A Chinese hamster ovary cell mutant with a heat-sensitive, conditional- lethal defect in vacuolar function

We describe a mutant derived from Chinese hamster ovary cells that is offt-sensitive for viability and for resistance to certain protein toxins. This mutant, termed G.7.1, grows normally at 34 degrees C but does not grow in Dulbecco's modified Eagle's medium at 39.5 degrees C. However, when this medium is supplemented with FeSO4, the mutant cells will grow at the elevated temperature. At 39.5 degrees C, G.7.1 cells acquire resistance to diphtheria toxin, modeccin, and Pseudomonas aeruginosa exotoxin A, all of which are protein toxins that require endocytosis and exposure to a low pH within vesicles before they can invade the cytosol and kill cells. The properties of mutant G.7.1 could result from a heat-sensitive lesion that impairs vacuolar acidification. We assayed the ATP-stimulated generation of pH gradients across the membrane of vesicles in cell-free preparations from mutant and parental cells by the partitioning of acridine orange into acidic compartments and found that the acidification response of the mutant cells was heat-labile. Altogether the evidence suggests that G.7.1 cells contain a heat-sensitive lesion that impairs vacuolar acidification and that they fail to grow in normal medium at 39.5 degrees C because they cannot extract Fe+3 from transferrin, a process that normally requires exposing transferrin to a low pH within endosomal vesicles.     

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.     

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.     

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.     

Low cytoplasmic pH inhibits endocytosis and transport from the trans- Golgi network to the cell surface

A fibroblast mutant cell line lacking the Na+/H+ antiporter was used to study the influence of low cytoplasmic pH on membrane transport in the endocytic and exocytic pathways. After being loaded with protons, the mutant cells were acidified at pH 6.2 to 6.8 for 20 min while the parent cells regulated their pH within 1 min. Cytoplasmic acidification did not affect the level of intracellular ATP or the number of clathrin-coated pits at the cell surface. However, cytosolic acidification below pH 6.8 blocked the uptake of two fluid phase markers, Lucifer Yellow and horseradish peroxidase, as well as the internalization and the recycling of transferrin. When the cytoplasmic pH was reversed to physiological values, both fluid phase endocytosis and receptor-mediated endocytosis resumed with identical kinetics. Low cytoplasmic pH also inhibited the rate of intracellular transport from the Golgi complex to the plasma membrane. This was shown in cells infected by the temperature-sensitive mutant ts 045 of the vesicular stomatitis virus (VSV) using as a marker of transport the mutated viral membrane glycoprotein (VSV-G protein). The VSV-G protein was accumulated in the trans-Golgi network (TGN) by an incubation at 19.5 degrees C and was transported to the cell surface upon shifting the temperature to 31 degrees C. This transport was arrested in acidified cells maintained at low cytosolic pH and resumed during the recovery phase of the cytosolic pH. Electron microscopy performed on epon and cryo-sections of mutant cells acidified below pH 6.8 showed that the VSV-G protein was present in the TGN. These results indicate that acidification of the cytosol to a pH less than 6.8 inhibits reversibly membrane transport in both endocytic and exocytic pathways. In all likelihood, the clathrin and nonclathrin coated vesicles that are involved in endo- and exocytosis cannot pinch off from the cell surface or from the TGN below this critical value of internal pH.     

Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin

In mammalian cells, nine conserved isoforms of the Na(+)/H(+) exchanger (NHE) are known to be important for pH regulation of the cytoplasm and organellar lumens. NHE1-5 are localized to the plasma membrane, whereas NHE6-9 are localized to distinct organelles. NHE6 is localized predominantly in endosomal compartments but is also found in the plasma membrane. To investigate the role of NHE6 in endocytosis, we established NHE6-knockdown HeLa cells and analyzed the effect of this knockdown on endocytotic events. The expression level of NHE6 in knockdown cells was decreased to ～15% of the level seen in control cells. Uptake of transferrin was also decreased. No effect was found on the endocytosis of epidermal growth factor or on the cholera toxin B subunit. Moreover, in the NHE6-knockdown cells, transferrin uptake was found to be affected in the early stages of endocytosis. Microscopic analysis revealed that, at 2 min after the onset of endocytosis, colocalization of NHE6, clathrin, and transferrin was observed, which suggests that NHE6 was localized to endocytotic, clathrin-coated vesicles. In addition, in knockdown cells, transferrin-positive endosomes were acidified, but no effect was found on cytoplasmic pH. In cells overexpressing wild-type NHE6, increased transferrin uptake was observed, but no such increase was seen in cells overexpressing mutant NHE6 deficient in ion transport. The luminal pH in transferrin-positive endosomes was alkalized in cells overexpressing wild-type NHE6 but normal in cells overexpressing mutant NHE6. These observations suggest that NHE6 regulates clathrin-dependent endocytosis of transferrin via pH regulation.     

