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At least 73 records · Page 4Linked to original sources
PubMed1995

Selective activation of the rat hepatic endosomal insulin receptor kinase. Role for the endosome in insulin signaling.

Insulin administration activates the insulin receptor kinase (IRK) in both plasma membrane (PM) and endosomes (ENs) raising the possibility of transmembrane signaling occurring in the endosomal compartment. Peroxovanadium compounds activate the IRK by inhibiting IR-associated phosphotyrosine phosphatase(s). Following the administration of the phosphotyrosine phosphatase inhibitor bisperoxo(1,10-phenanthroline)-oxovanadate (V) anion (bpV(phen)) activation of the hepatic IRK in ENs preceded that in PM by 5 min. When colchicine treatment preceded bpV(phen) administration IRK activation in ENs was unaffected but was totally abrogated in PM. Insulin receptor substrate-1 tyrosine phosphorylation followed the kinetics of IRK activation in ENs not PM and a hypoglycemic response similar to that achieved with a pharmacological dose of insulin ensued. These studies demonstrate that ENs constitute a site for IR-mediated signal transduction.

Animals↗
PubMed1992

Inhibition of endosomal proteolytic activity by leupeptin blocks surface expression of MHC class II molecules and their conversion to SDS resistance alpha beta heterodimers in endosomes.

The biosynthesis of MHC Class II molecules starts with the assembly of the alpha and beta subunits and the invariant chain. Intracellular transport of Class II molecules was followed in pulse-chase experiments of a human Epstein-Barr virus-transformed B lymphoblastoid cell line. Entry of Class II molecules into the endocytotic pathway and their cell surface appearance were monitored using neuraminidase as a fluid endocytotic marker and as a surface probe, respectively. In the course of intracellular transport, the Class II associated invariant chain is removed by proteases located in the endosomal pathway. Here, we show that leupeptin inhibits not only invariant chain breakdown, but also surface deposition of newly synthesized Class II molecules. Class II molecules display remarkable resistance to SDS at ambient temperature when occupied by peptide. We exploit this property to show that peptide binding precedes surface expression, and takes place in the course of intracellular transport through an endosomal compartment. Leupeptin blocks the conversion of Class II molecules to an SDS resistant complex.

Animals↗
PubMed2006

Endosomal and non-endosomal functions of ESCRT proteins.

The three endosomal sorting complexes required for transport (ESCRTs) are integral to the degradation of endocytosed membrane proteins and multivesicular body (MVB) biogenesis. Here, we review evidence that ESCRTs have evolved as a specialized machinery for the degradative sorting of ubiquitinated membrane proteins and we highlight recent studies that have shed light on the mechanisms by which these complexes mediate protein sorting, MVB biogenesis, tumour suppression and viral budding. We also discuss evidence that some ESCRT subunits have evolved additional functions that are unrelated to membrane trafficking.

Animals↗
PubMed2000

Inhibition of endosome fusion in primary hepatocytes prevents asialoglycoprotein degradation but not uptake of transferrin iron demonstrating that intracellular iron release occurs from early endosomes.

A comparison of the effects of inhibitors of membrane fusion on the uptake of asialoglycoprotein and transferrin by primary rat hepatocytes was made. This showed that while high potassium medium inhibited the degradation but not the uptake of asialoorosomucoid, both transferrin endocytosis and iron delivery to the cells were unaffected. This difference between the two pathways was also observed with an inhibitor of phospholipase A2, bromophenacyl bromide. With the latter, it was found that the asialoglycoproteins failed to traverse from a low-density to a high-density intracellular compartment, implying a role for phospholipase A2 in the trafficking of asialoglycoprotein receptor but not that for transferrin or iron. This demonstrates that, after its release from transferrin, iron is transported to the cytoplasm directly from the early endosome without the need for fusion of the iron-containing vesicle with a lysosome.

Acetophenones↗
PubMed2000

Inactivation of lmpA, encoding a LIMPII-related endosomal protein, suppresses the internalization and endosomal trafficking defects in profilin-null mutants.

Profilin is a key phosphoinositide and actin-binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. Using biochemical assays and microscopic approaches, we demonstrate that profilin-null cells are defective in macropinocytosis, fluid phase efflux, and secretion of lysosomal enzymes but are unexpectedly more efficient in phagocytosis than wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging to the CD36/LIMPII family suppressed, to different degrees, most of the profilin-minus defects, including the increase in F-actin, but did not rescue the secretion defect. Immunofluorescence microscopy indicated that DdLIMP, which is also capable of binding phosphoinositides, was associated with macropinosomes but was not detected in the plasma membrane. Also, inactivation of the lmpA gene in wild-type strains resulted in defects in macropinocytosis and fluid phase efflux but not in phagocytosis. These results suggest an important role for profilin in regulating the internalization of fluid and particles and the movement of material along the endosomal pathway; they also demonstrate a functional interaction between profilin and DdLIMP that may connect phosphoinositide-based signaling through the actin cytoskeleton with endolysosomal membrane trafficking events.

