Lysosomal transport of small molecules

BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

48,

179-193 (1992)

MINIREVIEW Lysosomal Transport of Small Molecules HSU-FANG CHOU, JAYDUTT VADGAMA, AND ADAM J. JONAS Division

of Medical

Genetics,

Department Torrance,

of Pediatrics, Harbor-UCLA California 90502

Medical

Center,

Received June 3, 1992

The lysosomal disassembly of macromolecules involves multiple steps which can be grouped into three phases: (1) uptake of extracellular and intracellular macromolecules into lysosomes, (2) coordinated enzymatic digestion of these materials, and (3) release of the products of digestion from the lysosome. Numerous inherited defects of macromolecular disassembly have been identified which have markedly different sequelae depending upon the nature of the material that is no longer properly degraded. For instance, LDL receptor defects and associated impairment of cholesterol ester metabolism result in atherosclerosis while absence of lysosomal cy-iduronidase activity causes accumulation of heparan sulfate and dermatan sulfate with progressive neurologic and visceral damage. Defects in the third phase of macromolecular disassembly, the transport of small molecules across the lysosomal membrane, have also been recognized. In the two such characterized disorders, cystinosis and sialic acid storage disease, lysosomal storage of small molecules is caused by defects in specific transport systems for cystine (1,2) and acidic sugars (3,4), respectively. A variety of studies suggest that a third condition, cobalamin F disease, is an analogous disorder with lysosomal storage of vitamin Br2 due to defective lysosomal transport of this vitamin (5). These defects have provided the impetus for further studies and the description of a variety of newly recognized lysosomal transport systems. PROTON

TRANSLOCATING

ATPase

The lysosomal ATPase is one of a number of vacuolar ATPases that are responsible for maintaining the acidic interior of a variety of organelles. The lysosomal Mg/ATPase is electrogenic and is strongly inhibited by N-ethylmaleimide and nitrate. Oligomycin, azide, ouabain, and vanadate (68), all of which are strong inhibitors of plasma membrane or mitochondrial Mg/ATPases, have lesser effects on the lysosomal system. The lysosomal ATPase has been well characterized, but whether it has a direct role in the transport of other substances is not clear. Proton translocation occurs 179 08854505192 $5.00 Copyright 8 lm by Academic Press, Inc. All rights of reproduction in any form reserved.

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TABLE 1 Summary of Lysosomal Amino Acid Transport System

Substrate

(c) Cationic

L-lys, arg

(d) Dicarboxylic (e) Small neutral (f) Small neutral

L-glu, asp L-ala, ser, thr L-pro, ala

(p) Small neutral

L-pro, 3,4-dehydrot-pro L-fyr, leu, ileu, try, phe, his, val, met r.-cysteine L-cystine

(h) Large neutral Cysteine tyrosine Cystine

External pH optima

Competitors

KdmM)

L-om, cysteaminecysteine disulfide L-a-aminoadipate L-leu P-Amino acids (<5 carbons long), N-methyl-L-ala, sarcosine -

0.32 (arg)

0.07

n-try, monodotyrosine”

0.02 (phe)

8>5

Cysteamine L-Selenocystine

0.053 0.3-0s

6-7.5 5.5-6.5

0.04-0.12 0.01

7>5 8 > 5.5 (?) at 7.0 6-7

6-7

a Also a substrate, K,,, = 0.0018, pH 7.5 (max uptake).

in the absence of permeant anions in reconstituted lysosomal membranes, suggesting that acidification of the lysosome is not coupled to other ionic species (7). However, Schneider (9) demonstrated that acidification of rat liver lysosomal vesicles by ATP was electroneutral and was inhibited by the anion transport inhibitors, consistent with the cotransport of protons and anions. In these studies, ATP was required for phosphate transport and ATP-dependent acidification of lysosomal vesicles was stimulated by phosphate. Recently, Pisoni (10) incubated human fibroblast lysosomes with radiolabeled ATP and found that radioactivity was accumulated in lysosomes only if [Y-~~P] ATP, but not if [WEEP] ATP was used. These data indicated that hydrolysis of ATP by ATPase provides energy for the proton pump and provides Pi as a substrate for phosphate transport. It has also been suggested that Cl- conductance plays an important role in the regulation of ATP-dependent acidification of vesicles. Entry of Cl- into vesicles increases vesicle acidification, and acidification is strongly inhibited by the Clconductance inhibitor 4,4’-dinitrostilbene-2,2’-disulphonic acid (DNDS) (11-13). It is likely that chloride shunts the interior positive diffusion potential produced by active proton pumping and/or directly stimulates proton pump activity. AMINO ACID TRANSPORT Transport of amino acids across the lysosomal membrane is complex. Thus far, eight discrete carrier-mediated transport systems have been identified and characterized, mainly in lysosomes from human fibroblasts and rat liver. For detailed information regarding each system, readers are referred to previous reviews (1416). The amino acid transport systems include the following: (1) system c for cationic amino acids, (2) system d for dicarboxylic amino acids, (3) systems e, f, and p

