Decreased endo-lysosomal acidification capacity in methylene diphosphonate-resistant mutants of Dictyostelium discoideum

Abstract The in vivo capacity for endo-lysosomal acidification has been monitored in Dictyostelium discoideum amoebae with acridine orange, a fluorescent weak base dye commonly used to probe transmembrane pH gradients. In the presence of aerobic amoebae, the initial rate of fluorescence quenching was found to be proportional to cell density between 5 × 10⁵ and 2.5 × 10⁶ cells ml⁻¹ and independent of acridine orange concentration in the 1.5 to 7.5 μM range. The dye response was sensitive to agents that perturb endo-lysosomal acidification such as NaN₃, nigericin or imidazole. Several mutant cell lines whose growth was resistant to methylene diphosphonate were found to be partially deficient in the acridine orange quenching test, suggesting that endo-lysosomal acidification was altered in these mutants.     

Endocytosis is required for the growth of vacuolar H(+)-ATPase- defective yeast: identification of six new END genes

Yeast mutants that are defective in acidification of the lysosome-like vacuole are able to grow at pH 5.5, but not at pH 7. Here, we present evidence that endocytosis is required for this low pH-dependent growth and use this observation to develop a screen for mutants defective in endocytosis. By isolating mutants that cannot grow when they lack the 60-kD vacuolar ATPase subunit (encoded by the VAT2 gene), we isolated a number of vat2-synthetic lethal (Vsl-) mutant strains. Seven of the Vsl- mutants are defective in endocytosis. Four of these mutant strains (end8-1, end9-1, end10-1, and end11-1) show altered uptake of the endocytosed ligand, alpha-factor, and three (end12-1, end12-2, and end13-1) are probably defective in transfer of internalized material to the vacuole. Most of the mutations also confer a strong Ts- growth defect. The mutants defective in uptake of alpha-factor sort newly synthesized vacuolar proteins correctly, while those which may be defective in subsequent transport steps secrete at least a fraction of the newly synthesized soluble vacuolar proteins. The mutations that result in a defect in alpha-factor uptake are not allelic to any of the genes previously shown to encode endocytic functions.     

Acidification of morphologically distinct endosomes in mutant and wild- type Chinese hamster ovary cells

In the preceding paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2713-2721), we have shown that there is rapid acidification of endosomal compartments to pH 6.3 by 3 min in wild-type Chinese hamster ovary (CHO) cells. In contrast, early acidification of endosomes is markedly reduced in the CHO mutants, DTF 1-5-4 and DTF 1-5- 1. Since these CHO mutants are pleiotropically defective in endocytosis (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308), our results are consistent with a requirement for proper acidification of early endocytic compartments in many pH-regulated endocytic processes. In this paper, by measuring the pH of morphologically distinct endosomes using fluorescence microscopy and digital image analysis, we have determined in which of the endocytic compartments the defective acidification occurs. We found that the acidification of both the para- Golgi recycling endosomes and lysosomes was normal in the CHO mutants DTG 1-5-4 and DTF 1-5-1. The mean pH of large endosomes containing either fluorescein-labeled alpha 2-macroglobulin or fluorescein- isothiocyanate dextran was only slightly less acidic in the mutant cells than in wild-type cells. However, when we examined the pH of individual large (150-250 nm) endosomes, we found that there was an increased number of endosomes with a pH greater than 6.5 in the CHO mutants when compared with wild-type cells. Heterogeneity in the acidification of large endosomes was also seen in DTF 1-5-1 by a combined null point pH method and digital image analysis technique. In addition, both CHO mutants showed a marked decrease in the acidification of the earliest endosomal compartment, a diffusely fluorescent compartment comprised of small vesicles and tubules. We suggest that the defect in endosome acidification is most pronounced in the early, small vesicular, and tubular endosomes and that this defect partially carries over to the large endosomes that are involved in the sorting and processing of ligands. The proper step-wise acidification of the different endosomes along the endocytic pathway may have an important role in the regulation of endocytic processes.     

Quantitative evaluation of the uptake capacity of intact thrombocytes using fluorescent dyes

A new approach to quantitative determination of fluorescent dye uptake by intact cells is suggested. Fluorescent amine acridine orange selectively accumulating in 5HT granules of platelets has been used. Fluorescence signal analysis allows the estimation of a relative granule volume and the ratios of acridine orange transfer over cytoplasmic and granule membranes. The following results were obtained in human and rabbit platelets: a relative granule size was 14 +/- 1 % and 29 +/- 2 % of the total cell volume, intra-granule to extra-granule dye concentration ratios were 2260 +/- 382 and 30000 +/- 5550, while intra-cytoplasm to extra-cytoplasm concentration ratios were 375 +/- 60 and 225 +/- 60, respectively.     

Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts.

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.     

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP- dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild- type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.     

Kinetics of alpha 2-macroglobulin endocytosis and degradation in mutant and wild-type Chinese hamster ovary cells

The production of Chinese hamster ovary (CHO) cell mutants which are defective in endocytosis has led to a greater understanding of the process by which cells sort ligands and their receptors. Robbins and coworkers have obtained CHO mutants which are resistant to diphtheria toxin, defective in the delivery of endocytosed lysosomal enzymes to lysosomes, and have a decreased uptake of iron from transferrin (Robbins et al.: J. Cell Biol. 96:1064-1071, 1983). We have previously shown that these CHO mutants are markedly deficient in the acidification of early endocytic compartments (Yamashiro and Maxfield: J. Cell Biol. 105:2713-2721, 1987). In this study we examined the endocytosis of alpha 2-macroglobulin (alpha 2M) to determine whether the defects in early endosome acidification would alter the processing of this ligand. We found that the CHO mutants DTG 1-5-4 and DTF 1-5-1 bind, internalize, and degrade 125I-alpha 2M in a manner similar to the wild-type cells. We also found that the CHO mutants retain the ability to recycle the receptors for alpha 2M. Since the binding of alpha 2M is greatly reduced at mildly acidic pH (approximately 6.8), only slight acidification of the endosomal compartment should be sufficient to achieve sorting of alpha 2M from its receptor. In contrast, lysosomal enzymes require more acidic conditions (pH less than 6.0) for dissociation. The different behavior of the two ligands provides biochemical evidence for a partial (but not complete) defect in early endosome acidification in the mutants. The data also indicate that pH regulation in a relatively narrow range can achieve differential sorting of various ligands.     

Lysosomal origin of the ferric iron required for cell killing by hydrogen peroxide

The lysosomotropic amines methylamine (40 mM) and chloroquine (125 mM) prevented the killing of cultured hepatocytes by hydrogen peroxide generated in the medium by glucose oxidase. Maximum protection required several hours preincubation with either amine. Sensitivity of the hepatocytes to H2O2 was restored either by the addition of ferrous or ferric iron to the culture medium, or by incubating the cells for 4 hours in the absence of either amine prior to treatment with H2O2. Neither methylamine nor chloroquine had any effect on the cell killing by t-butyl hydroperoxide, a hepatotoxin that does not require iron. The protective effect of the lysosomotropic amines was distinguished from that of the ferric iron chelator deferoxamine in two ways: 1) deferoxamine protected hepatocytes from H2O2 toxicity but did not require a pretreatment period; and 2) in contrast to methylamine or chloroquine, deferoxamine had no effect on lysosomal pH as assessed by the fluorescent probe acridine orange. The data suggest that a lysosomal pool is the source of the ferric iron necessary for the killing of hepatocytes by H2O2.     

Isolation of rat liver endocytic vesicles using the proton pump as a marker

ATP-driven acidification visualized by the delta pH indicator acridine orange was used as marker for isolation of endocytic vesicles from rat liver. By differential and Percoll density gradient centrifugation, a vesicle fraction was obtained with an approx. 80-fold enriched H+-pump activity. The preparation contained vesicles that had taken up fluorescein isothiocyanate-labeled dextran or horseradish peroxidase injected into rats in vivo, proving the presence of endosomes. The H+-pump in these vesicles showed: (a) strict preference for ATP; (b) stimulation by Mg2+ and Mn2+, but not by monovalent cations; (c) stimulation by Cl-, I- and Br-; (d) electrogenicity; (e) insensitivity to vanadate, slight inhibition by oligomycin and strong inhibition by N-ethylmaleimide (NEM) and N,N'-dicyclohexylcarbodimide (DCCD). The vesicles exhibited an ouabain-, oligomycin- and levamisole-resistant ATPase activity, which was slightly stimulated by Cl-, unaffected by vanadate and inhibited by NEM and DCCD. Thus, a simple and efficient high-speed centrifugation method is available for isolation of endocytic vesicles from mammalian liver.     

Volume regulation in Mycoplasma gallisepticum: evidence that Na+ is extruded via a primary Na+ pump.

