Lysosomal Membrane Vesicles Research Articles

Modulation of intracellular chloride channels by ATP and Mg2+

We report the effects of ATP and Mg2+ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n=112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5–2mmol/l) modulated and/or inhibited the chloride channel activities (n=38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n=5/93) or reversible (n=15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n=6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5–2mmol/l) without the presence of Mg2+ decreased the chloride channel current (n=12/14), whereas Mg2+ significantly reversed the effect (n=4/4). We suggest that ATP-intracellular chloride channel interactions and Mg2+ modulation of these interactions may regulate different physiological and pathological processes.

Dual control of cytosolic metals by lysosomal transporters in lobster hepatopancreas

This study describes the membrane transport mechanisms used by lobster (Homarus americanus) hepatopancreatic epithelial lysosomes to accumulate and sequester heavy metals from the cytosol, and thereby aid in the regulation of these ions entering the animal from dietary constituents. The present investigation extends previous work describing lysosomal metal uptake by cation exchange with protons and suggests that a second, parallel, lysosomal transport process involving metal-thiol conjugates may work in conjunction with the cation antiporter to control cytoplasmic metal concentrations. Transport of (65)Zn(2+) by lysosomal membrane vesicles (LMV) incubated in 1 mmol l(-1) glutathione (GSH) was not significantly different from metal transport in the absence of the tripeptide. However, preloading LMV with 1 mmol l(-1) alpha-ketoglutarate (AKG), and then incubating in a medium containing 1 mmol l(-1) GSH, more than doubled metal uptake, compared with vesicles equilibrated with chloride or possessing an outwardly directed chloride gradient. Kinetic analysis of lysosomal (65)Zn(2+) influx as a function of zinc concentration, in vesicles containing 1 mmol l(-1) AKG and incubated in 1 mmol l(-1) GSH, revealed the presence of a sigmoidal, low affinity, high capacity carrier process transporting the metal into the organelle. These data indicated the possible presence of an organic anion exchanger in lobster lysosomal membranes. Western blot analysis of LMV with a rabbit anti-rat OAT1 antibody showed the presence of an orthologous OAT1-like protein (approximate molecular mass of 80 kDa) signal from these membranes. These results, and those published previously, suggest the occurrence of two metal transporters on hepatopancreatic membranes, a high affinity, low capacity cation antiporter and a low affinity, high capacity organic anion exchanger. Together these two systems have the potential to regulate cytoplasmic metals over a wide concentration range.

Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo

The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

Heavy metal detoxification in crustacean epithelial lysosomes: role of anions in the compartmentalization process

Crustacean hepatopancreatic lysosomes are organelles of heavy metal sequestration and detoxification. Previous studies have shown that zinc uptake by lysosomal membrane vesicles (LMV) occurred by a vanadate- and thapsigargin-sensitive ATPase that was stimulated by a transmembrane proton gradient established by a co-localized V-ATPase associated with this organelle. In the present study, hepatopancreatic LMV from the American lobster Homarus americanus were prepared by standard centrifugation methods and 65Zn2+, 36Cl-, 35SO(4)2- and 14C-oxalate2- were used to characterize the interactions between the metal and anions during vesicular detoxification events. Vesicles loaded with SO4(2-) or PO(4)3- led to a threefold greater steady-state accumulation of Zn2+ than similar vesicles loaded with mannitol, Cl- or oxalate2-. The stimulation of 65Zn2+ uptake by intravesicular sulfate was SO(4)2- concentration dependent with a maximal enhancement at 500 micromol l(-1). Zinc uptake in the presence of ATP was proton-gradient enhanced and electrogenic, exhibiting an apparent exchange stoichiometry of 1Zn+/3H+. 35SO4(2-) and 14C-oxalate2- uptakes were both enhanced in vesicles loaded with intravesicular Cl- compared to vesicles containing mannitol, suggesting the presence of anion countertransport. 35SO4(2-) influx was a sigmoidal function of external [SO(4)2-] with 25 mmol l(-1) internal [Cl-], or with several intravesicular pH values (e.g. 7.0, 8.0 and 9.0). In all instances Hill coefficients of approximately 2.0 were obtained, suggesting that 2 sulfate ions exchange with single Cl- or OH- ions. 36Cl- influx was a sigmoidal function of external [Cl-] with intravesicular pH of 7.0 and 9.0. A Hill coefficient of 2.0 was also obtained, suggesting the exchange of 2 Cl- for 1 OH-. 14C-oxalate influx was a hyperbolic function of external [oxalate2-] with 25 mmol l(-1) internal [Cl-], suggesting a 1:1 exchange of oxalate2- for Cl-. As a group, these experiments suggest the presence of an anion exchange mechanism exchanging monovalent for polyvalent anions. Polyvalent inorganic anions (SO4(2-) and PO4(3-)) are known to associate with metals inside vesicles and a detoxification model is presented that suggests how these anions may contribute to concretion formation through precipitation with metals at appropriate vesicular pH.