LYSOSOMAL

181

TRANSPORT

for small neutral amino acids, (4) system h for large neutral amino acids, (5) a system for cystine, and (6) a system for cysteine. The important features of each system are summarized in Table 1. Based on kinetic analysis and substrate specificity, it has been suggested that lysosomal amino acid transport systems are discrete from analogous plasma membrane systems (17). At present, no information is available regarding the protein structure and molecular biology of the lysosomal transporters. In light of recent advances in the understanding of gene and protein structure of plasma membrane transport systems, the situation regarding lysosomal transport systems is likely to change. 1. System c (Lysine, Arginine) Information related to this transport system came mainly from studies of lysosomal arginine uptake (17) and from trans-stimulation studies of lysine exodus from human fibroblasts (18). Analogue inhibition studies showed that this transporter can carry cationic amino acids with a minimum of five carbon atoms in the side chain and monomethylation of the a-amino groups or a distal quaternary amino group. This system has relatively broader substrate specificity and higher sensitivity to pH (uptake rate was greater at pH 7 than at pH 5) than the plasma membrane y+ cation system. The K,,, for arginine uptake by system c is 0.32 mM, eight times greater than that for the y+ system. MgATP increased lysine efflux but decreased arginine uptake indicating that proton gradient/proton pump has opposite effects on influx and efflux. System c has relatively broad specificity and can accept the mixed disulfide of cysteamine and cysteine as a substrate. This accounts for the ability of cysteamine, a lysosomotropic amine used in the therapy of cystinosis, to deplete cystinotic cells of stored lysosomal cystine. 2. System d (Dicarboxylic

Amino Acid, Glutamate,

and Aspartate)

System d for transport of anionic amino acids was recently characterized in human fibroblast lysosomes (19). This system has a K,,, of 4-12 PM and can recognize glutamate, aspartate, and L-cr-aminoadipate. Anionic amino acid uptake increases as extralysosomal pH increases from 5.5 to 8 although the mechanism for this effect is not understood. 3. Systems e (Alanine, Serine, Threonine), (Proline, minor Route, Dehydroproline)

f (Proline, major Route), and p for Small Neutral Amino Acids

At least three separate routes for the passage of small neutral amino acids have been elucidated in human fibroblast lysosomes (20). System e, the major route for the transport of alanine, serine, and threonine, is strongly inhibited by leucine, but not by proline or other small neutral amino acids with a secondary amino in a-amino group (sarcosine, cw-N-methyl-L-alanine). System f, which is a minor route for alanine, serine, and threonine transport, turns out to be the major route for proline transport. Proline transport is inhibited by small neutral amino acids with short (up to three carbon atoms) unbranched chains such as alanine. Kinetic studies of lysosomal proline uptake indicate that at least two pathways are involved with K, values of 0.01 and 0.07 mM. The higher affinity system f accounts for 60-70% of total proline uptake. The lower affinity transport responsible for the

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remaining 25% of proline uptake was designated as system p. This system is highly specific for proline and dehydroproline transport and is not inhibited by alanine. 4. System h (Large Neutral, Hydrophobic Isoleucine, Tryptophan, Phenylalanine,

Amino Acids: Tyrosine, Leucine, Histidine, Valine, Methionine)