The primary extrusion of Na+ from Mycoplasma gallisepticum cells was demonstrated by showing that when Na+-loaded cells were incubated with both glucose (10 mM) and the uncoupler SF6847 (0.4 microM), rapid acidification of the cell interior occurred, resulting in the quenching of acridine orange fluorescence. No acidification was obtained with Na+-depleted cells or with cells loaded with either KCl, RbCl, LiCl, or CsCl. Acidification was inhibited by dicyclohexylcarbodiimide (50 microM) and diethylstilbesterol (50 microM), but not by vanadate (100 microM). By collapsing delta chi with tetraphenylphosphonium (200 microM) or KCl (25 mM), the fluorescence was dequenched. The results are consistent with a delta chi-driven uncoupler-dependent proton gradient generated by an electrogenic ion pump specific for Na+. The ATPase activity of M. gallisepticum membranes was found to be Mg2+ dependent over the entire pH range tested (5.5 to 9.5). Na+ (greater than 10 mM) caused a threefold increase in the ATPase activity at pH 8.5, but had only a small effect at pH 5.5. In an Na+-free medium, the enzyme exhibited a pH optimum of 7.0 to 7.5, with a specific activity of 30 +/- 5 mumol of phosphate released per h per mg of membrane protein. In the presence of Na+, the optimum pH was between 8.5 and 9.0, with a specific activity of 52 +/- 6 mumol. The Na+-stimulated ATPase activity at pH 8.5 was much more stable to prolonged storage than the Na+-independent activity. Further evidence that two distinct ATPases exist was obtained by showing that M. gallisepticum membranes possess a 52-kilodalton (kDa) protein that reacts with antibodies raised against the beta-subunit of Escherichia coli ATPase as well as a 68-kDa protein that reacts with the anti-yeast plasma membrane ATPases antibodies. It is postulated that the Na+ -stimulated ATPases functions as the electrogenic Na+ pump.     

Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line

Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4. (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0.) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37 degrees C. At 23 degrees C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.     

Identification and characterization of ATP-dependent proton transport by rat liver multivesicular bodies

Multivesicular bodies (MVB), prelysosomal organelles in the endocytic pathway, were prepared from estrogen-treated rat livers and examined for the presence of ATP-dependent proton transport. Vesicle acidification, assessed by acridine orange fluorescence quenching, was ATP dependent (ATP much greater than GTP, UTP), was enriched 25-fold over homogenate, was abolished by pretreatment with protonophores or a nonionic detergent, exhibited a pH optimum of 7.5, was inhibited by N-ethylmaleimide (NEM) (IC50 approximately 5 microM) and N,N'-dicyclohexylcarbodiimide (IC50 approximately 5 microM), and was resistant to inhibition by vanadate, ouabain, and oligomycin. Acidification exhibited no specific cation requirement; however, maximal rates of acidification depended upon the presence of Cl- (Km approximately 20 mM). Other anions were less effective in supporting acidification (Cl- greater than Br- greater than much greater than gluconate, NO-3, SO2-4, and mannitol), and indeed NO-3 inhibited acidification even in the presence of 150 mM Cl-. The proton transport mechanism appeared to be electrogenic based on: (a) enhancement of acidification by valinomycin in the presence of K gluconate, and (b) ATP-dependent fluorescence quenching of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol, a membrane potential-sensitive anionic dye. Furthermore, the magnitude of the pH and electrical gradients generated by the proton transport mechanism appeared to vary inversely in the presence and absence of Cl-. Finally, MVB exhibited ATPase activity that was resistant to ouabain and oligomycin, but was inhibited 32.3% by 1 mM NEM, 33.7% by 200 microM dicyclohexylcarbodiimide, and 18.7% by KNO3. In isolated MVB, therefore, the NEM-sensitive ATPase activity may represent the enzymatic equivalent of a proton pump. These studies identify and characterize an ATP-dependent electrogenic proton transport process in rat liver MVB which shares many of the properties of the proton pump described in clathrin-coated vesicles, endosomes, lysosomes, Golgi, and endoplasmic reticulum from liver and other tissues. Acidification of MVB differed somewhat from that of rat liver clathrin-coated vesicles in response to Br- and NO-3, suggesting that membrane properties of these two organelles might differ.     