Biological Transport↗
PubMed1989

Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants.

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH less than or equal to 6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH less than or equal to 5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH less than or equal to 6.2 in early endosomes with a t1/2 of 5 min. Although a fraction of the virus reached a pH of less than or equal to 5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t1/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41 degrees C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH less than or equal to 5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H+ pump to fluctuations in the endosomal membrane potential.

Animals↗
PubMed1991

Tubular early endosomal networks in AtT20 and other cells.

Using horseradish peroxidase (HRP) as a fluid-phase endocytic tracer, we observed through the electron microscope numerous tubular endosomes with a diameter of 30-50 nm and lengths of greater than 2 microns in thick sections (0.2-0.5 microns) of AtT20 cells. These tubular endosomes are multibranching and form local networks but not a single reticulum throughout the cytoplasm. They are sometimes in continuity with vesicular endosomal structures but have not been observed in continuity with AtT20 cell late endosomes. Tubular endosomal networks are not uniformly distributed throughout the cytoplasm, but are particularly abundant in growth cones, in patches below the plasma membrane of the cell body, and surrounding the centrioles and microtubule organizing center (MTOC). Tubular endosomes at all these locations receive HRP within the first 5 min of endocytosis but approximately 30 min of endocytosis are required to load the tubular endosomal networks with HRP so that their full extent can be visualized in the electron microscope. After 10 min of endocytosis, complete unloading occurs within 30 min of chase, but between 30 and 60 min are required to chase out all the tracer from the tubular endosomes loaded to steady state during 60 min endocytosis of 10 mg/ml HRP. In interphase cells, neither the loading nor unloading of tubular endosomes depends on microtubules but in cells blocked in mitosis by depolymerization of the mitotic spindle with nocodazole, HRP does not chase out of tubular endosomes. The thread-like shape of tubular endosomes is not dependent on microtubules. Furthermore, HRP is delivered to AtT20 tubular endosomes at 20 degrees C. All these properties indicate that AtT20 cell tubular endosomes are an early endocytic compartment distinct from late endosomes. Tubular endosomes like those in AtT20 cells have been seen in cells of the following lines: PC12, HeLa, Hep2, Vero, MDCK I and II, CCL64, RK13, and NRK; they are particularly abundant in the first three lines. In contrast, tubular endosomes are sparse in 3T3 and BHK21 cells. The tubular endosomes we have observed appear to be identical to the endosomal reticulum observed in the living Hep2 cells by Hopkins, C. R., A. Gibson, H. Shipman, and K. Miller. 1990.

3T3 Cells↗
PubMed1995

Imaging of endosome fusion in BHK fibroblasts based on a novel fluorimetric avidin-biotin binding assay.

A fluorescence assay of in vivo endosome fusion was developed and applied to define the kinetics of endosome fusion in baby hamster kidney (BHK) fibroblasts. The assay is based on an approximately 10-fold enhancement of the green fluorescence of BODIPY-avidin upon biotin binding. The BODIPY-avidin fluorescence enhancement occurred in < 25 ms, was pH-independent, and involved a BODIPY-tryptophan interaction. For endocytosis in vivo, BHK fibroblasts were pulse-labeled with BODIPY-avidin together with a red (rhodamine) fluorescent fusion-independent chromophore (TMR). After specified chase times in a nonfluorescent medium, a second cohort of endosomes was pulse-labeled with biotin-conjugated albumin, dextran, or transferrin. Fusion of biotin-containing endosomes with avidin-containing endosomes was quantified by ratio imaging of BODIPY-to-TMR fluorescence in individual endosomes, using imaging methods developed for endosome pH studies. Analysis of BODIPY-to-TMR ratio distributions in avidin-labeled endosomes exposed to zero and maximum biotin indicated > 90% sensitivity for detection of endosome fusion. In avidin pulse (10 min) -chase-biotin albumin pulse (10 min) studies, both fused and unfused endosomes were identified; the fractions of avidin-labeled endosomes that fused with biotin-labeled endosomes were 0.48, 0.21, 0.16, and 0.07 for 0-, 5-, 10-, and 20-min chase times. Fitting of fusion data to a mathematical model of in vivo endosome fusion required the existence of an intermediate fusion compartment. Pulse-chase studies performed with biotin-transferrin to label the early/recycling endosomes indicated that after a 10-min chase, avidin-labeled endosomes reached a compartment that was inaccessible to biotin-transferrin. The assay was also applied to determine whether endosome fusion was influenced by temperature, pH (bafilomycin A1), second messengers (cAMP agonists, phorbol 12-myristate 13-acetate, staurosporine), and growth-related factors (platelet-derived growth factor, genistein). The results establish a sensitive fluorescence assay to quantify the fusion of vesicular compartments in living cells.