Uptake of a fluorescent marker in plant cells is sensitive to brefeldin A and wortmannin

We assessed FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl)pyridinium dibromide] as a fluorescent endocytosis marker in intact, walled plant cells. At 4 degrees C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26 degrees C, FM1-43 labeled cytoplasmic vesicles and then the vacuole. Fluorimetric quantitation demonstrated dye uptake temperature sensitivity (approximately 65% reduction at 16 degrees C, >90% at 4 degrees C). FM1-43 uptake in suspension cells was stimulated more than twofold by brefeldin A and inhibited approximately 0.4-fold by wortmannin. FM1-43 delivery to the vacuole was largely inhibited by brefeldin A, although overall uptake was stimulated, and brefeldin A treatment caused the accumulation of large prevacuolar endosomal vesicles heavily labeled with FM1-43. Three-dimensional time lapse imaging revealed that FM1-43-labeled vacuoles and vesicles are highly dynamic. Thus, FM1-43 serves as a fluorescent marker for imaging and quantifying membrane endocytosis in intact plant cells.     

Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts

It has been shown that endocytic vesicles in BALB/c 3T3 cells have a pH of 5.0 (Tycko and Maxfield, Cell, 28:643-651). In this paper, a method for measuring the effect of various agents, including weak bases and ionophores, on the pH of endocytic vesicles is presented. The method is based on the increase in fluorescein fluorescence with 490-nm excitation as the pH is raised above 5.0. Intensities of cells were measured using a microscope spectrofluorometer after internalization of fluorescein-labeled alpha 2-macroglobulin by receptor-mediated endocytosis. The increase in endocytic vesicle pH was determined from the increase in fluorescence after addition of various concentrations of the test agents. The following agents increased endocytic vesicle pH above 6.0 at the indicated concentrations: monensin (6 microM), FCCP (10 microM), chloroquine (140 microM), ammonia (5 mM), methylamine (10 mM). The ability of many of these agents to raise endocytic vesicle pH may account for many of their effects on receptor-mediated endocytosis. Dansylcadaverine caused no effect on vesicle pH at 1 mM. The observed increases in vesicle pH were rapid (1-2 min) and could be reversed by removal of the perturbant. This reversibility indicates that the vesicles themselves contain a mechanism for acidification. The increase in vesicle pH due to these treatments can be observed visually using an SIT video camera. Using this method, it is shown that endocytic vesicles become acidic at very early times (i.e., within 5-7 min of continuous uptake at 37 degrees C).     

Acidification of endocytic compartments and the intracellular pathways of ligands and receptors

Several hormones, serum proteins, toxins, and viruses are brought into the cell by receptor-mediated endocytosis. Initially, many of these molecules and particles are internalized into a common endocytic compartment via the clathrin-coated pit pathway. Subsequently, the ligands and receptors are routed to several destinations, including lysosomes, the cytosol, or the plasma membrane. We have examined the mechanism by which sorting of internalized molecules occurs. A key step in the process is the rapid acidification of endocytic vesicles to a pH of 5.0-5.5 This acidification allows dissociation of several ligands from their receptors, the release of iron from transferrin, and the penetration of diphtheria toxin and some viral nucleocapsids into the cytoplasm. Transferrin, a ligand that cycles through the cell with its receptor, has been used as a marker for the recycling receptor pathway. We have found that in Chinese hamster ovary (CHO) cells transferrin is rapidly segregated from other ligands and is routed to a complex of small vesicles and/or tubules near the Golgi apparatus. The pH of the transferrin-containing compartment is approximately 6.4, indicating that it is not in continuity with the more acidic endocytic vesicles which contain ligands destined to be degraded in lysosomes.     