Role of polyvalent anions in lysosomal heavy metal detoxification

Crustacean hepatopancreatic lysosomes are organelles of heavy metal sequestration and detoxification. In this study hepatopancreatic lysosomal membrane vesicles (LMV) from the American lobster (Homarus americanus) were prepared by standard centrifugation methods and 65Zn2+, 36Cl−, and 35SO42− were used to characterize the transport processes responsible for vesicular accumulation of these ions. 65Zn2+ influx into LMV was a hyperbolic function of external [Zn] and followed Michaelis-Menten kinetics (Km = 32 ±10 μM; Jmax = 21 ± 3 pmol/mg protein x sec). This transport was stimulated by 1 mM ATP and abolished by the simultaneous addition of 1 mM ATP + 250 μM vanadate. Thapsigargin (10 μM) was also a significant inhibitor of zinc influx, but not as effective as vanadate. Cadmium and copper were competitive inhibitors of 65Zn2+ influx (Cd2+ Ki = 68 ± 3 μM; Cu2+ Ki = 33 ± 2 μM). An outwardly-directed proton gradient, in the absence of ATP, stimulated 65Zn2+ influx, while an inwardly-directed proton gradient inhibited metal uptake. Vesicles loaded with SO42− or PO43− led to a 3-fold greater steady-state accumulation of Zn2+ than similar vesicles loaded with mannitol, Cl−, or oxalate2−. 35SO42− influx was a hyperbolic function of internal [Cl] and 36Cl− influx appeared to occur by diffusion in the absence of an antiport substrate. These results suggest that 65Zn2+ influx occurred by a vanadate- and thapsigargin-sensitive ATPase transporter that was stimulated by an outwardly-directed proton gradient. Polyvalent inorganic anions (SO42− and PO43−) associate with the metal inside the vesicles and may result in concretion formation through precipitation at appropriate vesicular pH. Supported by NSF grant IBN04-21986.

65Zn2+ transport by lobster hepatopancreatic lysosomal membrane vesicles

In crustaceans, the hepatopancreas is the major organ system responsible for heavy metal detoxification, and within this structure the lysosomes and the endoplasmic reticulum are two organelles that regulate cytoplasmic metal concentrations by selective sequestration processes. This study characterized the transport processes responsible for zinc uptake into hepatopancreatic lysosomal membrane vesicles (LMV) and the interactions between the transport of this metal and those of calcium, copper, and cadmium in the same preparation. Standard centrifugation methods were used to prepare purified hepatopancreatic LMV and a rapid filtration procedure, to quantify 65Zn2+ transfer across this organellar membrane. LMV were osmotically reactive and exhibited a time course of uptake that was linear for 15-30 sec and approached equilibrium by 300 sec. 65Zn2+ influx was a hyperbolic function of external zinc concentration and followed Michaelis-Menten kinetics for carrier transport (Km = 32.3 +/- 10.8 microM; Jmax = 20.7 +/- 2.6 pmol/mg protein x sec). This carrier transport was stimulated by the addition of 1 mM ATP (Km = 35.89 +/- 10.58 microM; Jmax = 31.94+/-3.72 pmol/mg protein/sec) and replaced by an apparent slow diffusional process by the simultaneous presence of 1 mM ATP+250 microM vanadate. Thapsigargin (10 microM) was also a significant inhibitor of zinc influx (Km = 72.87 +/- 42.75 microM; Jmax =22.86 +/- 4.03 pmol/mg protein/sec), but not as effective in this regard as was vanadate. Using Dixon analysis, cadmium and copper were shown to be competitive inhibitors of lysosomal membrane vesicle 65Zn2+ influx by the ATP-dependent transport process (cadmium Ki = 68.1 +/- 3.2 microM; copper Ki = 32.7 +/- 1.9 microM). In the absence of ATP, an outwardly directed H+ gradient stimulated 65Zn2+ uptake, while a proton gradient in the opposite direction inhibited metal influx. The present investigation showed that 65Zn2+ was transported by hepatopancreatic lysosomal vesicles by ATP-dependent, vanadate-, thapsigargin-, and divalent cation-inhibited, carrier processes that illustrated Michaelis-Menten influx kinetics and was stimulated by an outwardly directed proton gradient. These transport properties as a whole suggest that this transporter may be a lysosomal isoform of the ER Sarco-Endoplasmic Reticulum Calcium ATPase.