Studies of lysosomal tyrosine uptake in thyroid epithelial cells (FRTLJ) led Bernar et al. (21) to identify system h’ for the transport of large, neutral amino acids. This system h recognizes L-forms of tyrosine, leucine, isoleucine, tryptophan, phenylalanine, and histidine, and also weakly recognizes valine and methionine. This system is not sensitive to MgATP or N-ethylmaleimide and has a K,,, of 20 PM for tyrosine countertransport. The same group of investigators further demonstrated that monoiodotyrosine (MIT), a metabolite of thyroglobulin degradation in FRTL-5 cells, can also be transported by system h (22,23). The K,,, of MIT uptake was 1.8 PM, about lofold less than that for tyrosine uptake. Countertransport of MIT by system h was evident by the fact that preloading of lysosomes with MIT resulted in transstimulation of uptake of tyrosine, leucine, phenylalanine, and MIT. Rapid efflux of tyrosine (21) and MIT (23) from FRTL-5 cell lysosomes was also demonstrated. Transport of tyrosine and MIT were enhanced several fold when 0.1 nM TSH was present in FRTLJ cell incubation medium for 2 days. TSH also stimulated transport of phenylalanine and leucine in thyroid cell lysosomes probably via a mechanism which requires cyclic AMP and protein synthesis (22,23). Thus, the lysosomal system h may be part of the complex regulatory system for the synthesis and degradation of thyroid hormone and thyroglobulin. A relatively low level of lysosomal uptake of large neutral amino acid has been demonstrated in fetal human skin fibroblasts (24). Kinetic studies revealed a nonsaturable pathway at high concentrations of substrates (mM), and a saturable uptake at lower concentrations with a K,,, of 5 to 15 PM (for phenylalanine), similar to that found in FRTL-5 cells. This suggests that multiple routes for the transport of large neutral amino acids are present in fibroblast lysosomes. In contrast to the case in thyroid cells, the fibroblast lysosomal system h appears to lack stereospecificity. 5. Cystine Transport Lysosomal cystine transport has been studied more extensively than any other amino acid transport system. In fact, recognition that lysosomal cystine transport is defective in the human inherited disorders in cystinosis served as an impetus for the study of other lysosomal transport systems (1,2). Cystine transport has been studied in a number of cells including human fibroblasts, lymphoblasts, leukocytes, mouse fibroblast, rat liver cells, and FRTL-5 thyroid cells. Carrier-mediated countertransport of cystine has been characterized in lysosomes of human leukocytes (1,25), FRTL-5 cells (21), and mouse fibroblasts (26). Preloading of lysosomes with cystine results in trans-stimulation of uptake of “Scystine in these cells. The system recognizes L-isomers of cystine, selenocystine and to a lesser extent, cystathionine, cystamine, leucine, and cysteamine-cysteine mixed disulfide. In human leukocytes, optimal pH for cystine uptake is between

LYSOSOMAL

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183

pH 5.5 and 6.5 (27). At pH 7.0, lysosomes of human leukocytes (25) and mouse fibroblasts (26) have a K,,, of 0.3-0.5 mu for cystine. Exodus of cystine from lysosomes occurs with a tllz of 25-45 min at 37°C and pH 7.0. MgATP stimulates cystine efflux (1,2,27,28) and this effect is probably due to proton-pump-mediated acidification of the lysosome, the effect of divalent ions (27) or both. In rat liver lysosomes, polyamines can substitute for the stimulatory effect of divalent cations on cystine transport (29). Entry of K+ into lysosomes results in cystine efflux from human leukocyte lysosomes (30) suggesting a possible effect of membrane potential on cystine release. The effect of membrane potential on cystine transport has been confirmed by Smith et al. (31). A K+ effect was found in leukocytes of cystinotic patients, suggesting the existence of an additional transport system which is different from the one missing (or abnormal) in the cystinotic patient (30). In fact, in mouse fibroblast lysosomes, an unsaturable pathway, in addition to the carrier-mediated system, was observed for cystine transport (26). Lemons et al. (32) also found a route of cystine efflux from several cystinotic cell lines which was sensitive to temperature change from 37” to 43°C. 6. Cysteine Transport System A single, highly specific transport system for L-cysteine has been demonstrated in human fibroblast lysosomes (33). This system has a K,,, of 0.053 mM, and is so specific that only the decarboxylated analog of cysteine, cysteamine, can strongly inhibit cysteine uptake. Serine, alanine, and homocysteine are weak inhibitors of cysteine transport. This system is highly pH dependent with maximal uptake observed at pH 7.5. Very little uptake occurs at acidic pH (4.6-6.0) suggesting that in vivo, the environment favors cysteine uptake rather than release by lysosomes. It has been suggested that cysteine release from lysosomes may use the systems e and f as described above. Cysteine uptake by lysosomes provides a thiol source for certain proteinases and allows for reduction of protein disulfide bridges during proteolysis. This system is also important in transporting cysteamine into the lysosome. TAURINE

