of 50 pM competing amino acids. Results are expressed as a percent of control CT in which no competing amino acid was present. Differences in the concentration of transport ligands and competing amino acids between MIT and tyrosine CT were necessary because the K,,, for tyrosine is 12 times that for MIT. Results of 8 pM [3HJtyrosine CT are from Bernar et al. (4); effectively identical values of competition for the various ligands were obtained in the present study for MIT CT as was found for tyrosine CT. or (3H]cystine in 0.25 M sucrose, 10 mM Hepes for 5 min at 37 "C. One-ml aliauots were collected and washed in ice-cold phosphate-buffered saline-on GF/B filters. Countertransport was calculated as described under "Experimental Procedures," and expressed as femtomales/unit of hexosaminidase/min. Bars give the standard error of the mean of three of four separate experiments; cystine transport was performed twice. was 3.2 pM and the KI against tyrosine CT was 3.5 /*hi.
We assessed the structural requirements for ligand recognition by the MIT carrier by using various MIT analogues (10 pM) to compete against 0.1 pM ['251]MIT CT (Table II). 3-(p-Hydroxyphenyl)-propionic acid, which resembles tyrosine but lacks an amino group in the o-position to the car-boxy& did not compete against ['*'I] MIT CT, suggesting that the o-amino acid configuration is necessary for ligand recog- MIT-loaded and nonloaded Percoll-purified cell lysosomes were exposed to 0.1 (IM [*2Si]MiT in the presence of IO PM competing ligand for 5 min at 37 "C. Countertransport was calculated as described under "Experimental Procedures." The effect of competing ligands was expressed as a percent of control MIT countertransport with no competing ligand present. Results are the means of duplicate or triplicate determinations in two or three separate experiments. Competing ligand (10 PM nition. Tyramine also failed to compete, suggesting that the oc-carboxyl is also required. Replacing the amino with a hydroxyl (as in 2-hydroxy-3-phenylpropionic acid) or placing the amino in the position fi to the carboxyl (as in 3-amino-3phenylpropionic acid) also resulted in weak competition of MIT CT, compared to phenylalanine.
Addition or removal of a methylene from the aliphatic chain had little effect on competition for MIT CT, ilhrstrated by the relatively similar competition of homophenylalanine and 2-amino-2-phenylacetic acid. The exact configuration of the aliphatic chain is not critical for recognition since structures as disparate as norleucine, isoleucine, leucine, and cyclohexylalanine gave similar competition. Phenylalanine derivatives with substituents on the aromatic ring which confer less hydrophobicity relative to phenylalanine competed less than phenylalanine; the most obvious example was 3,4-dihydroxyphenylalanine.
As noted earlier, the iodine substituent decreased the K, of tyrosine 12-fold but only minimally affected the V,,,,,. Since all halogenated phenylalanines competed to a greater extent than phenylalanine (Table II), the effect of the iodine substituent did not appear to be specific. No effect of halogen size was observed since all halogenated phenylalanines competed approximately to the same degree. meta-, para-, and ortho-substituted fluorophenylalanine competed equally, suggesting that the position of the halogen substituent was not critical for recognition by the carrier.

DISCUSSION
A primary function of lysosomes is to degrade macromolecules to allow salvage of the breakdown products. Transport of these small molecules out of the lysosome makes them available for reutilization via cytosolic pathways. In the past 7 years, lysosomal membrane transport systems have been elucidated for amino acids, carbohydrates, nucleosides and Nacetylhexosamines in several cell types (14, 15). Different lysosomal membrane transport systems for amino acids display different specificities for various ligands. The cystine carrier has been demonstrated in leukocyte (16), fibroblast (17), and lymphoblast (18) lysosomes, and its impairment in cystinosis results in intralysosomal accumulation of cystine in most tissues of the body (19). System c is a carrier for cationic amino acids elucidated in human fibroblast lysosomes (16). Tyrosine and neutral, hydrophobic amino acids are carried by system h in rat thyroid cell lysosomes (4) and transport by this carrier is stimulated by thyrotropin (5). Lysosomal transport systems for small neutral amino acids in human fibroblasts demonstrate some overlap of ligand specificities (13). Proline, for example, is transported by a low affinity system (p) as well as a high affinity system (f).
