pH gradient across the lysosomal membrane generated by selective cation permeability and donnan equilibrium

The pH within isolated Triton WR 1339-filled rat liver lysosomes was determined by measuring the distribution of [¹⁴C]methylamine between the intra- and extralysosomal space. The intralysosomal pH was found to be approximately one pH unit lower than that of the surrounding medium. Increasing the extralysosomal cation concentration lowered the pH gradient by a cation exchange indicating the presence of a Donnan equilibrium. The lysosomal membrane was found to be significantly more permeable to protons than to other cations. The relative mobility of cations through the lysosomal membrane is H⁺ ? Cs⁺ > Rb⁺ > K⁺ Na⁺ > Li⁺ ? Mg²⁺, Ca²⁺. The presented data suggest that the acidity within isolated Triton WR 1339-filled lysosomes is maintained by: (1) a Donnan equilibrium resulting from the intralysosomal accumulation of nondifussible anions and (2) a selective permeability of the lysosomal membrane to cations.     

Relationship between medium pH and that of the lysosomal matrix as studied by two independent methods.

1. The method of estimating the intralysosomal pH by measuring the distribution of [14C]methylamine in lysosomes isolated from the livers of Triton WR 1339-treated rats has been critically examined. 2. In lysed lysosomes, methylamine is bound to the membrane fragments, but this binding can be completely suppressed by increasing the concentration of monovalent cations in the medium. 3. In intact lysosomes, the binding of [14C]methylamine is only partly inhibited by monovalent cations at 25 degrees C. 4. THe accumulation of [14C]methylamine in intact lysosomes is progressively inhibited as the concentration of methylamine is increased. A similar inhibition of [14C]methylamine accumulation is obtained with NH4Cl. 5. Similar values for the intralysosomal pH were obtained from measurements of the distribution of methylamine, dimethylamine and trimethylamine, which are accumulated in the lysosomes, and of 5,5-dimethyloxazolidinedione-2,4, which is excluded. 6. The breakdown of endocytosed 123I-labelled bovine serum albumin by intact isolated lysosomes is much less sensitive to the pH of the medium than the breakdown of added protein by lysed lysosomes. 7. The intralysosomal pH has been estimated by comparing the rate of breakdown of endocytosed 125I-labelled albumin in intact lysosomes as a function of medium pH with that of added 125I-labelled albumin by lysed lysosomes at different pH values. The values obtained agree well with those calculated from the distribution of [14C]methylamine. 8. Methylamine and NH4Cl inhibit the breakdown of 125I-labelled albumin in intact lysosomes, particularly at high medium pH, but have no effect on the breakdown by lysed lysosomes. 9. It is concluded that a pH difference across the lysosomal membrane (more acidic inside than outside) is maintained by the presence of indiffusible negatively charged groups within the lysosomes, and by the permeation across the lysosomal membrane of protons together with permeant anions (or of OH- in exchange for anions).     

Membrane lipids of rat liver lysosomes prepared by freeflow electrophoresis

Rat liver lysosomes were isolated by free-flow electrophoresis and were examined morphologically and enzymatically for purity. Their membrane fraction was prepared by osmotic shock and analyzed for cholesterol, phospholipids and fatty acids. The results were compared with the membrane fraction of Triton WR 1339-filled lysosomes and with mitochondria. The cholesterol content (0.269 M cholesterol per M lipid phosphorus), the sphingomyelin concentration (7.9% of total lipid phosphorus) and the degree of unsaturation of fatty acids (38–45%) were found to be intermediate between those of membranes of Triton WR 1339-filled lysosomes (“plasma membrane-like”) and mitochondria (“endoplasmic reticulum-like”). The similarity of these results with corresponding data for the Golgi apparatus support the present view concerning the formation of primary lysosomes via the Golgi apparatus. The drastic changes in the lipid composition found after overloading with Triton WR 1339 confirm that the plasma membrane participates in the formation of the secondary lysosomal membrane. The data presented here underline the significance of the analysis of membrane lipids in evaluating correlations between morphologically different but functionally closely related membrane types.     