Characterization of a heavy metal ion transporter in the lysosomal membrane

Lysosomes are thought to play a role in various aspects of heavy metal metabolism. In the present study we demonstrate for the first time the presence of a heavy metal ion transport protein in the lysosomal membrane. Uptake of radioactive silver both in highly purified lysosomal membrane vesicles and in purified intact lysosomes showed the typical kinetics of a carrier-mediated process. This transport was stimulated by ATP hydrolysis, and showed specificity for Ag +, Cu 2+, and Cd 2+. All biochemical properties of this lysosomal metal ion transporter could classify it as a heavy metal transporting P-type ATPase. Long Evans Cinnamon (LEC) rats, an animal model for the copper transport disorder Wilson disease, showed normal lysosomal silver transport.

An Active Mechanism for Completion of the Final Stage of Protein Degradation in the Liver, Lysosomal Transport of Dipeptides

Accumulation of products of proteolysis (e.g. dipeptides) in lysosomes may have pathological consequences. In the present experiment we have investigated the existence of a dipeptide transporter in a membrane preparation of liver lysosomes using Gly-3H-Gln as the probe. The results showed that (a) there was transport of Gly-Gln into an osmotically reactive space inside the lysosomal membrane vesicles; (b) transport was stimulated by acidification (pH 5.0) of the external medium; (c) there was a coupling between transport of protons and Gly-Gln with a stoichiometry of 1:1; (d) the presence of both acidic pH and membrane potential was necessary for uphill transport of Gly-Gln; (e) a single transporter with a Km of 4.67 mM mediated the uptake of Gly-Gln; and (f) Gly-Gln uptake was inhibited by dipeptides and tripeptides but not by amino acids. The results suggest the presence of a low affinity proton-coupled oligopeptide transporter in the liver lysosomal membrane which mediates transfer of dipeptides from a region of low dipeptidase activity (intralysosome) to a region of high dipeptidase activity (cytosol). In this manner, the transporter provides an active mechanism for completion of the final stage of protein degradation.

Evidence for an essential histidine residue located in the binding site of the cysteine-specific lysosomal transport protein

Previously, we observed that the activity of the cysteine-specific lysosomal transport system increases 7–10-fold between pH 6 and 7.3 to be maximally active in the neutral pH range. To understand what factors contribute to this pH dependence, different chemical modifying agents were used to probe the nature of amino acid residues residing in the transport protein binding site. Diethyl pyrocarbonate (1 mM) and N-ethylmaleimide (5 mM) each strongly inactivated lysosomal cysteine uptake ≥ 88%, whereas dicyclohexylcarbodiimide (2.5 mM), phenylisothiocyanate (2 mM), N-acetylimidazole (33 mM), and phenylglyoxal (2 mM) had a moderate to small effect. Maximal inactivation by DEPC occurs within 12–15 min upon exposure to DEPC concentrations ≥ 1 mM. DEPC inactivation is consistent with modification of a histidine residue, displaying no inactivation at pH < 6, half-maximal inactivation at pH 6.6, and maximal inactivation at pH ≥ 7.3. The close correspondence of DEPC inactivation to the pH activity curve of cysteine uptake suggests the large increase in lysosomal cysteine transport activity between pH 6 and 7.3 reflects deprotonation of an essential histidine residue. The substrate, l-cysteine (4 mM), fully protects the transport protein from DEPC inactivation suggesting that this histidine residue is located in the carrier's substrate binding site. Finally, part of the pH dependence of the lysosomal cysteine carrier appears to be due to responsiveness to the lysosomal transmembrane proton gradient as indicated by lysosomal membrane vesicles which display a 1.5-fold greater rate of cysteine uptake when pH 7.4 out > pH 5.3 in than when pH 7.4 out = pH 7.4 in.

