Lysosomal cystine transport. Effect of intralysosomal pH and membrane potential

The regulation of lysosomal cystine transport was studied using cystine dimethyl ester-loaded lysosomes isolated from human diploid fibroblasts. Net efflux from normal fibroblast lysosomes was compared to that from lysosomes of cystinotic fibroblasts, which contain an inherited mutation decreasing lysosomal cystine transport. This exodus of cystine from normal fibroblast lysosomes was greater than from cystinotic fibroblast lysosomes. When lysosomes were incubated with both 5 mM MgCl2 and 2 mM ATP (Mg/ATP), the amount of lysosomal cystine lost from normal lysosomes doubled, but the amount of cystine lost from cystinotic lysosomes remained small. This effect of Mg/ATP on cystine loss from lysosomes isolated from normal fibroblasts was abolished when either carbonyl cyanide m-chlorophenylhydrazone or N-ethylmaleimide was present, suggesting that the effect of Mg/ATP was mediated by the action of a lysosomal proton-translocating ATPase. Addition of KCl, RbCl, or NaCl to normal lysosomes caused smaller increases in cystine exodus. A variety of experimental conditions altered lysosomal pH, membrane potential, and the cystine lost from normal fibroblast lysosomes. These same experimental conditions produced similar alterations in the lysosomal pH and membrane potential of cystinotic fibroblast lysosomes without a comparable alteration in cystine loss. These results have led us to propose a model in which the transport of cystine out of the normal lysosome is regulated by both the lysosomal membrane potential gradient and the transmembrane pH gradient.     

Proton-translocating ATPase and lysosomal cystine transport

A proton-translocating ATPase was identified in highly purified lysosomes from Epstein-Barr virus-transformed human lymphoblasts. Activity of this ATPase caused acidification of highly purified, fluorescein isothiocyanate dextran-loaded lysosomes and correlated with the ATP-dependent efflux of lysosomal cystine. The lysosomal ATPase was distinct from mitochondrial F1-ATPase in its responses to a variety of inhibitors. Although ATP-dependent lysosomal cystine efflux is not demonstrable in cultured lymphoblasts from individuals with nephropathic cystinosis, ATPase activity and acidification in lysosomes from these cells is comparable to that in noncystinotic lysosomes. ATPase activity in lymphoblasts from normal individuals was 543 +/- 79 nmol/mg/min while in lymphoblasts from cystinotic individuals this activity was 541 +/- 25 nmol/mg/min. ATP-dependent acidification of lysosomes from normals was -0.5 +/- 0.1 pH units compared to -0.5 +/- 0.1 pH units in cystinotic lysosomes. Activity of the lysosomal proton-translocating ATPase is a necessary, but not sufficient, condition for lysosomal cystine efflux.     

Impaired clearance of free cystine from lysosome-enriched granular fractions of I-cell-disease fibroblasts.

Cultured fibroblasts from patients with I-cell disease (mucolipidosis II) accumulate excessive amounts of free cystine, similarly to cells from patients with nephropathic cystinosis, a disorder of lysosomal cystine transport. To clarify whether the intralysosomal accumulation of cystine in I-cell-disease fibroblasts was due to a defective disposal mechanism, we measured the rates of clearance of free [35S]cystine from intact normal, cystinotic and I-cell-disease fibroblasts. Loss of radioactivity from the two mutant cell types occurred slowly (t 1/2 = 500 min) compared with the rapid loss from normal cells (t 1/2 = 40 min). Lysosome-rich granular fractions isolated from three different cystine-loaded normal, cystinotic and I-cell-disease fibroblast strains were similarly examined for non-radioactive cystine egress. Normal granular fractions lost cystine rapidly (mean t 1/2 = 43 min), whereas cystinotic granular fractions did not lose any cystine (mean t 1/2 = infinity). I-cell-disease granular fractions displayed prolonged half-times for cystine disposal (mean = 108 min), suggesting that I-cell-disease fibroblasts, like cystinotic cells, possess a defective carrier mechanism for cystine transport.     