TRANSPORT

Taurine (2-aminoethanethanesulfonic acid) is a sulfur amino acid derivative which has several biochemical and physiological functions. It is known that in most cells intracellular taurine concentrations are greater than those in plasma, suggesting that some intracellular mechanisms must exist to regulate osmotic effects caused by high intracellular taurine concentrations. Indeed, recently, Vadgama et al. (34) have demonstrated a distinct lysosomal transport system for taurine influx and exodus. Kinetic studies of taurine uptake into rat liver lysosomes revealed a biphasic uptake with K,,, values of 30.9 and 198 mM, and V,, values of 1.2 and 12.5 pmol/min/unit of hexosaminidase, respectively. Taurine uptake is independent of Naf concentration characteristic of lysosomal amino acid transport systems. The uptake of taurine has a pH optimum of 6.5. It is stimulated by the K+ ionophore valinomycin, but decreased by 2 mM MgATP suggesting that K+ gra-

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dients and acidification of the lysosomal interior inhibit taurine uptake. Taurine effhrx from lysosomes is rapid with a t rj2 of 5 min at 37°C. Uptake of taurine is also rapid with an overshoot between 5 and 10 min. These fairly rapid fluxes of taurine across the lysosomal membrane may help regulate intracellular osmotic changes caused by taurine influx from the cell membrane. Whether taurine has any function in the lysosome remains unclear. Analog inhibition studies indicate that p-alanine and hypotaurine share the same transport system as taurine in lysosomes while taurine is not a substrate for neutral amino acid transport systems in human fibroblast lysosomes (20). The lysosomal taurine carrier appears to be discrete from the p-alanine carrier found in plasma membranes. This latter system is Na+ dependent and has a smaller K,,, than that for the lysosomal system. SUGAR TRANSPORT Lysosomal degradation of glycoproteins, glycosaminoglycans, glycosphingolipids, and glycogen leads to the production of a wide array of monosaccharides. Similar to the case for amino acids, accumulation of these sugars would cause a state of osmotic imbalance and possible secondary inhibition of lysosomal processes. Three transport systems that have been identified in rat liver lysosomes that account for the efflux of a wide array of sugars are: one for acidic sugars such as sialic and glucuronic acids (35), one for the acetylated sugars N-acetylglucosamine and N-acetylgalactosamine (36,37), and one for neutral D-hexoses such as glucose, galactose, and mannose (38,39). Studies of countertransport have helped to delineate the substrate specificity of these systems. Analogous to the case for lysosomal cystine transport, human disease has been attributed to the defective transport of acidic sugars. It is possible that defects in the other systems exist as unrecognized causes of human disease. For instance, accumulation of lysosomal free glucose due to defective transport could cause lysosomal glycogen storage by secondary inhibition of cw-glucosidase. 1. Acidic System Of the sugar transport systems, only the acidic sugar system has been convincingly demonstrated to be dependent upon the lysosomal proton gradient. This system is highly specific for a variety of acidic sugars including gluconate, glucaronate, galactonate, and galactuonate. It has a Kd of 250 PM for sialic acid and exhibits countertransport (35). A similar transport system has also been identified in lysosomes from human fibroblasts (4). Inherited dysfunction of the human transport system results in the related disorders, Salla disease and infantile sialic acid storage disease which are both characterized by the lysosomal storage of sialic acid (3,4,40,42). The infantile form is more severe and correlates with approximately lo-fold increased lysosomal storage of sialic acid over that observed for Salla disease. Since a variety of acidic sugars are substrates for the acidic sugar carrier, it is not surprising that lysosomal storage of glucuronic acid is also a feature of these disorders.