Formation of triton WR 1339-filled rat liver lysosomes : II. Involvement of autophagy and of pre-existing lysosomes

The participation of endocytosis in the formation of Triton-filled lysosomes was followed after injecting Triton WR 1339 simultaneously with colloidal gold or horseradish peroxidase. According to cytomorphometric measurements Triton WR 1339 also significantly enhances autophagy. The analysis of the influence of Triton WR 1339 subfractions shows that autophagy is only augmented by the polymer component (‘macrotriton’) but not by the low molecular component (‘microtriton’) alone. When the latter was injected combined with either macrotriton or colloidal gold particles, autophagy increased to a level observed with Triton WR 1339 and beyond the levels determined for either component alone. Thus, autophagy appears to be stimulated by endocytosis. Autolysosomes are transformed within a few hours to peribiliary ‘dense bodies’. These do not display any significant turnover rate within a period of several days; therefore, dense bodies could be prelabelled with colloidal gold in order to show that they are the target organelles ingesting (macro-)Triton. According to difference spectra autophagy leads to a considerable concentration of microsomal and mitochondria! cytochromes in (macro-)Triton-filled lysosomes far beyond the level detected in ‘normal’ lysosomes. Because of the participation of both hetero- and autophagy in the formation of Triton WR 1339-filled lysosomes they have to be classified as telolysosomes.     

The permeability of the lysosomal membrane to small ions.

The permeability of the lysosomal membrane to small anions and cations was studied at 37 degrees C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K+ in the presence of valinomycin, and cation influx was coupled to an efflux of H+ using the protonophore 3-tert-butyl-5,2'-dichloro-4'-nitrosalicilylanilide. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases. The order of permeability of the lysosomal membrane to anions was found to be SCN- greater than I- greater than CH3COO- greater than Cl- approximately Pi greater than SO24- and that to cations Cs+ greater than K+ greater than Na+ greater than H+. These orders are largely in agreement with the lyotropic series of anions and cations. The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.     

The effect of Triton WR-1339 on the subcellular distribution of Trypan Blue and 125I-labelled albumin in rat liver

1. The density-gradient distribution patterns of acid phosphatase, Trypan Blue and denatured (125)I-labelled albumin were studied by discontinuous sucrose- and isopycnic sucrose-density-gradient centrifugation on combined heavy and light mitochondrial (M+L) fractions of liver isolated from normal rats and from rats injected with Triton WR-1339. 2. The results obtained from the subfractionation of the M+L pellet of normal animals indicate that the equilibrium density of Trypan Blue and acid-insoluble radioactivity is the same as that for acid phosphatase, which suggests they are bound by a common membrane to form a distinct subcellular population of lysosomal nature. 3. In contrast, the analysis of the isopycnic gradients obtained on subfractionation of M+L pellets of liver isolated from rats treated with Triton WR-1339 show that the acid-insoluble radioactivity has an equilibrium density around 1.21, whereas the acid hydrolases, including cathepsin D, show the characteristic shift to an equilibrium density of around 1.12. Trypan Blue is distributed along the gradient with distinct peaks at densities 1.22 and 1.12. 4. Similar equilibrium-density distribution patterns were obtained with M+L pellets isolated from rats pretreated with Triton WR-1339 but not injected with Trypan Blue. 5. Treatment of the rats with Triton WR-1339 does not affect albumin digestion of isolated intact lysosomes despite the fact that most of the cathepsin D and the albumin ingested by phagocytosis are located in different vacuoles. 6. It is concluded from these experiments that in the liver of animals treated with Triton WR-1339 (125)I-labelled albumin is located within heterophagosomes which do not fuse with heterolysosomes containing the non-ionic detergent Triton WR-1339. The inability of these two lysosomal populations to fuse is not due to Trypan Blue.     