The effect of chloroquine on the metabolism of [35S]cystine in normal and cystinotic human skin fibroblasts.

The present study concerns the effect of the lysosomotropic drug chloroquine on the uptake and metabolism of [35S]cystine in vitro by normal human fibroblasts and those from patients suffering from the lysosomal storage disease cystinosis. When the cells were cultured with [35S]cystine for periods in excess of 4 h, it was found that chloroquine considerably increased (up to 30-fold) the labelling of the intracellular cystine pool in cystinotic cells, with no increase or a much smaller increase in normal cells. For this effect chloroquine had an optimum concentration of 20 microM, with a small effect still being noticeable at 1 microM. A quinoline analogue, 4-(dimethylaminoethylamino)-7-iodoquinoline, had a similar effect to chloroquine. However, NH4Cl at concentrations of between 100 microM and 50 mM showed either no effect (at the lower concentrations) or a depression of intracellular cystine labelling (at the higher concentrations). The differences between the effects of the quinolines on cystinotic acid normal cells were not due to differences in total cell uptake of drug.     

Upregulation of the Rab27a-Dependent Trafficking and Secretory Mechanisms Improves Lysosomal Transport,Alleviates Endoplasmic Reticulum Stress,and Reduces Lysosome Overload in Cystinosis

Cystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes cystinosin, the lysosomal cystine transporter. Although many cellular dysfunctions have been described in cystinosis, the mechanisms leading to these defects are not well understood. Here, we show that increased lysosomal overload induced by accumulated cystine leads to cellular abnormalities, including vesicular transport defects and increased endoplasmic reticulum (ER) stress, and that correction of lysosomal transport improves cellular function in cystinosis. We found that Rab27a was expressed in proximal tubular cells (PTCs) and partially colocalized with the lysosomal marker LAMP-1. The expression of Rab27a but not other small GTPases, including Rab3 and Rab7, was downregulated in kidneys from Ctns^−/− mice and in human PTCs from cystinotic patients. Using total internal reflection fluorescence microscopy, we found that lysosomal transport is impaired in Ctns^−/− cells. Ctns^−/− cells showed significant ER expansion and a marked increase in the unfolded protein response-induced chaperones Grp78 and Grp94. Upregulation of the Rab27a-dependent vesicular trafficking mechanisms rescued the defective lysosomal transport phenotype and reduced ER stress in cystinotic cells. Importantly, reconstitution of lysosomal transport mediated by Rab27a led to decreased lysosomal overload, manifested as reduced cystine cellular content. Our data suggest that upregulation of the Rab27a-dependent lysosomal trafficking and secretory pathways contributes to the correction of some of the cellular defects induced by lysosomal overload in cystinosis, including ER stress.     

Renal cell culture using autopsy material from children with cystinosis

Renal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr. Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coon's modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE1, and fibronectin. These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive. Both tight junctions and microvilli were seen by electron microscopy. Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coon's F12 with 10% fetal bovine serum. The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients. Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine. However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine. Study of cultured renal material may have practical significance in pharmacologic considerations.     

Renal cell culture using autopsy material from children with cystinosis

Summary Renal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr. Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coon's modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE₁, and fibronectin. These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive. Both tight junctions and microvilli were seen by electron microscopy. Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coon's F12 with 10% fetal bovine serum. The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients. Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine. However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine. Study of cultured renal material may have practical significance in pharmacologic considerations. This work was supported by Grants AM 01074-01, AM 18434, and GM 17702 from the National Institutes of Health, Bethesda, MD.     