LYSOSOMAL

TRANSPORT

185

2. Acetylated System The acetylated sugar transport system has a K,,, of approximately 4.4 mM for its two primary substrates, N-acetylglucosamine and N-acetylgalactosamine. In contrast to findings with the acidic transport system, the acetylated system does not appear to be particularly responsive to pH and is not stimulated by Mg/ATP. It exhibits countertransport and can be inhibited by cytochalasin B (36,37). Additional studies using membrane vesicles derived from rat liver lysosomes have revealed that orientation of the acetyl group is an important feature for substrate recognition. Phosphorylation or sulfation impairs substrate recognition suggesting the importance of charge. Interestingly, transport by membrane vesicles is not nearly as sensitive to inhibition by cytochalasin B as is transport by intact lysosomes. This may reflect the random membrane orientation of the membrane vesicles or the destruction of a cytochalasin B binding site. 3. Neutral System A number of investigators using osmotic protection studies have suggested that lysosomes exhibit stereospecific transport of neutral hexoses that is inhibited by cytochalasin B (43-45). This system has now been further characterized using more direct methods. Using membrane vesicle preparations, Mancini et al. (38) have demonstrated that o-glucose uptake has a K,,, of 75 mM at pH 7.4 and that the system demonstrates countertransport properties when vesicles are preloaded with high concentrations of substrate (100 mM). Jonas et al. (39) have obtained similar results using intact rat liver lysosomes. However, they were unable to demonstrate counter-transport using intact lysosomes that were loaded with high concentrations of substrate by equilibration. They reported a K,,, of 25 mM for the uptake of b-glucose and 75 mM for L-fucose. Both groups found that many neutral hexoses competed for transport and that transport was not dependent upon proton gradients. n-ribose did not appear to be a primary substrate for this system (46). VITAMIN

TRANSPORT

Vitamin B12 A number of complex extracellular and intracellular steps are required for the human metabolism of vitamins. These steps include absorption across the gastrointestinal tract, circulation, delivery to cells, and intracellular conversion to forms that are useful as enzyme cofactors. In the case of vitamin Br2, newly absorbed cobalamin appears in the circulation as a complex with the protein, transcobalamin II. This complex is taken up intact by endocytosis but dissociates before Bi2 is converted via a series of steps into the enzyme cofactors methylcobalamin and adenosylcobalamin. Dissociation of the B,,/transcobalamin II protein complex apparently takes place in lysosomes where transcobalamin II is degraded to amino acids (47,48). It has been suggested that in some tissues, such as kidney, lysosomes are reservoirs of free Bi2 (49). Clearly, in order to allow for further metabolism, free B12 must be released to the cytoplasm. Uptake of [57Co]cyanocobalamin by rat liver lysosomal membrane vesicles (in-

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JONAS

ternal pH of 7.0) is highly dependent upon extravesicular pH with much greater uptake under more acidic conditions (50). This is consistent with in vivo conditions where free Bn is released to the cytoplasm from the acidic interior of lysosomes. The Br2 carrier exhibits the property of countertransport. Studies with the BIz metabolites adenosylcobalamin and methylcobalamin revealed that when on the same side of the membrane these compounds inhibit uptake of Bi2. However, when on the opposite side of the membrane they exchange with B,, and thus can serve as substrates for transport. These derivatives are less effective at promoting countertransport than cyanocobalamin and thus do not appear to be preferred substrates for transport. Cobainamide dicyanide, an analog lacking the normal nucleotide component, does not support countertransport although it does inhibit uptake of Bi2. In contrast, the nucleotide adenosine neither inhibits uptake nor supports countertransport. These observations are consistent with a carrier recognition site for the corrin ring moiety of Bi2. Interestingly, individuals have been identified with abnormalities in the metabolism of both methyl malonate and homocystine associated with lysosomal storage of free Bi2 (5152). The lysosomal localization of Blz in this disorder has recently been confirmed by autoradiography (51). Based on complementation studies involving other disorders of methylmalonate metabolism, this condition has been classified as cobalamin F disease. Presumably defective transport of free Bi2 across the lysosomal membrane results in secondary deficiency of the Bi2 enzyme cofactors and disturbed intermediary metabolism. BlOTiN Studies of pyruvate carboxylase indicate that biotinylated proteins are degraded in the lysosome (53). Whether free biotin, the lysine/biotin complex biocytin, or small biotinylated peptides exit the lysosome is currently unknown. Recent preliminary evidence suggests that lysosomal membrane vesicles exhibit pH and divalent cation-dependent, saturable transport of free biotin (54). However, biotinidase is presumably required for generation of free biotin from biotinylated proteins and this enzyme does not appear to be concentrated within lysosomes (55). The role of lysosomes in biotin metabolism still remains to be resolved. FOLATE DERIVATIVES TRANSPORT In cytoplasm, folic acid is usually converted to folypolyglutamates (FPGs) by adding more glutamate groups to the terminal glutamyl moiety of the vitamin. It is known that inside the lysosome, an enzyme called FPG hydrolase can hydrolyze FPGs presumably after uptake by the lysosomes. Recently, a lysosomal transport system for FPGs has been described in S180 tumor cells by using methotrexate (MTX) + Gl as the substrate (41). MTX is a folic acid analog and a highly effective antitumor agent which is also converted to polyglutamates (MTX PGs) in cytoplasm. In order to measure MTX PGs transport, an incubation mixture was used containing no sulfhydryl, which is required for FPG hydrolase activity. Uptake of [3H]MTX + Gl by lysosomes exhibits saturation kinetics with a K, of 346 PM and V,,,, of 2.8 pmol/min/unit of P-hexosaminidase activity. However, at higher substrate concentrations, a nonsaturable component became apparent,