Plasma membrane proton ATPase from human kidney

Distal urinary acidification is thought to be mediated by a proton ATPase (H+-ATPase). We isolated a plasma membrane fraction from human kidney cortex and medulla which contained H+-ATPase activity. In both the cortex and medulla the plasma membrane fraction was enriched in alkaline phosphatase, maltase, Na+,K+-ATPase and devoid of mitochondrial and lysosomal contamination. In the presence of oligomycin (to inhibit mitochondrial ATPase) in the presence of ouabain (to inhibit Na+,K+-ATPase) and in the absence of Ca (to inhibit Ca2+-ATPase) this plasma membrane fraction showed ATPase activity which was sensitive to dicyclohexylcarbodiimide and N-ethylmaleimide. This ATPase activity was also inhibited by vanadate, 4,4'-diisothiocyano-2,2'-disulfonic stilbene and ZnSO4. In the presence of ATP, but not GTP or UTP, the plasma membrane fraction of both cortex and medulla was capable of quenching of acridine orange fluorescence, which could be dissipated by nigericin indicating acidification of the interior of the vesicles. The acidification was not affected by presence of oligomycin or ouabain indicating that it was not due to mitochondrial ATPase or Na+,K+-ATPase, respectively. Dicyclohexylcarbodiimide and N-ethylmaleimide completely abolished the acidification by this plasma membrane fraction. In the presence of valinomycin and an outward-directed K gradient, there was increased quenching of acridine orange, indicating that the H+-ATPase is electrogenic. Acidification was not altered by replacement of Na by K, but was critically dependent on the presence of chloride. In summary, the plasma membrane fraction of the human kidney cortex and medulla contains a H+-ATPase, which is similar to the H+-ATPase described in other species, and we postulate that this H+-ATPase may be involved in urinary acidification.     

Selective degradation of insulin within rat liver endosomes

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.     

Degradation of insulin in isolated liver endosomes is functionally linked to ATP-dependent endosomal acidification

The degradation of insulin in isolated liver endosomes and the relationships of this process with ATP-dependent endosomal acidification have been studied. Incubation of endosomal fractions containing 125I-insulin in isotonic KCl at 30 degrees C resulted in a rapid loss of insulin integrity as judged from trichloroacetic acid precipitability, Sephadex G-50 chromatography, immunoreactivity and receptor binding ability, with a maximum at pH 5-6 (t1/2: 10, 10, 6 and 6 min, respectively). On a log/log plot, the amount of acid-soluble products generated was linearly related to the amount of insulin associated with endosomes (slope, 0.80). Upon incubation, virtually all acid-soluble products diffused out of endosomes as judged from their solubility in aqueous poly(ethyleneglycol). In permeabilized endosomes, intact insulin was also released in part extraluminally, but only when degradation was inhibited did this release increase with lowering pH. ATP shifted the pH for maximal insulin degradation to about 7.5-8.5 and caused endosomal acidification as judged from the uptake of acridine orange and the fluorescence of internalized fluorescein-labeled dextran and galactosylated bovine serum albumin (delta pH about 0.8-0.9). GTP, ITP and UTP exerted comparable effects but with lower potencies. The ability of ATP to alter the pH dependence of insulin degradation was maximal in the presence of Cl-, other anions being less effective (Br- greater than gluconate = SO4(2-) greater than NO3- = sucrose = mannitol) and/or inhibitory (NO3-). Na+, K+ and Li+ supported more effectively ATP-dependent insulin degradation than did choline. Divalent cations were required for the ATP effect (Mg2+ = Mn2+ greater than Co2+ greater than Ni2+ = Zn2 greater than Ca2+). Little or no effects of ATP occurred in the presence of proton ionophores such as monensin and carbonyl cyanide chlorophenylhydrazone, and inhibitors of the proton ATPase such as N-ethylmaleimide. The abilities of nucleotides, ions and inhibitors to support or inhibit ATP-dependent insulin degradation were well correlated with their abilities to affect ATP-dependent acidification. The acidotropic agents chloroquine and quinacrine caused a leftward shift in the pH dependence of insulin degradation and a decrease in maximal degradation; in the presence of ATP, chloroquine almost completely inhibited degradation at pH 5-9. It is concluded that ATP-dependent acidification, in part by enhancing the dissociation of the insulin-receptor complex, is required for optimum degradation of insulin within liver endosomes.     