Disorders of lysosomal membrane transport--cystinosis and Salla disease

Two lysosomal storage diseases are now known to result from impaired transport of small molecules across the lysosomal membrane. In cystinosis, the disulfide amino acid, cystine, accumulates and in free sialic acid storage disorders, N-acetylneuraminic acid is stored. The lysosomal cystine carrier exhibits saturability, counter-transport, temperature dependence, and stereospecificity; it is highly specific for molecules resembling cystine. Less is known about sialic acid transport, but its temperature dependence and deficiency in certain autosomal-recessive human mutations strongly suggests that it is a carrier-mediated process. Cystine and sialic acid serve as prototypes for amino acids and sugars transported by specific lysosomal membrane carriers, whose impairment results in lysosomal storage disorders.     

pH effects on cystine transport in lysosome-rich leucocyte granular fractions.

Inhibitors of lysosomal acidification (4,4'-di-isothiocyanostilbene-2,2'-disulphonate, NN'-dicyclohexylcarbodi-imide, carbonyl cyanide m-chlorophenylhydrazone, NH4Cl and methylamine hydrochloride) did not alter cystine egress or countertransport in polymorphonuclear-leucocyte lysosome-rich granular fractions at pH 7.0. Together, 2 mM-MgCl2/MgATP and 90 mM-KCl stimulated cystine egress 2-fold, but this effect also was not influenced by inhibitors of ATP-dependent lysosomal acidification. MgCl2/MgATP stimulated cystine transport at pH 5.5, but the effect also occurred with MgCl2, MgSO4 or MnCl2 alone, was prevented by chelation, and was not seen with NaATP; therefore, it was considered a bivalent-cation, not an ATP, effect. Proton-pump-mediated acidification of lysosomes does not appear to be required for cystine transport in normal polymorphonuclear-leucocyte granular fractions, as reported for lymphoblast lysosomes.     

Polyamines stimulate lysosomal cystine transport

Lysosomal cystine transport is a carrier-dependent process that, in isolated lysosomes, is stimulated by proton gradients, membrane potential, and millimolar concentrations of divalent cations. The importance of these regulatory factors in vivo is not well established. Polyamines were found to stimulate cystine transport in Percoll gradient purified rat liver lysosomes with spermidine greater than putrescine = cadaverine greater than spermine in order of effectiveness. Maximal stimulation was achieved with 500 microM spermidine. The effects of optimal concentrations of polyamines and divalent cations on cystine transport were not additive. Spermidine stimulated cystine efflux from lysosomes of cultured human diploid fibroblasts, but had no effect on lysosomes of cystinotic fibroblasts which have defective cystine transport. Spermidine did not accumulate within lysosomes in exchange for cystine, had no effect on lysosomal pH, had only slight effects on the lysosomal membrane potential, and had little effect on either methionine or tyrosine efflux. Polyamines are cellular cytoplasmic components that, in physiologic concentrations, stimulate lysosomal cystine transport.     

Lysosomal turnover of GABARAP-phospholipid conjugate is activated during differentiation of C2C12 cells to myotubes without inactivation of the mTor kinase-signaling pathway

Although conjugation of overexpressed GABARP to phospholipid has been reported during starvation-induced autophagy, it is unclear whether endogenous GABARAP-phospholipid conjugation is also activated under starvation conditions. We observed little accumulation of GABARAP-phospholipid conjugate (GABARAP-PL) in mouse liver and kidney under starvation conditions, whereas endogenous LC3-phospholipid conjugate (LC3-II) accumulated. A small amount of endogenous GABARAP-PL was observed in the heart, independent of starvation. In rapamycin-treated HEK293 cells, there was little accumulation of endogenous GABARAP-PL, even in the presence of lysosomal protease-inhibitors, whereas there was significant accumulation of endogenous LC3-II, together with inactivation of the mTor kinase-signaling pathway. In HeLa and C2C12 cells, GABARAP-PL accumulation in the presence of lysosomal protease inhibitors was independent of starvation-induced autophagy, whereas LC3-II accumulation was significant during starvation-induced autophagy. Interestingly, we observed activation of lysosomal turnover of GABARAP-PL during the differentiation of C2C12 cells to myotubes, along with increased lysosomal turnover of LC3-II. Under these conditions, S6 ribosomal protein was still phosphorylated, suggesting that the mTor kinase-signaling pathway is active during the differentiation of C2C12 cells to myotubes, in contrast to starvation-induced autophagy. These results indicated that lysosomal turnover of GABARAP-PL was activated during the differentiation of C2C12 cells to myotubes without inactivation of the mTor kinase-signaling pathway, whereas little lysosomal turnover of GABARAP-PL was activated during starvation-induced autophagy.     