LYSOSOMAL

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187

suggesting a passive diffusion pathway may be involved in addition to the carriermediated transport. Uptake of MTX + Gl was enhanced by higher temperature (37°C) and stimulated by di- or monocations especially by magnesium and/or potassium. The presence of these two cations in the medium increased uptake by sevenfold. The mechanisms involved in magnesium- and potassium-stimulated transport are not clear. It is very interesting to note that transport of MTX + Gl is competitively inhibited by longer chain polyglutamates with increasing effectiveness as the number of glutamates increased from one to four. This increased affinity of MTX PGs of increasing chain length differs from the case for plasma membrane where permeability decreases with increasing chain length (56). CALCIUM

TRANSPORT

An ATP-dependent calcium pump with a high affinity for Ca*+ (K,,, = 107 nM) was first described in human neutrophil lysosomes (57). Transport is optimal at 37°C and pH 7.0-7.5. Uptake of Ca*’ by neutrophil lysosomes is not affected by azide and antimycin, but is inhibited by chemotactic peptide (FMLP) which activates polymorphonuclear leukocytes. Another distinct calcium transport system was described in human fibroblast lysosomes (58). Uptake of Ca*+ is a carrier-mediated process with a K,,, of 5.7 mM and V,,,, of 10 pmol/min/unit hexosaminidase. Ca*+ uptake increases as extra lysosomal pH increases from 5.0 to 8.5. Ca*+ influx is strongly inhibited by divalent cations in the periodic table group IIa and IIb with Cd*’ > Hg*+ > Zn*+ > Mg*+ > Ba*+ > S?‘. Mono- and trivalent cations have no effect on Ca*+ transport. ATP at a concentration of 1 mM inhibits Ca*+ uptake by 80%, which is strikingly different from the ATP-dependent Ca pump found in neutrophil lysosomes. Chloroquine and L-cystine also inhibit Ca*+ uptake while cysteamine, N-ethylmaleimide, and the anions Cl-, sulfate, and acetate have no effect on uptake. Inositol triphosphate, a potent stimulus for calcium uptake in several subcellular organelles, does not affect lysosomal Ca*+ transport. Ca*+ released by lysosomes is thought to use the same transport system. Maintenance of a constant low cytosolic Ca” is essential for normal cellular function. Calcium transport across plasma membrane, mitochondria, and endoplasmic reticulum membranes is known to play an important role in intracellular calcium homeostasis. It is possible that lysosomal Ca*+ transport is also important for regulation of cytosolic Ca*+ concentrations especially in tissues which have few mitochondria. Lysosomal Ca* + transport is probably involved in intestinal Ca*’ absorption. Despite a great deal of work, the mechanisms involved in vitamin D-dependent intestinal Ca*+ absorption are still unclear. One postulated mechanism suggests that Ca*+ first moves across the brush border in endocytic vesicles, which then fuse with lysosomes, and finally deliver the cation to the basal lateral membrane by exocytosis. Evidence to support this theory has been provided (59). With in vivo ligated loop technique, 45Ca transport and consequent subcellular localization of 45Ca were studied in vitamin D-deficient and -treated chicks. Among the subcellular organelles examined, lysosomes had the highest level of 45Ca activity and

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this 45Cauptake was sensitive to 1,25(OH)* D3 treatment. Lysosomes also contained the greatest level of calbindin-D28K (calcium-binding protein). Chloroquine which inhibited lysosomal function also inhibited intestinal 45Caabsorption. These data suggestthat lysosomal Ca*+ transport is required for 1,25(OH)* D,-mediated intestinal calcium transport. PHOSPHATE