MIT transport in rat FRTL-5 cell lysosomes displays saturation kinetics and countertransport (3), providing strong evidence that transport was carrier-mediated. We now show that the temperature characteristics of MIT CT are also consistent with carrier-mediated transport, which requires a relatively high energy of activation, and typically have a Qlo of approximately 2.0. In contrast, noncarrier-mediated transport systems, such as membrane channels, have a Qio closer to 1.0 (20). We also demonstrated that MIT transport is cation-independent and does not require ATP or a transmembrane gradient of K+ or protons.
MIT has a high affinity for its carrier as evidenced by an apparent K, of 1.8 PM, approximately 12-fold less than the K,,, of tyrosine for system h by our determination in Percollpurified lysosomes. Tyrosine competes with MIT for uptake into loaded lysosomes (3) and lysosomal MIT transport is stimulated by thyrotropin, a characteristic which has thus far been attributed only to the system h carrier (5). These findings raised the possibility that MIT was transported by the neutral, hydrophobic amino acid carrier, system h. If the two carriers were the same, they should have identical ligand specificities as demonstrated by competition studies. The relative amount of competition of various amino acids against MIT CT was the same as found by Bernar et al. (3)  and leucine CT) supports the identity of the MIT and system h carriers.
The system h carrier recognizes a wide range of compounds, provided they contain the a-amino configuration.
Chemical moieties which decrease the hydrophobicity of the molecule are unfavorable for recognition. The carrier also recognizes a variety of different structures, such as leucine, phenylalanine, and cyclohexylalanine, to a similar degree. This finding is highlighted by the fact that cyclohexylalanine's ring, which is capable of the "boat" and "chair" conformation, is much larger and capable of greater steric hindrance than the planar ring of phenylalanine.
Halogenated phenylalanine derivatives compete with MIT countertransport to a greater degree than phenylalanine. MIT also competes more strongly than tyrosine for MIT CT, and has a higher affinity than tyrosine for system h as judged by a lower Km value of MIT CT. The position of the halogen substitution or halogen size has no significant effect on MIT CT. Previous studies of the competitive effect of DIT, triiodothyronine, and thyroxine on MIT CT showed no significant difference between MIT and DIT in their ability to compete against MIT CT, but triiodothyronine and thyroxine competed much better than MIT or DIT against MIT CT (3). Whether this shows an additive effect of halogens on carrier affinity, or is related to the considerable difference in structures is unclear. The favorable influence of halogens, but not other substitutions, in all ring positions suggests that the carrier possesses a binding site which is specific for the halogen-ring complex. The characteristics of MIT transport suggest that lysoso-ma1 system h is essential for the salvage of iodine in the thyroid cell cytoplasm. Rousset et al. (1) showed that of approximately 42 iodine atoms/molecule of thyroglobulin present in the colloid, 11 iodine atoms/molecule of thyroglobulin remain in mature lysosomes after selective hydrolytic release of triiodothyronine and thyroxine, of which approximately one-third was MIT and two-thirds was DIT. While lysosomal transport of DIT remains to be demonstrated, we have shown that DIT competes strongly against MIT uptake (3). Preliminary experiments in our laboratory suggest that ['251]DIT countertransport can be shown in MIT-loaded lysosomes using low temperatures (20 "C) and short incubation times (30-60 s), perhaps because transport in unloaded lysosomes occurs so quickly that this background uptake must be reduced to demonstrate the effect of loading. System h seems to be responsible for transporting MIT, and possibly DIT, across the lysosomal membrane for iodine salvage.