Lysosomal Turnover of GABARAP-Phospholipid Conjugate is Activated During Differentiation of C2C12 Cells to Myotubes without Inactivation of the mTor Kinase-Signaling Pathway

Although a conjugation of overexpressed GABARAP to phospholipid has been reported to be activated during starvation-induced autophagy, it is unclear whether endogenous GABARAP-conjugation is also activated under starvation conditions. We observed that little GABARAP-phospholipid conjugate (GABARAP-PL) accumulated in mouse liver and kidney under starvation conditions, while endogenous LC3-phospholipid conjugate (LC3-II) accumulated. A small amount of endogenous GABARAP-PL was observed in the heart independent of starvation. In rapamycin-treated HEK293 cells, there was little accumulation of endogenous GABARAP-PL, even in the presence of lysosomal protease-inhibitors, whereas there was significant accumulation of endogenous LC3-II together with inactivation of the mTor kinase-signaling pathway. In HeLa, and C2C12 cells, the accumulation of GABARAP-PL in the presence of lysosomal protease inhibitors is independent of starvation-induced autophagy, whereas the accumulation of LC3-II in their presence is significantly activated during starvation-induced autophagy. Interestingly, we observed that the lysosomal turnover of GABARAP-PL is activated during the differentiation of C2C12 cells to myotubes. Under these conditions, S6 ribosomal protein is still phosphorylated, suggesting that mTor kinase-signaling pathway is active during the differentiation of C2C12 cells to myotubes, different from the case during starvation-induced autophagy. These results indicated that the lysosomal turnover of GABARAP-PL is activated during the differentiation of C2C12 cells to myotubes without inactivation of mTor kinase-signaling pathway, while little is activated during starvation-induced autophagy.     

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.     

Characteristics of cystine counter-transport in normal and cystinotic lysosome-rich leucocyte granular fractions.

Normal leucocyte lysosome-rich granular fractions exhibited counter-transport of cystine, confirming that cystine transport across the lysosomal membrane is carrier-mediated. The trans-activation of cystine transport was temperature-dependent but relatively independent of the external Na+ or K+ concentration in phosphate buffer. Counter-transport, measured as uptake of exogenous [3H]cystine, increased with increasing intralysosomal cystine content up to approx. 3 nmol of half-cystine/unit of hexosaminidase activity. The amount of [3H]cystine entering lysosomes loaded with unlabelled cystine decreased when unlabelled cystine was added to the extralysosomal medium. Lysosomal cystine counter-transport was stereospecific for the L-isomer. Cystathionine, cystamine and cysteamine-cysteine mixed disulphide gave evidence of sharing the lysosomal cystine-transport system, although at lower activity than cystine. Other tested amino acids, including arginine, glutamate and homocystine, were inactive in this system. Nine leucocyte lysosome-rich preparations from eight different cystinotic patients displayed virtually no counter-transport of cystine, conclusively establishing that a carrier-mediated system for cystine transport is dysfunctional in cystinotic lysosomes.     

Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro

Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100-400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNS(Red) transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.     

Cystinosin, the protein defective in cystinosis, is a H(+)-driven lysosomal cystine transporter.