Lysosomal sulfate efflux following glycosaminoglycan degradation: measurements in enzyme-supplemented Maroteaux-Lamy syndrome fibroblasts and isolated lysosomes

Studies using lysosomal membrane vesicles have suggested that efflux of the sulfate that results from lysosomal glycosaminoglycan degradation is carrier-mediated. In this study, glycosaminoglycan degradation and sulfate efflux were examined using cultured skin fibroblasts and lysosomes deficient in the lysosomal enzyme N-acetylgalactosamine-4-sulfatase. Such fibroblasts store dermatan sulfate lysosomally, which could be labelled biosynthetically with Na2(35)SO4. The addition of recombinant N-acetylgalactosamine-4-sulfatase to the media of 35S labelled fibroblasts degraded up to 82% of the stored dermatan [35S] sulfate over a subsequent 96 h chase and released inorganic [35S] sulfate into the medium. In the presence of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), sulfate was reused to a minor extent in newly synthesized proteoglycan. Isolated granules from recombinant enzyme supplemented fibroblasts degraded stored dermatan [35S]sulfate to sulfate which was rapidly released into the medium at a rate that was reduced by the extra-lysosomal presence of the lysosomal sulfate transport inhibitors SITS, Na2SO4 and Na2MoO4. SITS also inhibited dermatan sulfate turnover, although it had no effect on the action of purified recombinant enzyme in vitro. These data imply that sulfate clearance occurred concomitantly with dermatan sulfate turnover in the lysosome even at high substrate loading, and that lysosome-derived sulfate, while available, is reutilized minimally in synthetic pathways.

Nucleotide-activated chloride channels in lysosomal membranes

Lysosomal membrane vesicles purified from rat liver contain a basal chloride conductance that was enhanced in the presence of ATP, non-hydrolysable ATP-analogs and, to a lesser extent, GTP. Other nucleotides, including AMP, ADP and cAMP, as well as CTP and UTP were not effective. Following fusion of the vesicles with an artificial phosphatidylethanolamine/phosphatidylserine bilayer, we found that ATP gamma S dramatically increased the incidence of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS)-sensitive chloride channels with a unitary slope conductance of approx. 40 pS in 300 mM/50 mM KCl buffers and 120 pS in symmetrical 300 mM KCl buffers. Since similar results were obtained with AMP-PNP, the results indicate that lysosomes contain a chloride permeable ion channel that is activated by ATP through allosteric interaction.

Sialic acid storage diseases. A multiple lysosomal transport defect for acidic monosaccharides.

A defective efflux of free sialic acid from the lysosomal compartment has been found in the clinically heterogeneous group of sialic acid storage disorders. Using radiolabeled sialic acid (NeuAc) as a substrate, we have recently detected and characterized a proton-driven carrier for sialic acid in the lysosomal membrane from rat liver. This carrier also recognizes and transports other acidic monosaccharides, among which are uronic acids. If no alternative routes of glucuronic acid transport exist, the disposal of uronic acids can be affected in the sialic acid storage disorders. In this study we excluded the existence of more than one acidic monosaccharide carrier by measuring uptake kinetics of labeled glucuronic acid [( 3H]GlcAc) in rat lysosomal membrane vesicles. [3H]GlcAc uptake was carrier-mediated with an affinity constant of transport (Kt) of 0.3 mM and the transport could be cis-inhibited or trans-stimulated to the same extent by sialic acid or glucuronic acid. Human lysosomal membrane vesicles isolated from cultured fibroblasts showed the existence of a similar proton-driven transporter with the same properties as the rat liver system (Kt of [3H]GlcAc uptake 0.28 mM). Uptake studies with [3H]NeuAc and [3H]GlcAc in resealed lysosome membrane vesicles from cultured fibroblasts of patients with different clinical presentation of sialic acid storage showed defective carrier-mediated transport for both sugars. Further evidence that the defective transport of acidic sugars represents the primary genetic defect in sialic acid storage diseases was provided by the observation of reduced, half-normal transport rates in lymphoblast-derived lysosomal membrane vesicles from five unrelated obligate heterozygotes. This study reports the first observation of a human lysosomal transport defect for multiple physiological compounds.

Glucose transport in lysosomal membrane vesicles. Kinetic demonstration of a carrier for neutral hexoses.