A Chinese Hamster Ovary Cell Mutant Resistant to Phosphatidylserine Is Defective in Transbilayer Movement of Cell Surface Phosphatidylserine

A mammalian plasma membrane protein(s) which catalyzes ATP-dependent transbilayer movement (flip-flop) of phosphatidylserine (PS) has been suggested to be involved in the formation and maintenance of membrane lipid asymmetry. Flip-flop of PS in the cell surface of nucleated cells was first described by O. C. Martin and R. E. Pagano (1987,J. Biol. Chem.262, 5890–5898). It has been suggested that flip-flop is involved in the internalization of exogenous PS in cultured cells. In the present study we report that incubation with an excess amount of PS is cytotoxic to Chinese hamster ovary (CHO) cells, while the same amount of phosphatidylcholine gives no effect. This effect allowed us to obtain PS-resistant cells among mutagenized CHO cells. Endocytosis-independent internalization of exogenous fluorescent PS analog was defective in 40% of the PS-resistant mutants. One of the mutants, PSR (phosphatidylserine resistant) 406 was further characterized. Unlike wild-type CHO cells, this mutant did not transport fluorescent PS significantly at 15°C. Fluorescent PS was not metabolized at 15°C in either wild-type or mutant cells. These results suggest that transbilayer movement of cell surface PS is defective in PS-resistant cells.     

An L-proline-dependent proton flux is located at the apical membrane level of the eel enterocytes

This study has demonstrated the existence of an L-proline-dependent (Na independent) proton flux at the apical membrane level of the eel intestinal absorbing cells. Using isolated eel enterocytes and the pH-sensitive fluorescent dye 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF), it was shown that a 20 mM concentration of the imino acid L-proline in the extracellular medium determined an intracellular acidification of approximately 0.28 pH units. However, neither sucrose nor other amino acids were able to significantly acidify the resting intracellular pH. A hyperbolic relationship between extracellular proline concentration and intracellular proton accumulation was observed. Using both isolated brush-border and basolateral membrane vesicles, it was demonstrated that this proline-proton cotransport mechanism was located at the apical membrane level only. In addition, the existence of a coupling mechanism between proline and proton fluxes was demonstrated by the observation that, in brush-border membrane vesicles, the presence of a pH gradient (pH(in) > pH(out)) stimulated the uptake of L-proline.     

Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane

Digital, video-enhanced fluorescence microscopy and spectrofluorometry were used to follow the internalization into the yeast Saccharomyces cerevisiae of phosphatidylcholine molecules labeled on one acyl chain with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). Two pathways were found: (1) transport by endocytosis to the vacuole and (2) transport by a non-endocytic pathway to the nuclear envelope and mitochondria. The endocytic pathway was inhibited at low temperature (< 2 degrees C) and by ATP depletion. Mutations in secretory (SEC) genes that are necessary for membrane traffic through the secretory pathway (including SEC1, SEC2, SEC4, SEC6, SEC7, SEC12, SEC14, SEC17, SEC18, and SEC21) almost completely blocked endocytic uptake. In contrast, mutations in the SEC63, SEC65, or SEC11 genes, required for translocation of nascent secretory polypeptides into the ER or signal peptide processing in the ER, only slightly reduced endocytic uptake. Phospholipid endocytosis was also independent of the gene encoding the clathrin heavy chain, CHC1. The correlation of biochemical analysis with fluorescence microscopy indicated that the fluorescent phosphatidylcholine was degraded in the vacuole and that degradation was, at least in part, dependent on the vacuolar proteolytic cascade. The non-endocytic route functioned with a lower cellular energy charge (ATP levels 80% reduced) and was largely independent of the SEC genes. Non-endocytic transport of NBD-phosphatidylcholine to the nuclear envelope and mitochondria was inhibited by pretreatment of cells with the sulfhydryl reagents N-ethylmaleimide and p- chloromercuribenzenesulfonic acid, suggesting the existence of protein- mediated transmembrane transfer (flip-flop) of phosphatidylcholine across the yeast plasma membrane. These data establish a link between lipid movement during secretion and endocytosis in yeast and suggest that phospholipids may also gain access to intracellular organelles through non-endocytic, protein-mediated events.     