TRANSPORT

A thorough description of a lysosomal phosphate transport system has recently been reported in human fibroblasts (10). This system exhibits saturable kinetics with a K,,,of 5 PM at pH 7.0 and 37°C. Inhibitory studies revealed that this system is highly specific. Uptake of phosphate was competitively inhibited by arsenate, the phosphate analog, and noncompetitively inhibited by pyridoxal phosphate, glucosed-phosphate, MgATP, Na2CTP, and the nonhydrolyzable ATP analog, AMP-PNP. Maximum uptake of phosphate occurred at acidic pH (4.5 and 5.5) and only the monobasic form of phosphate was recognized by this route. The phosphate transport systemin human fibroblast lysosomesis different from that in mitochondrial membranes or rat liver lysosomes in various aspects, such as K,,, (60) or sensitivity to inhibitors (9). Efflux of phosphate is difficult to demonstrate in intact lysosomes because phosphate that accumulates in lysosomesis quickly converted into polyphosphate. This high molecular weight polyphosphate consists of long chains of inorganic phosphate ranging from 100 to 600 phosphate residues in length has recently been characterized in human fibroblasts (61). High molecular weight 32P-labeledlysosomal material can be partially hydrolyzed under alkaline conditions, but is resistant to degradation by proteinase K, ribonuclease, and deoxyribonuclease. When incubated with 1 N HCl at lOO”C, this material is converted into orthophosphate. The biological significance of the polyphosphate material in lysosomes is, however, not clear at the present time. SULFATE TRANSPORT

At least seven sulfatases are responsible for the generation of free sulfate from more complex structures within lysosomes. Sulfate appears to be the primary substrate for a lysosomal anion transporter that has some functional analogy to the erythrocyte band 3 anion transporter (62). The sulfate transporter has a K, of 150 PM and exhibits the properties of saturation kinetics, pH dependence, temperature dependence, and counter-transport. Under specified conditions of pH this systemis sensitive to changes in membrane potential. Evidence suggeststhat this system is sensitive to proton titration and possibly may promote the electroneutral exchange of sulfate and a proton for chloride. The systemdoes not appear to carry phosphate or bicarbonate, although it can carry molybdate. Both the lysosomal and band 3 anion transport systemsare inhibited by impermeant site blockers such as DIDS and phenylglyoxal, translocation inhibitors such as DNFB and niflumic acid, and channel blockers such as cyclohexanedione. Interestingly, while the band 3 transporter is sensitive to dipyridamole, the lysosomal transporter is not. This is the only difference in patterns of inhibition that has yet been noted between the two transporters (63).

LYSOSOMAL

CHLORIDE

TRANSPORT

189

TRANSPORT

Chloride conductance has been shown to be important for control of vesicular pH. The existence of a Cl- channel or Cl- transporter has been suggested, however, detailed characteristics of the lysosomal Cl- transporter are not yet available. Recently, it was shown that in cystic fibrosis cells, lysosomal Cl- conductance and acidification are abnormal (64). With an in vivo labeling technique, Cl- influx in endocytic vesicles from proximal tubules was inhibited by the stilbene inhibitor (DNDS) but unchanged by institution of a transmembrane Na+ or HC03/pH gradient. Chloride conductance was also shown to be activated by a cyclic AMP-dependent protein kinase (11). NUCLEOSIDE