Evidence against a MgATP-dependent proton pump in rat-liver lysosomes.

1. The effect of MgATP has been studied on the accumulation of the lipid-soluble anion thiocyanate, the accumulation of the lipid-soluble base methylamine, and the fluorescence of bound anilinonaphthalene sulphonate in rat-liver lysosomes. The lysosomes used were isolated from the livers of rats pretreated with Triton WR 1339. 2. The accumulation of thiocyanate is stimulated by the addition of valinomycin in the presence of K+ but not by the addition of MgATP. 3. The fluorescence of anilinonaphthalene sulphonate bound to lysosomes is enhanced by valinomycin in the presence of K+, the extent of the enhancement being dependent on the concentration of K+. In contrast, MgATP has no effect on the fluorescence. 4. The intralysosomal pH, as estimated from the distribution of methylamine, is not affected by the addition of MgATP in media with or without K+, Na+ or HCO3-. 5. These data strongly suggest that there is no MgATP-dependent proton pump in rat-liver lysosomes.     

Involvement of a non-proton pump factor (possibly Donnan-type equilibrium) in maintenance of an acidic pH in lysosomes.

Change of the internal pH of isolated lysosomes was measured with fluorescein isothiocyanate-dextran. In buffer of pH 7.0, isolated lysosomes had an acidic pH of about 5.5, which decreased to pH 5.2 on addition of ATP. Addition of bafilomycin inhibited the acidification by H(+)-ATPase and resulted in an increase of the internal pH to 5.5 due to passive diffusion of protons across the lysosomal membrane. However, no further alkalization was observed. The acidic pH (pH 5.5) of isolated lysosomes could be maintained for at least 48 h in the absence of ATP, but increased gradually to pH 5.9-6.4 upon incubation with monovalent cations (K+ or Na+), amines, or ionophores. These results suggest that a non-proton pump factor (possibly Donnan equilibrium) is involved in maintaining the acidic pH of isolated lysosomes.     

A proteomic analysis of lysosomal integral membrane proteins reveals the diverse composition of the organelle

Lysosomes are endocytic subcellular compartments that contribute to the degradation and recycling of cellular material. Using highly purified rat liver tritosomes (Triton WR1339-filled lysosomes) and an ion exchange chromatography/LC-tandem MS-based protein/peptide separation and identification procedure, we characterized the major integral membrane protein complement of this organelle. While many of the 215 proteins we identified have been previously associated with lysosomes and endosomes, others have been associated with the endoplasmic reticulum, Golgi, cytosol, plasma membrane, and lipid rafts. At least 20 proteins were identified as unknown cDNAs that have no orthologues of known function, and 35 proteins were identified that function in protein and vesicle trafficking. This latter group includes multiple Rab and SNARE proteins as well as ubiquitin. Defining the roles of these proteins in the lysosomal membrane will assist in elucidating novel lysosomal functions involved in cellular homeostasis and pathways that are affected in various disease processes.     

A critical examination of the evidence for an MgATP-dependent proton pump in rat liver lysosomes

(1) When lysosomes isolated from the livers of Triton WR 1339-treated rats were incubated for 30 min in the presence of 100 mM KCl and 14CH3NH2, a stimulation by MgATP of the calculated accumulation of the base was observed, in agreement with previous results (Schneider, D.L. (1979) Biochem. Biophys. Res. Commun. 87, 559-565). A similar stimulation was seen with MgITP. Excess EDTA had very little effect on the stimulation by MgATP. (2) There was little effect of MgATP or MgITP on the calculated accumulation of 14CH3NH2 if the base was added to the incubation medium 1, 3, 4 or 5 min before terminating the incubation instead of being present for the total incubation period of 30 min. (3) The accumulation of the basic dye, acridine orange, by a crude lysosomal preparation isolated from the livers of untreated rats was found to be stimulated by MgATP, in agreement with earlier results (Dell'Antone, P. (1979) Biochem. Biophys. Res. Commun. 86, 180-189). Similar results were obtained with a crude lysosomal preparation isolated from the livers of Triton WR 1339-treated rats. In both cases, the stimulation was partly oligomycin-sensitive. (4) There was very little or no effect of MgATP on the accumulation of acridine orange by preparations of pure lysosomes isolated from the livers of Triton WR 1339-treated rats. (5) Our data do not acquire us to postulate the existence of an MgATP-dependent proton pump in lysosomes.     