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.     

Cholesterol controls lipid endocytosis through Rab11

Cellular cholesterol increases when cells reach confluency in Chinese hamster ovary (CHO) cells. We examined the endocytosis of several lipid probes in subconfluent and confluent CHO cells. In subconfluent cells, fluorescent lipid probes including poly(ethylene glycol)derivatized cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol, and fluorescent sphingomyelin analogs were internalized to pericentriolar recycling endosomes. This accumulation was not observed in confluent cells. Internalization of fluorescent lactosylceramide was not affected by cell confluency, suggesting that the endocytosis of specific membrane components is affected by cell confluency. The crucial role of cellular cholesterol in cell confluency-dependent endocytosis was suggested by the observation that the fluorescent sphingomyelin was transported to recycling endosomes when cellular cholesterol was depleted in confluent cells. To understand the molecular mechanism(s) of cell confluency- and cholesterol-dependent endocytosis, we examined intracellular distribution of rab small GTPases. Our results indicate that rab11 but not rab4, altered intracellular localization in a cell confluency-associated manner, and this alteration was dependent on cell cholesterol. In addition, the expression of a constitutive active mutant of rab11 changed the endocytic route of lipid probes from early to recycling endosomes. These results thus suggest that cholesterol controls endocytic routes of a subset of membrane lipids through rab11.     

Chloride concentration in endosomes measured using a ratioable fluorescent Cl- indicator: evidence for chloride accumulation during acidification.

A novel long wavelength fluorescent Cl(-) indicator was used to test whether endosomal Cl(-) conductance provides the principal electrical shunt to permit endosomal acidification. The green fluorescent Cl(-)-sensitive chromophore 10,10'-bis[3-carboxypropyl]-9,9'-biacridinium dinitrate (BAC) was conjugated to aminodextran together with the red fluorescent Cl(-)-insensitive chromophore tetramethylrhodamine (TMR). BAC fluorescence is pH-insensitive and quenched by Cl(-) with a Stern-Volmer constant of 36 m(-1). Endosomes in J774 and Chinese hamster ovary (CHO) cells were pulse-labeled with BAC-TMR-dextran by fluid-phase endocytosis. Endosomal [Cl(-)] increased over 45 min from 17 to 53 mm in J774 cells and from 28 to 73 mm in CHO cells, during which time endosomal pH decreased from 6.95 to 5.30 (J774) and 6.92 to 5.60 (CHO). The acidification and increased [Cl(-)] were blocked by bafilomycin. Together with ion substitution and buffer capacity measurements, we conclude that Cl(-) transport accounts quantitatively for the electrical shunt during vacuolar acidification. Measurements of relative endosomal volume by a novel ratio imaging method involving fluorescence self-quenching indicated a 2.5-fold increase in volume during early acidification and Cl(-) accumulation, which was blocked by bafilomycin. These experiments provide the first direct measurement of endosomal [Cl(-)] and indicate that endosomal acidification is accompanied by significant Cl(-) entry and volume increase.     

Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal

After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles.     