Animals↗
PubMed2004

Ca2+ and N-ethylmaleimide-sensitive factor differentially regulate disassembly of SNARE complexes on early endosomes.

The endosome-associated protein Hrs inhibits the homotypic fusion of early endosomes. A helical region of Hrs containing a Q-SNARE motif mediates this effect as well as its endosomal membrane association via SNAP-25, an endosomal receptor for Hrs. Hrs inhibits formation of an early endosomal SNARE complex by displacing VAMP-2 from the complex, suggesting a mechanism by which Hrs inhibits early endosome fusion. We examined the regulation of endosomal SNARE complexes to probe how Hrs may function as a negative regulator. We show that although NSF dissociates the VAMP-2.SNAP-25.syntaxin 13 complex, it has no effect on the Hrs-containing complex. Whereas Ca(2+) dissociates the Hrs-containing complex but not the VAMP-2-containing SNARE complex. This is the first demonstration of differential regulation of R/Q-SNARE and all Q-SNARE-containing SNARE complexes. Ca(2+) also reverses the Hrs-induced inhibition of early endosome fusion in a tetanus toxin-sensitive manner and removes Hrs from early endosomal membranes. Moreover, Hrs inhibition of endosome fusion and its endosomal localization are sensitive to bafilomycin, implying a role for luminal Ca(2+). Thus, Hrs may bind a SNARE protein on early endosomal membranes negatively regulating trans-SNARE pairing and endosomal fusion. The release of Ca(2+) from the endosome lumen dissociates Hrs, allowing a VAMP-2-containing complex to form enabling fusion.

Calcium↗
PubMed2007

Endosomal receptor kinetics determine the stability of intracellular growth factor signalling complexes.

There is an emerging paradigm that growth factor signalling continues in the endosome and that cell response to a growth factor is defined by the integration of cell surface and endosomal events. As activated receptors in the endosome are exposed to a different set of binding partners, they probably elicit differential signals compared with when they are at the cell surface. As such, complete appreciation of growth factor signalling requires understanding of growth factor-receptor binding and trafficking kinetics both at the cell surface and in endosomes. Growth factor binding to surface receptors is well characterized, and endosomal binding is assumed to follow surface kinetics if one accounts for changes in pH. Yet, specific binding kinetics within the endosome has not been examined in detail. To parse the factors governing the binding state of endosomal receptors we analysed a whole-cell mathematical model of epidermal growth factor receptor trafficking and binding. We discovered that the stability of growth factor-receptor complexes within endosomes is governed by three primary independent factors: the endosomal dissociation constant, total endosomal volume and the number of endosomal receptors. These factors were combined into a single dimensionless parameter that determines the endosomal binding state of the growth factor-receptor complex and can distinguish different growth factors from each other and different cell states. Our findings indicate that growth factor binding within endosomal compartments cannot be appreciated solely on the basis of the pH-dependence of the dissociation constant and that the concentration of receptors in the endosomal compartment must also be considered.

Endosomes↗
PubMed2003

Receptor-mediated endocytosis by the asialoglycoprotein receptor: effect of ethanol administration on endosomal distribution of receptor and ligand.