Formation of triton WR 1339-filled rat liver lysosomes : I. Properties and intracellular distribution of [H]Triton WR 1339

Triton WR 1339 was found to contain a high molecular weight fraction with globular polymers of ˜ 10⁵ D and a diameter of ˜80 Å (‘macrotriton’) and a low molecular weight fraction (‘microtriton’). The intracellular distribution of subfractions of [³H]Triton WR 1339 was followed by cell fractionation and by gel chromatography techniques in parallel with electron microscopy and autoradiography. Macrotriton is selectively stored in lysosomes and all evidence supports a slowly working endocytotic uptake mechanism. Microtriton permeates quickly into the cells and is rapidly and efficiently released into the bile. The data presented suggest some intracellular leaking of lysosomal contents due to the action of Triton WR 1339.     

Properties of binding sites for chloroquine in liver lysosomal membranes

Chloroquine (CQ) is an antimalarial and antirheumatic drug that accumulates in lysosomes. We purified liver lysosomal membranes of tritosomes from albino mice injected with Triton WR 1339. The membranes were used for the binding assay with CQ in 0.01 M Tris-HCl buffer (pH 7.4). This binding was saturable, with a KD value of 6.2 microM. To understand the nature of CQ affinity, the binding was done under conditions that alter membrane structure and composition. Changes in pH, high ionic strength, and bivalent cations reversibly decreased the binding, while the effect of non-ionic detergents was partially reversed. The cationic detergent Hyamine strongly decreased the binding, and its effect was trypsin and neuraminidase had no effect. The results indicate the existence of binding sites for CQ in liver lysosomal membranes, which were strongly affected by changes of charge in the molecules involved in the binding. The treatment with the enzymes suggests that loss of polar groups of phospholipids increases the affinity of CQ by exposing protein sites located deep in the membrane, or by permiting a closer interaction between the drug and membrane lipids. CQ lysosomotropism and other effects of CQ on the lysosomal apparatus studied by other authors may be due not only to its accumulation inside the acid milieu of the lysosomes, in the same manner as other weak bases, but also to the affinity of CQ for binding sites in the lysosomal membrane.     

Turnover studies on proteins of rat liver lysosomes.

The turnover of rat liver lysosomal proteins was studied by a double isotope-labeling technique. The cellular fractions investigated included soluble lysosomal proteins, lysosomal membrane proteins, highly purified lysosomal beta-glucuronidase, and for comparison, microsomal proteins and soluble cytoplasmic proteins. Both "normal" lysosomes and Triton WR-1339-filled lysosomes (tritosomes) were studied, with similar results. It was found that (a) the turnover rate of lysosomal proteins, of both the soluble and membranous compartments, was very similar to that of the proteins of the microsomal and soluble cytoplasmic fractions, and (b) the turnover rate of lysosomal proteins was asynchronous. The latter conclusion was based on two lines of evidence: (a) lysosomal beta-glucuronidase had a distinctly slower turnover rate than the average rate of the soluble lysosomal proteins, and (b) subunits of the proteins of the soluble lysosomal fraction as separated by sodium dodecyl sulfate. Sephadex G-200 gel filtration showed different rates of degradation.     

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.     