ATP11C mutation is responsible for the defect in phosphatidylserine uptake in UPS-1 cells

Type IV P-type ATPases (P4-ATPases) translocate phospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. We and others previously showed that ATP11C, a member of the P4-ATPases, translocates phosphatidylserine (PS) at the plasma membrane. Twenty years ago, the UPS-1 (uptake of fluorescent PS analogs) cell line was isolated from mutagenized Chinese hamster ovary (CHO)-K1 cells with a defect in nonendocytic uptake of nitrobenzoxadiazole PS. Due to its defect in PS uptake, the UPS-1 cell line has been used in an assay for PS-flipping activity; however, the gene(s) responsible for the defect have not been identified to date. Here, we found that the mRNA level of ATP11C was dramatically reduced in UPS-1 cells relative to parental CHO-K1 cells. By contrast, the level of ATP11A, another PS-flipping P4-ATPase at the plasma membrane, or CDC50A, which is essential for delivery of most P4-ATPases to the plasma membrane, was not affected in UPS-1 cells. Importantly, we identified a nonsense mutation in the ATP11C gene in UPS-1 cells, indicating that the intact ATP11C protein is not expressed. Moreover, exogenous expression of ATP11C can restore PS uptake in UPS-1 cells. These results indicate that lack of the functional ATP11C protein is responsible for the defect in PS uptake in UPS-1 cells and ATP11C is crucial for PS flipping in CHO-K1 cells.     

Functional water channels and proton pumps are in separate populations of endocytic vesicles in toad bladder granular cells

Functional water channels are retrieved by endocytosis from the apical membrane of toad bladder granular cells in response to vasopressin [Shi, L.-B., & Verkman, A.S. (1989) J. Gen. Physiol. 94, 1101-1115]. To examine whether endocytic vesicles which contain the vasopressin-sensitive water channel fuse with acidic vesicles for entry into a lysosomal pathway, ATP-dependent acidification and osmotic water permeability were measured in endosomes from control bladders and bladders treated with vasopressin (VP) and/or phorbol myristate acetate (PMA). Endosomes were labeled with the fluid-phase markers 6-carboxyfluorescein or fluorescein-dextran. Osmotic water permeability (Pf) was measured by stopped-flow fluorescence quenching and proton ATPase activity by ATP-dependent, N-ethylmaleimide-inhibitable acidification. In a microsomal pellet, Pf was low (less than 0.002 cm/s, 20 degrees C) in labeled endocytic vesicles from control bladders but high (0.05-0.1 cm/s) in a subpopulation (50-70%) of vesicles from VP- and PMA-treated bladders. Following ATP addition, the average drop in pH was 0.1 (control), 0.3 (VP), and 0.2 (PMA) unit. Measurement of pH in individual endocytic vesicles by quantitative image analysis showed that less than 20% of vesicles from VP-treated bladders acidified by greater than 0.5 pH unit. To examine whether water channels and proton pumps were present in the same endocytic vesicles, the pH of endosomes with high and low water permeability was measured from the effect of ATP on the amplitude of the fluorescence quenching signal in response to an osmotic gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infectious Cell Entry Mechanism of Influenza Virus

Interaction between influenza virus WSN strain and MDCK cells was studied by using spin-labeled phospholipids and electron microscopy. Envelope fusion was negligibly small at neutral pH but greatly activated in acidic media in a narrow pH range around 5.0. The half-time was less than 1 min at 37°C at pH 5.0. Virus binding was almost independent of the pH. Endocytosis occurred with a half-time of about 7 min at 37°C at neutral pH, and about 50% of the initially bound virus was internalized after 1 h. Electron micrographs showed binding of virus particles in coated pits in the microvillous surface of plasma membrane and endocytosis into coated vesicles. Chloroquine inhibited virus replication. The inhibition occurred when the drug was added not later than 10 min after inoculation. Chloroquine caused an increase in the lysosomal pH 4.9 to 6.1. The drug did not affect virus binding, endocytosis, or envelope fusion at pH 5.0. Electron micrographs showed many virus particles remaining trapped inside vacuoles even after 30 min at 37°C in the presence of drug, in contrast to only a few particles after 10 min in vacuoles and secondary lysosomes in its absence. Virus replication in an artificial condition, i.e., brief exposure of the inoculum to acidic medium followed by incubation in neutral pH in the presence of chloroquine, was also observed. These results are discussed to provide a strong support for the infection mechanism of influenza virus proposed previously: virus uptake by endocytosis, fusion of the endocytosed vesicles with lysosome, and fusion of the virus envelope with the surrounding vesicle membrane in the secondary lysosome because of the low pH. This allows the viral genome to enter the target cell cytoplasm.