Using the asialoglycoprotein receptor (ASGP-R) and a representative ligand, asialoorosomucoid (ASOR), we have previously shown ethanol-induced impairment of endosomal acidification, receptor recycling and ligand binding, internalization, and degradation. In the current study, we further investigated ethanol-induced alterations in receptor/ligand trafficking by labeling endosomes in vivo with either Texas-Red-ASOR or 125I-ASOR, and then assessing the receptor/ligand content of endosomes. We assessed two fractions after both 5 and 25 min of labeling: 'early endosomes' (EEs; endosomes from the cell periphery) and 'late endosomes' (LEs; endosomes farther into the cell interior). At both time points, significantly more ligand was found in EE fractions isolated from chow- and pair-fed controls (3:1, EE to LE, respectively). However, endosomes isolated from ethanol-fed animals showed a shift over time toward a more equal ligand distribution between endosome fractions (P < or = 0.05). Analysis of the ASGP-R content revealed a distribution pattern between the endosome fractions similar to that observed for ligand distribution. Impairment of receptor-ligand dissociation was assessed in endosome fractions by determining bound/free ligand ratios. Analysis showed that most of the ligand present in both endosome fractions was free (56-99%), although more was bound to receptor in EE vs LE of both control and ethanol animals (P < or = 0.05). At 5 min, more ligand remained bound in endosomes from ethanol-fed animals compared with control endosomes (P < or = 0.05), and the same pattern was observed at the latter time point. These results suggest that delayed dissociation may cause the receptor ligand complexes to travel farther into the cell interior, which may impair proper trafficking of the ligand to lysosomes and alter the receptor recycling.

Animals↗
PubMed1994

Energy transfer assays of rat renal cortical endosomal fusion: evidence for superfusion.

The complex of components necessary to allow endosomal fusion includes both membrane-bound receptors and several soluble proteins. Although these factors have been isolated from cultured cell lines, and endosomal fusion has been reconstituted in vitro for vesicular systems from yeast to synaptosomes, there is a paucity of data from mammalian systems. To investigate fusion in rat renal cortical endosomes, we began by developing a fusion assay. As the immunoglobulin and avidin-based probes almost universally employed in fusion assays are excluded by the glomerular ultrafiltration barrier, it was necessary to begin by finding ultrafilterable probes which could serve as a fusion assay. We labeled the apical endosomal pathway of the renal proximal tubule by intravenous infusion of ultrafilterable fluorescent dextrans. Energy transfer from entrapped fluorescein-dextran to rhodamine-dextran had a narrow concentration dependence but allowed fluorometric assay of endosomal fusion. The "spectroscopic ruler" property of energy transfer, whereby it will only occur at < 60 A, makes fusion measurements unequivocal. The energy transfer efficiency of fluorometric (48 +/- 1%) and flow cytometry (57 +/- 1%) assays were close to the theoretical optimum (57%). Energy transfer is detected as a decrease in fluorescence of the fluorescein donor and an increase in fluorescence of the rhodamine acceptor. Our endosomal fusion assay was utilized to determine the optimal conditions for fusion of rat renal cortical light endosomes and heavy endosomes. Independent measurements of fluorescein-dextran and rhodamine-dextran on an endosome-by-endosome basis using dual-beam two-color flow cytometry demonstrated that each fusion event involves multiple endosomes rather than a single pair of endosomes. Electron microscopy analysis demonstrated that the average vesicle diameter was five times larger in the fused heavy endosomal fractions compared with control fractions without fusion. Hence, fusion of mammalian renal cortical endosomes reconstituted in vitro is consistent with multiple fusion events dubbed superfusion.

Adenosine Triphosphatases↗
PubMed1986

Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures.

A protocol employing discontinuous sucrose gradient centrifugation was developed to prepare light mitochondrial (L) and Golgi fraction endosomes from simultaneously prepared parent L and microsomal fractions. As judged by the concentration of labeled hormone postinjection, L intermediate and heavy endosome subfractions were 40- to 175-fold purified and Golgi intermediate and heavy endosome subfractions were 30- to 45-fold purified. On electron microscopy, L endosomal fractions contained a predominance of lipoprotein-filled vesicles and were less heterogeneous than corresponding Golgi endosomal fractions. All endosomal fractions were enriched in receptors for insulin and prolactin but binding sites for the former were more broadly distributed in other subfractions than those for the latter. On Percoll gradient centrifugation, L endosomal fractions yielded one peak (rho 1.057) corresponding to the heavier of two peaks seen in Golgi endosomal fractions. The protein composition of high density L and Golgi endosomes, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar. The bulk of marker enzymes assayed did not migrate with the endosomal components. Combined acid phosphatase cytochemistry and electron microscope radioautography established that about 80% of the L endosomes contained no acid phosphatase. By affinity labeling and immunological titration with insulin receptor antibody, insulin receptors were identical in L and Golgi endosomes. Insulin-stimulable receptor kinase was demonstrable in both L and Golgi endosome fractions. Following in vivo insulin administration, the insulin receptor kinase in both L and Golgi endosomes was significantly activated. This activated state was not inhibited by a large excess of antiserum to insulin and thus not due to insulin contaminating the partially purified receptor preparation. These observations are compatible with the maintenance and/or initiation of hormone-dependent phosphorylations intracellularly.