The lysosomal H+ pump: 8-azido-ATP inhibition and the role of chloride in H+ transport

Lysosomes (tritosomes) were purified from the livers of rats injected with Triton WR 1339. The lysosomes developed an Mg2+-ATP-dependent pH gradient as measured by Acridine orange accumulation. H+ transport was supported by chloride, but not sulfate, and was independent of the cation used. H+ transport and Mg2+-stimulated ATPase was inhibited by diethylstilbesterol (K0.5 = 2 microM). N-Ethylmaleimide inhibited H+ transport (K0.5 = 30 microM). At low concentrations of N-ethylmaleimide, ATP partially protected H+ transport from inhibition with N-ethylmaleimide. Photolysis with 8-azido-ATP inhibited H+ transport and Mg2+-stimulated ATPase activity. Under these same conditions, 8-azido-[alpha-32P]ATP reacted with a number of polypeptides of the intact lysosome and lysosomal membranes. Pump-dependent potentials were measured using the fluorescent potential-sensitive dye, DiSC3(5) (3,3'-dipropylthiocarbocyanine) and ATP-dependent potential generation was inhibited by diethylstilbesterol. Chloride, but not sulfate reduced the magnitude of the ATP-dependent membrane potential, as measured using merocyanine 540. The chloride conductance, independent of ATP, was of sufficient magnitude to generate a H+ gradient driven by external chloride in the presence of tetrachlorosalicylanilide. In Cl- free media, ATP-dependent H+ transport was restored to control levels by outwardly directed K+ gradients in the presence of valinomycin. The role of cell Cl- is to provide the necessary conductance for supporting lysosomal acidification by the electrogenic proton pump.     

The acidification of rat liver lysosomes in vitro: a role for the membranous ATPase as a proton pump.

Lysosomes were purified from the livers of rats which had been treated with Triton WR-1339. The ATPase activity of these lysosomes was stimulated by preincubation with NaCl or KCl, conditions which diminish the proton gradient due to Donnan equilibrium. Subsequent to this preincubation measurements of methylamine uptake by lysosomes showed an ATP-dependent enhancement. Simultaneous measurements of the internal volumes of lysosomes confirmed that ATP-dependent methylamine uptake is due to acidification of lysosomes by 0.3 to 0.5 pH units. Because the conditions which stimulated ATP-dependent methylamine uptake also stimulated the ATPase activity it is concluded that acidification of lysosomes requires an ATPase which functions as a proton pump.     

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.     

Effect on lysosomes of invertase endocytosed by rat-liver

The intracellular localization of invertase endocytosed by rat liver was investigated by analytical centrifugation in sucrose and Percoll gradients of mitochondrial fractions originating from rats killed 15 h after injection. After isopycnic centrifugation in a sucrose gradient, invertase is located in higher density zones than acid hydrolases. The difference between the distribution of invertase and that of acid hydrolases increases with the amount of invertase injected. When the invertase dose is sufficiently high, a change of lysosomal enzyme distribution is clearly visible. It consists in the shift of a proportion of these enzymes to higher density regions where invertase is located. The proportion of hydrolase activity affected by invertase is different for each enzyme measured; it is the least pronounced for acid phosphatase, and most for acid deoxyribonuclease and arylsulfatase. A pretreatment of the rat with Triton WR 1339 considerably decreases the equilibrium density of structures bearing invertase. Nevertheless invertase distribution is quite distinct from that of the bulk of lysosomal enzymes that are recovered in lower density zones of the gradient; on the other hand the invertase injection to rats treated with Triton WR 1339 causes a spreading of the acid hydrolase distribution towards higher density zones. The distribution of acid hydrolases and invertase in a Percoll gradient depends on the sucrose concentration of the solvent. It is shifted towards higher densities when the sucrose concentration increases. The phenomenon is more important for invertase. These results are best explained by supposing that invertase accumulates in a distinct population of lysosomes that can be individualized as a result of the density increase they are subjected to by the invertase they accumulate. It is proposed that these lysosomes mainly originate from non-parenchymal cells of the liver.     

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.