TRANSPORT

Lysosomes play an important role in controlling cytoplasmic RNA turnover, simply by enzymatic degradation of nucleic acids to nucleosides and inorganic phosphate (65). Pisoni and Thoene (66) have demonstrated carrier-mediated transport for passage of nucleosides across the lysosomal membrane in human fibroblasts. Transport of adenosine into lysosomes is a saturable process, with a K,,, of 9 mM, at pH 7.0, 37°C and a V,,,,, of 21 pmol/min/unit of hexosaminidase. Rapid efflux of nucleosides from human fibroblast lysosomes was also demonstrated with the half-time of 6, 7.5, and 7 min for uridine, inosine, and adenosine, respectively. This system recognizes both purine and pyrimidine nucleosides but has a lower affinity for pyrimidine nucleosides. Recognition appears to be determined by the base portion, but not the sugar moiety of nucleosides. The extraordinarily high K,,, and transport capacity of this system allow it to handle the large quantities of nucleosides generated by the disassembly of RNA. Transport is strongly inhibited by the nucleoside analog, nitrobenzylthioinosine. The same investigators (66) have also reported a nonsaturable passage for lysosomal transport of nucleobases such as adenine, which perhaps is distinct from that for nucleoside transport. PEPTIDE TRANSPORT Peptide transport across the lysosomal membrane has been studied mainly by the method of osmotic protection. With this method, the activity of a soluble lysosomal enzyme (usually N-acetyl$-n-glucosaminidase) is measured at various time intervals (0, 30, 60 min) after incubation of the lysosomes with a particular solute. If the solute is permeant, the entry of the solute gradually causes osmotic imbalance and may lead to lysosomal rupture with release of enzyme into the isotonic buffer. The rate of solute entry can be determined based on the rate of enzyme release, generally referred to as the loss of lysosomal latency. Using this method, Lloyd (67) showed, 20 years ago, that dipeptides are transported across the rat liver lysosome at faster rates than amino acids or tripeptides at neutral pH. In 1985, Mego (68) provided direct evidence to support Lloyd’s finding that dipeptides can cross lysosomal membranes. Investigation of the stimulatory effects of glutathione on proteolysis in mouse kidney lysosomes revealed

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that cysteinyl-glycine was transported into lysosomes. This degradation product rather than glutathione itself exerted a stimulatory effect on proteolysis activity. Recently, Bird and Lloyd (69) reexamined transport of a series of dipeptides and their analogs in rat liver lysosomes.They found that L-peptides are taken up faster than n-peptides, suggesting that the lysosomal peptide transport is stereospecific. Their observation does not exclude movement through a pathway by simple diffusion. Peptides were categorized into three groups by rate of movement: slow (Gly-Asn, Gly-Val), medium (Gly-Ala, Gly-Leu, Gly-Ser, Gly-Thr), and rapid (Ala-Ala, Ala-Gly, Gly-Gly). Direct measurement of uptake or release of labeled dipeptides across the lysosomal membrane may be necessaryto clarify and characterize the transport system for dipeptides. CONCLUSION

Transport of small molecular weight materials is a critical function of the lysosomal membrane. It is now clear that a variety of transport systemswork in concert to regulate osmotic balance and allow for reutilization of materials derived from macromolecular disassembly. While it appears that, in general, materials are moved from the interior of the lysosome to the cytoplasm, some of the transport systemsmediate the import of materials required to maintain the lysosomal environment. It is conceivable that lysosomesmay serve as storage depots for certain metabolites. Little is known regarding the regulation of lysosomal transport. However, it would not be surprising if the lysosomal systems are regulated in concert with plasma membrane transport systemsto provide cells with critical nutrients. Studies of regulation will be greatly facilitated when the genes for lysosomes transport systemsare identified. Gene identification will be an important area of study for a number of other issues including the topic of human disease. At present, defects in the lysosomal transport of small molecules have been associated with three inherited human disorders: cystinosis, sialic acid storage disease, and cobalamin F disease. There is no reason to assume that this list of disorders is all inclusive. It is entirely possible that additional disorders will be identified that are related to dysfunction of these systems. REFERENCES 1. Gahl WA, Bashan N, Tietze F, Bemardini I, Schulman JD. Cystine transport is defective in isolated leukocyte lysosomes from patients with cystinosis. Science 217:X263-1265, 1982. 2. Jonas AJ, Smith ML, Schneider JA. ATP-dependent lysosomal cystine effluz is defective in cystinosis. J Biol Chem 257:13185-13188, 1982. 3. Renhmd M, Tietze F, Gahl WA. Defective siahc acid egress from isolated fibroblast lysosomes of patients with Salla disease. Science 2X&759-762, 1986. 4. Mancini GMS, Beerens EMT, Aula PP, Verheijen FW. Sialic acid storage diseases: A multiple lysosomal transport defect for acidic monosaccharides. I Clin Invest 87:1329-1335, 1991. 5. Rosenblatt DS, Hosack A, Matiaezuk NV, Cooper BA, Laframboise R. Defect in vitamin B1, release from lysosomes: Newly described inborn error of vitamin Blz metabolism. Science Z&13191321, 1985. 6. Harikumar P, Reeves JP. The lysosomal proton pump is electrogenic. I Biol Chem 25&1040310410, 1983.

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