Cystinosis is an inherited lysosomal storage disease characterized by defective transport of cystine out of lysosomes. However, the causative gene, CTNS, encodes a seven transmembrane domain lysosomal protein, cystinosin, unrelated to known transporters. To investigate the molecular function of cystinosin, the protein was redirected from lysosomes to the plasma membrane by deletion of its C-terminal GYDQL sorting motif (cystinosin-DeltaGYDQL), thereby exposing the intralysosomal side of cystinosin to the extracellular medium. COS cells expressing cystinosin-DeltaGYDQL selectively take up L-cystine from the extracellular medium at acidic pH. Disruption of the transmembrane pH gradient or incubation of the cells at neutral pH strongly inhibits the uptake. Cystinosin-DeltaGYDQL is directly involved in the observed cystine transport, since this activity is highly reduced when the GYDQL motif is restored and is abolished upon introduction of a point mutation inducing early-onset cystinosis. We conclude that cystinosin represents a novel H(+)-driven transporter that is responsible for cystine export from lysosomes, and propose that cystinosin homologues, such as mammalian SL15/Lec35 and Saccharomyces cerevisiae ERS1, may perform similar transport processes at other cellular membranes.     

Lysosomal cystine counter-transport in heterozygotes for cystinosis.

Heterozygotes for cystinosis exhibited approximately half the normal rate of cystine counter-transport into isolated leukocyte lysosomes. This gene-dosage effect strongly supports previous findings demonstrating that the basic defect in cystinosis is impaired cystine transport across the lysosomal membrane. The method was used to determine the cystinosis carrier status for siblings of affected children in two families with cystinosis.     

Degradation of methoxysuccinyl-phe-leu-phe-7-amido-4-trifluoromethyl coumarin (FLF) in cultured myotubes and HepG2 cells is proteasome- and calpain/calcium-dependent

During recent years, it has become increasingly clear that the ubiquitin-proteasome proteolytic pathway regulates intracellular protein degradation in various physiological and pathophysiological conditions. Substrates specifically degraded by the proteasome are important tools to assess the involvement of the proteasome in cellular proteolysis. It was recently proposed that the membrane permeable substrate methoxysuccinyl-phenylalanine-leucine-phenylalanine-7-amido-4- trifluoromethyl coumarin (FLF) is degraded specifically by the proteasome. The role of other proteolytic pathways in the degradation of FLF, however, is not fully understood. In the present study, we tested the role of different proteolytic pathways in the degradation of FLF in cultured myotubes and HepG2 cells by treating the cells with inhibitors of lysosomal, calpain and proteasome activity. In addition, we tested the hypothesis that insulin blocks proteasome-dependent degradation of FLF in myotubes and HepG2 cells. Results suggest that degradation of FLF in both myotubes and HepG2 cells is regulated by proteasome and calpain activity but not by lysosomal activity. Insulin inhibited proteasome-dependent but not calpain-dependent degradation of FLF in both myotubes and HepG2 cells. The results are important because they suggest that FLF degradation does not specifically reflect proteasome activity.     

Proteolytic enzyme activity in rat hindlimb muscles in fetus and during post-natal development

The enzymatic activity of two lysosomal enzymes, acid phosphatase and cathepsin D, was determined in fetus and during post-natal development of the rat gastrocnemius muscle in comparison to the histological differentiation of this muscle. The specific activity of cathepsin D and acid phosphatase was 7 and 2.5 fold higher in the muscle during development until 20 days after birth, than that of mature muscle, respectively. A trend of gradual decrease in the activity of these enzymes was observed concomitantly with the differentiation and maturation of the muscle from mononucleated cells in the fetus to myotubes formation at day 1 after birth, followed by the formation of "young" and then striated myofibers in 10- and 20-day old neonates, respectively. However, no correlation could be found between the lysosomal enzyme activity and the developmental stages of the muscle until 20 days after birth. It is suggested that the elevated activity of lysosomal acid hydrolases may be associated with late developmental processes from young to mature myofibers in normal skeletal muscle and not only in various pathological conditions.