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.     

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.     

Evidence for lysosomal reduction of cystine residues.

Previously reported evidence for the existence of a thiol: protein disulphide oxidoreductase in rat liver lysosomes has been re-examined and ambiguous results obtained. However, incubation of purified rat liver lysosomes with ¹²⁵I-labelled insulin at pH 5.5 shows that cathepsin D and a thiol-dependent enzyme other than cathepsin B or L are important in its digestion. The latter enzyme is most probably a thiol: protein disulphide oxidoreductase.     

The efflux of lysosomal cholesterol from cells

To gain insight into the transport of sterol from lysosomes to the plasma membrane, we studied the efflux of lysosomal free cholesterol from intact Fu5AH rat hepatoma cells to high density lipoprotein (HDL) and other extracellular acceptors that promote sterol desorption from the plasma membrane. The procedures involved pulsing cells at 15 degrees C with low density lipoprotein that had been reconstituted with [3H]cholesteryl oleate and then incubating the cells at 37 degrees C in the presence of a sterol acceptor, while monitoring both the hydrolysis of [3H]cholesteryl oleate in lysosomes and the efflux of the resulting [3H]free cholesterol to the acceptor. After warming cells to 37 degrees C, rapid hydrolysis of [3H]cholesteryl oleate began after 10-20 min, and the lysosomally generated [3H]free cholesterol became available for efflux after an additional delay of 40-50 min. The kinetics of hydrolysis and the delay between hydrolysis and efflux were unchanged over a wide range of HDL3 concentrations (10-1000 micrograms of protein/ml), and with acceptors that do not interact with HDL-specific cell surface binding sites (phospholipid vesicles, dimethyl suberimidate cross-linked HDL). In addition, the delivery of lysosomal cholesterol to the plasma membrane was unaffected when cellular cholesterol content was elevated 2.6-fold above the normal control level, or when the activity of cellular acyl-coenzyme A/cholesterol acyltransferase (ACAT) was stimulated with exogenous oleic acid. We conclude that in the Fu5AH cell, a maximum of 40-50 min is required for the transport of cholesterol from lysosomes to the plasma membrane and that this transport is not regulated in response to either specific extracellular acceptors or the content of sterol in cells. The lack of effect of increased ACAT activity implies that the pathway for this transport does not involve passage of sterol through the rough endoplasmic reticulum, the subcellular location of ACAT.     

The formation of autophagosomes during lysosomal defect: A new source of cytotoxicity

Macroautophagy/autophagy comprises autophagosome synthesis and lysosomal degradation. It is well known that lysosomal defects cause toxicity to cells. However, it has not been investigated previously if cytotoxicity is conferred by autophagosome formation during lysosomal defect. Recently, we found that the formation of autophagosomes in such conditions also causes cytotoxicity, in addition to lysosomal defect insults. We revealed that a partial reduction in autophagosome synthesis was beneficial for cell survival in cells bearing the autophagosome formation-based toxicity. Our study suggests that production/accumulation of autophagosomes during lysosomal defect directly induces cellular toxicity, and this process may be implicated in the pathological conditions where lysosomes are defective.     

Stearylamine permeabilizes the lysosomal membrane to cystine and sialic acid

Cystine efflux from isolated rat liver lysosomes was enhanced by concentrations of stearylamine that were above the critical micellar concentration. Lysosomal latency, pH, and activity of the proton-translocating ATPase were largely unaffected under controlled experimental conditions. Loss of lysosomal latency was observed at higher stearylamine to protein ratios consistent with a detergent-like mechanism of action. Partially purified cultured fibroblast lysosomes with either defective cystine or sialic acid transport lost their stored material upon exposure to stearylamine. Concentrations of stearylamine which were effective for lysosomal efflux were highly toxic for cultured fibroblasts, thus limiting its use. Under specific conditions, stearylamine apparently selectively permeabilizes the lysosomal membrane. A similar acting, but less toxic agent may be of use in the treatment of lysosomal transport disorders.     