Lysosomal membrane vesicles isolated from rat liver were exploited to analyze the mechanism of glucose transport across the lysosomal membrane. Uptake kinetics of [14C]D-glucose showed a concentration-dependent saturable process, typical of carrier-mediated facilitated transport, with a Kt of about 75 mM. Uptake was unaffected by Na+ and K+ ions, membrane potentials, and proton gradients but showed an acidic pH optimum. Lowering the pH from 7.4 to 5.5 had no effect on the affinity of the carrier for the substrate but increased the maximum rate of transport about 3-fold. As inferred from the linearity of Scatchard plots, a single transport mechanism could account for the uptake of glucose under all conditions tested. As indicated by the transstimulation properties of the carrier, other neutral monohexoses, including D-galactose, D-mannose, D- and L-fucose were transported by this carrier. The transport rates and affinities of these sugars, measured by the use of their radiolabeled counterparts, were in the same range as those for D-glucose. Pentoses, sialic acid, and other acidic monosaccharides including their lactones, aminosugars, N-acetyl-hexosamines, and most L-stereoisomers, particularly those not present in mammalian tissues, were not transported by this carrier. Glucose uptake and transstimulation were inhibited by cytochalasin B and phloretin. The biochemical properties of this transporter differentiate it from other well-characterized lysosomal sugar carriers, including those for sialic acid and N-acetylhexosamines. The acidic pH optimum of this glucose transporter is a unique feature not shared with any other known glucose carrier and is consistent with its lysosomal origin.

Characterization of a proton-driven carrier for sialic acid in the lysosomal membrane: Evidence for a group-specific transport system for acidic monosaccharides

Abstract Highly purified lysosomal membrane vesicles, obtained from rat liver lysosomes, were used to study characteristics of NeuAc transport across the lysosomal membrane. Uptake of [14C]NeuAc was found to be strongly influenced by a pH gradient across the membrane. When a proton gradient (pHin greater than pHout) was generated by impermeable buffers, NeuAc uptake above equilibrium level (overshoot) was observed. The influence of membrane diffusion potentials was ruled out by experiments where K+ and valinomycin were present. The overshoot appeared to be specifically produced by protons, since gradients of other cations (Na+ and K+) did not give stimulation. Proton-driven uptake was saturable (Kt = 0.24 mM) and mediated by a single system, as shown by linearity of the Scatchard plot. Stimulation of transport was also obtained by preincubation of vesicles with MgATP and the effect was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by the protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Monocarboxylic sugars like glycuronic acids were competitive inhibitors of sialic acid transport. Transstimulation of [14C] NeuAc uptake was observed when vesicles were preloaded either with unlabeled NeuAc or with glucuronic acid. The data demonstrate that lysosomal membrane vesicles from rat liver are a suitable system for kinetic studies of solute transport events. The presence of a proton-driven carrier in the lysosomal membrane specific for sialic acid and other acidic sugars, including glucuronic acid, is shown. The possible physiological significance of these findings for the human lysosomal carrier and the patients with a sialic acid transport defect is discussed.

ATP-dependent acidification of membrane vesicles isolated from purified rat liver lysosomes. Acidification activity requires phosphate.

Membrane vesicles were isolated from purified liver lysosomes of rats treated with Triton WR-1339. In order to preserve ATP-dependent acidification activity, proteolysis of membranes was minimized by adding protease inhibitors and by centrifuging to form dilute bands of vesicles rather than highly concentrated pellets. The membrane vesicle fraction represented about 20% of the total lysosomal protein, 80% of the ATPase activity, and 3% of the solute proteins as marked by N-acetylglucosaminidase. About one-half of the membranes were oriented right side out. The space unavailable to [14C]sucrose corresponded to 3 microliters/mg of membrane protein which indicates that the membranes form vesicles about one-tenth the size of lysosomes. Uptake of either [14C]methylamine or [14C]chloroquine by lysosomal membrane vesicles was ATP-dependent, indicating acidification of the intravesicle space. The acidification activity was inhibited when either 1.5 microM carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, 100 microM dicyclohexylcarbodiimide, or millimolar concentrations of such permeant weak bases as ammonium sulfate and dansyl cadaverine were added. Acidification of lysosomal vesicles by ATP occurred electroneutrally. This acidification activity was not dependent on added salts but was inhibited by the anion transport inhibitors pyridoxal phosphate and diisothiocyanostilbene disulfonic acid, thus suggesting co-transport of protons and anions. Results which indicate that phosphate is the transported anion included (a) ATP-dependent uptake of [32P]phosphate by lysosomal membrane vesicles and (b) stimulation of ATP-dependent acidification of these vesicles by added phosphate. These observations provide further evidence that maintenance of the acid intralysosomal pH necessary for activation of lysosomal hydrolases is due to an ATP-driven proton pump located in the lysosomal membrane.