Characterization and intracellular trafficking pattern of vacuoles containing Chlamydia pneumoniae in human epithelial cells

Chlamydiae are obligate intracellular pathogens that reside within a membrane-bound vacuole throughout their developmental cycle. In this study, the intraphagosomal pH of Chlamydia pneumoniae ( Cpn ) was qualitatively assessed, and the intracellular fate of the pathogen-containing vacuole and its interaction with endocytic organelles in human epithelial cells were analysed using conventional immunofluorescence and confocal microscopy. The pH-sensitive probes acridine orange (AO), LysoTracker (LyT) and DAMP did not accumulate in the bacterial inclusion. In addition, exposure of cells to bafilomycin A1 (BafA1), a potent acidification inhibitor, did not inhibit or delay chlamydial growth. The chlamydial compartment was not accessible to the fluid-phase tracer Texas Red (TR)-dextran and did not exhibit any level of staining for the late endosomal marker cation-independent mannose-6-phosphate receptor (Ci-M6PR) or for the lysosomal-associated membrane proteins (LAMP-1 and -2) and CD63. In addition, transferrin receptor (TfR)-enriched vesicles were observed close to Cpn vacuoles, potentially indicating a specific translocation of these organelles through the cytoplasm to the vicinity of the vacuole. We conclude that Cpn , like other chlamydial spp., circumvents the host endocytic pathway and inhabits a non-acidic vacuole, which is dissociated from late endosomes and lysosomes, but selectively accumulates early endosomes.     

Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.

Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H(+)-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic (Bowman, E.M., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7972-7976). Effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 microM bafilomycin A1. The effect was revealed by washing the cells. Both studies using 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysomal pH of A431 cells increased from about 5.1-5.5 to about 6.3 in the presence of 1 microM bafilomycin A1. The pH increased gradually in about 50 min. In the presence of 1 microM bafilomycin A1, 125I-labeled epidermal growth factor (EGF) bound to the cell surface at 4 degrees C was internalized normally into the cells at 37 degrees C but was not degraded at all, in marked contrast to the rapid degradation of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irrespective of the addition of bafilomycin A1. These results suggest that the vacuolar type H(+)-ATPase plays a pivotal role in acidification and protein degradation in the lysosomes in vivo.     

Entry of feline calicivirus is dependent on clathrin-mediated endocytosis and acidification in endosomes

Feline calicivirus is a major causative agent of respiratory disease in cats. It is also one of the few cultivatable members of Caliciviridae. We have examined the entry process of feline calicivirus (FCV). An earlier study demonstrated that acidification in endosomes may be required. We have confirmed this observation and expanded upon it, demonstrating, using drugs to inhibit the various endocytic pathways and dominant-negative mutants, that FCV infects cells via clathrin-mediated endocytosis. We have also observed that FCV permeabilizes cell membranes early during infection to allow the co-entry of toxins such as alpha-sarcin. Inhibitors of endosome acidification such as chloroquine and bafilomycin A1 blocked this permeabilization event, demonstrating that acidification is required for uncoating of the genome and access to the cytoplasm.     

Phagocytized intracellular microsporidian blocks phagosome acidification and phagosome-lysosome fusion

This study examines the relationship between phagosome acidification and phagosome-lysosome fusion events using phagocytized Glugea hertwigi spores. The incidence of lysosome fusion with Glugea spores in phagosomes of mouse peritoneal macrophages and of Tetrahymena was monitored using colloidal gold and acridine orange as labels for secondary lysosomes. Over 80% of the Glugea phagosomes remained segregated from the labeled compartments in macrophages after 60 min; this inhibition of fusion was still evident after 4 h. In Tetrahymena, Glugea spores also showed a high capacity to block fusion with secondary lysosomes (67%); however, spores coated with cationized ferritin showed an 80% fusion rate with labeled acidic compartments (i.e. lysosomes) after 60 min with both Tetrahymena and macrophages. The pH of phagosome compartments was monitored by measuring the emissions of fluorescein isothiocyanate (FITC)-labeled Glugea ingested by Tetrahymena. Tetrahymena phagosomes with FITC-Glugea did not acidify within the first hour after phagocytosis; however, phagosomes with cationized ferritin-labeled Glugea underwent acidification during this time period. This acidification took place although the capability of the host cells' lysosomes to fuse was blocked by pretreatment with poly-D-glutamic acid. The cationized ferritin bound to Glugea spores was uncoupled from the spore wall prior to fusion with colloidal gold-labeled compartments. In vitro testing showed that ferritin dissociation requires an acid pH, indicating that phagosomes acidify prior to lysosome fusion.     

Apparent inhibition of Na+/H+ exchange by amiloride and harmaline in acridine orange studies

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na⁺ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na⁺ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K⁺ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I₅₀) of Na⁺/H⁺ exchange and K⁺ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I₅₀ for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na⁺/H⁺ exchange by these drugs.