Animals↗
PubMed1997

Free-flow electrophoretic analysis of endosome subpopulations of rat hepatocytes.

The separation of functional early and late endosomes from other cellular compartments by free-flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells. Here, using 125I-labeled anti-secretory component antibodies ([125I]SC Ab) and FITC-labeled asialoorosomucoid (FITC-ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16 degrees C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20 degrees C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37 degrees C, transcytotic endosomes carrying [125I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC-ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common (i.e. early endosomes) and two distinct endosome subpopulations (i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.

Animals↗
PubMed1996

Isolation of heavy endosomes from dog proximal tubules in suspension.

During the preparation of a suspension of dog kidney proximal tubules by collagenase treatment, an uptake of FITC-albumin was demonstrated. This process is attributed to the activation of receptor-mediated endocytosis leading to the appearance of FITC-albumin into intracellular vesicular structures. The isolation of brush border membrane vesicles (BBMV) from the dog kidney proximal tubules in suspension by the magnesium precipitation technique leads to the copurification of a large population of endosomes. These endosomes were separated from BBM vesicles by a technique involving wheat-germ agglutinin. The enrichment in BBM markers and in bafilomycin-sensitive ATPase activity was comparable in endosomes and BBM vesicles. However, the acridine orange acidification assay showed a V-type ATPase-dependent acidification in endosomes but not in BBMV, demonstrating a different orientation of the proton pumps in these structures. SDS-PAGE analysis also showed significant differences in protein pattern of vesicles and endosomes. The most notable difference was the presence of 42-44 kDa and 20-24 kDa proteins in BBMV and their complete absence in endosomes. Western blot analysis identified these proteins as actin and RhoA, among other small proteins, respectively. Western blot experiments also demonstrated a different distribution of beta-COP, beta-adaptin, and RhoGDI in vesicles and endosomes. The morphological aspect (electron microscopy) and sedimentation of endosomes in a 50% Percoll gradient identified these structures as "heavy endosomes" (buoyant density D = 1.036 g/ml). Flow cytometry analysis of heavy endosomes purified from tubules isolated in presence of FITC-albumin showed the presence of FITC-albumin in up to 92% of these intracellular organelles. Western blot analysis using anti-FITC and anti-collagenase antibodies allowed quantification of the FITC-albumin and collagenase A in the purified endosomes. Our results indicate that heavy endosomes are formed during the preparation of the proximal tubules following activation of receptor-mediated endocytosis, probably by soluble proteins. The suspension of tubules thus offers a experimental tool to study the protein reabsorption and traffic of endosomal vesicles in the proximal tubules.

Animals↗
PubMed2004

ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation.

We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [Cl-] in control versus ClC-3-deficient hepatocytes and in control versus ClC-3-transfected Chinese hamster ovary cells. Endosomes were labeled with pH or [Cl-]-sensing fluorescent transferrin (Tf), which targets to early/recycling endosomes, or alpha2-macroglobulin (alpha2M), which targets to late endosomes. In pulse label-chase experiments, [Cl-] was 19 mM just after internalization in alpha2M-labeled endosomes in primary cultures of hepatocytes from wild-type mice, increasing to 58 mM over 45 min, whereas pH decreased from 7.1 to 5.4. Endosomal acidification and [Cl-] accumulation were significantly impaired in hepatocytes from ClC-3 knock-out mice, with [Cl-] increasing from 16 to 43 mM and pH decreasing from 7.1 to 6.0. Acidification and Cl- accumulation were blocked by bafilomycin. In Tf-labeled endosomes, [Cl-] was 46 mM in wild-type versus 35 mM in ClC-3-deficient hepatocytes at 15 min after internalization, with corresponding pH of 6.1 versus 6.5. Approximately 4-fold increased Cl- conductance was found in alpha2M-labeled endosomes isolated from hepatocytes of wild-type versus ClC-3 null mice. In contrast, Golgi acidification was not impaired in ClC-3-deficient hepatocytes. In transfected Chinese hamster ovary cells expressing ClC-3A, endosomal acidification and [Cl-] accumulation were enhanced. [Cl-] in alpha2M-labeled endosomes was 42 mM (control) versus 53 mM (ClC-3A) at 45 min, with corresponding pH 5.8 versus 5.2; [Cl-] in Tf-labeled endosomes at 15 min was 37 mM (control) versus 49 mM (ClC-3A) with pH 6.3 versus 5.9. Our results provide direct evidence for involvement of ClC-3 in endosomal acidification by Cl- shunting of the interior-positive membrane potential created by the vacuolar H+ pump.

Acids↗