ERS1 encodes a functional homologue of the human lysosomal cystine transporter

Cystinosis is a lysosomal storage disease caused by an accumulation of insoluble cystine in the lumen of the lysosome. CTNS encodes the lysosomal cystine transporter, mutations in which manifest as a range of disorders and are the most common cause of inherited renal Fanconi syndrome. Cystinosin, the CTNS product, is highly conserved among mammals. Here we show that the yeast Ers1 protein and cystinosin are functional orthologues, despite sharing only limited sequence homology. Ers1 is a vacuolar protein whose loss of function results in growth sensitivity to hygromycin B. This phenotype can be complemented by the human CTNS gene but not by mutant ctns alleles that were previously identified in cystinosis patients. A genetic screen for multicopy suppressors of an ers1Delta yeast strain identified a novel gene, MEH1, which is implicated in regulating Ers1 function. Meh1 localizes to the vacuolar membrane and loss of MEH1 results in a defect in vacuolar acidification, suggesting that the vacuolar environment is critical for normal ERS1 function. This genetic system has also led us to identify Gtr1 as an Meh1 interacting protein. Like Meh1 and Ers1, Gtr1 associates with vacuolar membranes in an Meh1-dependent manner. These results demonstrate the utility of yeast as a model system for the study of CTNS and vacuolar function.     

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.     

Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase

The lipid droplet (LD) is the major site of cholesterol storage in macrophage foam cells and is a potential therapeutic target for the treatment of atherosclerosis. Cholesterol, stored as cholesteryl esters (CEs), is liberated from this organelle and delivered to cholesterol acceptors. The current paradigm attributes all cytoplasmic CE hydrolysis to the action of neutral CE hydrolases. Here, we demonstrate an important role for lysosomes in LD CE hydrolysis in cholesterol-loaded macrophages, in addition to that mediated by neutral hydrolases. Furthermore, we demonstrate that LDs are delivered to lysosomes via autophagy, where lysosomal acid lipase (LAL) acts to hydrolyze LD CE to generate free cholesterol mainly for ABCA1-dependent efflux; this process is specifically induced upon macrophage cholesterol loading. We conclude that, in macrophage foam cells, lysosomal hydrolysis contributes to the mobilization of LD-associated cholesterol for reverse cholesterol transport.     

Correction of I-cell defect by hybridization with lysosomal enzyme deficient human fibroblasts

I-cell fibroblasts with a multiple intracellular lysosomal enzyme deficiency were hybridized with cells from patients with different types of single lysosomal enzyme defects. Fusion with G_M2 gangliosidosis, type 2, (Sandhoff disease) fibroblasts resulted in a restoration of the hexosaminidase activity, in a normalization of the electrophoretic mobility of the isoenzymes, and in a decreased activity in the medium. Fusion of I-cells with fibroblasts from G_M1 gangliosidosis, type 1, led to enhancement of β-galactosidase (β-gal) activity. This complementation must be the result of the presence of normal polypeptide chains in I-cells, whereas the other cell types provide a factor that causes the intracellular retention of the enzymes. Restoration of β-gal was also observed in heterokaryons after fusion of I-cells with β-galactosidase/neuraminidase-deficient (β-gal⁻/neur⁻) variants, indicating that the neuraminidase(s) and the posttranslational modification of β-gal are affected in a different way in I-cell disease and in β-gal⁻/neur⁻ variants. Fusion of I-cells with mannosidosis fibroblasts resulted in a restoration of the acidic form of α-mannosidase and in a decrease of the extracellular activity of both this enzyme and the hexosaminidase enzyme, indicating that fusion of I-cells with different types of fibroblasts with a single lysosomal enzyme deficiency not only leads to complementation for one particular enzyme but also to a correction of the basic defect in I-cells.     

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.     

Lys05: A new lysosomal autophagy inhibitor

Lys05 is a previously undescribed dimeric chloroquine which more potently accumulates in the lysosome and blocks autophagy compared with HCQ. Lys05 produced more potent antitumor activity as a single agent both in vitro and in vivo in multiple human cancer cell lines and xenograft models compared with HCQ. Initial structure-activity relationship studies demonstrated that the increased activity associated with Lys05 was due to the bivalent aminoquinoline rings, C7-Chlorine, and a short triamine linker. While lower doses of Lys05 were well tolerated and associated with antitumor activity, at the highest dose tested, Lys05 produced Paneth cell dysfunction and intestinal toxicity, similar to what can be observed in mice and humans with genetic defects in the autophagy gene ATG16L1. Lys05 is therefore a new lysosomal autophagy inhibitor that has potential to be developed further into a drug for cancer and other medical applications.