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

Marnell, M H; Mathis, Lucia; Stookey, M; Shia, Shang Pwu; Stone, Dennis K.; Draper, Rockford K.

doi:10.1083/jcb.99.6.1907

Cited by 59 publications

(34 citation statements)

References 38 publications

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“…Several groups have obtained CHO mutants pleiotropically defective in endocytosis by selecting cells that were resistant to diphtheria toxin (2,9,14,21). These mutants are also resistant to other toxins and certain viruses that require an acidic pH for toxicity or infectivity.…”

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

Yamashiro

Maxfield

1987

The Journal of Cell Biology

103

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

Yamashiro

Maxfield

1987

The Journal of Cell Biology

103

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“…Some mutants were found to be defective in the uptake of iron from transferrin, a phenotype linked to their defect in vacuolar acidification, and some exhibited altered Golgi apparatus functions as well. Selection for resistance to both modeccin and diphtheria toxin enabled Draper and co-workers (7,22) to isolate Chinese hamster ovary cell lines that were temperature sensitive for viability, resistance to protein toxins (diphtheria toxin, modeccin, Pseudomonas exotoxin A), and ATP-stimulated acidification of endocytic vesicles. The temperature-sensitive lesion in viability could be overcome by the addition of FeSO4 to the growth media.…”

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A chloroquine-resistant Swiss 3T3 cell line with a defect in late endocytic acidification

Cain

1988

The Journal of Cell Biology

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Abstract. To investigate the role of acidification in cell proliferation, several cell lines resistant to chloroquine were isolated with the expectation that some would express altered endocytic acidification. The preliminary characterization of one of these lines, CHL60-64, is described. In contrast to endocytic mutants described previously, the initial phase of endocytic acidification, as measured by transferrin acidification, is normal in this cell line. However, a difference in subsequent endocytic acidification was observed in CHL60-64. In the parental cells, internalized dextran was fully acidified to approximately pH 5.5 within 1 h. In CHL60-64, the pH in the endocytic compartment was only 6.1 after 1 h and remained as high as 5.8 for at least 4 h. After an 8-h incubation, the pH decreased to 5.5, indicating that the second phase of acidification is only slowed in CHL60-64, and not blocked. Consistent with this retarded acidification, ATP-dependent acidification in vitro (as measured by acridine orange accumulation) was reduced in both the lysosomal fraction and the endosomal fraction isolated from CHL60-64. A decrease in the in vivo rate of acridine orange accumulation after perturbation with amine was also observed. In addition to amine resistance and defective acidification, CHL60-64 was found to be resistant to vacuolation in the presence of chloroquine and ammonium chloride, and was resistant to ouabain. Further studies on this new class of endocytosis mutant, in combination with existing mutants, should help to clarify the mechanisms responsible for the regulation of endocytic acidification. M^Nv viruses and toxins require internalization and exposure to an acidic pH in order to penetrate into the cytosol (8,20,24). A number of cell lines that are defective in endocytosis and/or endocytic acidification have been isolated by taking advantage of this fact. Robbins et al. (6,15,16,19) have described a number of cell lines, selected for diphtheria toxin resistance and impaired mannose-6-phosphate receptor activity, that are resistant to infection by Sindbis virus and vesicular stomatitis virus, as well as protein toxins. Some mutants were found to be defective in the uptake of iron from transferrin, a phenotype linked to their defect in vacuolar acidification, and some exhibited altered Golgi apparatus functions as well. Selection for resistance to both modeccin and diphtheria toxin enabled Draper and co-workers (7, 22) to isolate Chinese hamster ovary cell lines that were temperature sensitive for viability, resistance to protein toxins (diphtheria toxin, modeccin, Pseudomonas exotoxin A), and ATP-stimulated acidification of endocytic vesicles. The temperature-sensitive lesion in viability could be overcome by the addition of FeSO4 to the growth media. Merion et al. (9) have characterized Chinese hamster ovary acidification mutants resistant to Pseudomonas exotoxin A, which were found to be cross-resistant to diphtheria toxin and several animal viruses. Monensin-resistant mutants have been isolated th...

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“…Several lines of evidence point to the importance of a low endosomal pH in the effective delivery of iron: the increase in the rate of iron release from transfemn upon acidification in vitro (6); the inhibition of iron release caused by lysosomotropic amines known to raise intravesicular pH in vivo (1,(7)(8)(9); and the existence of Chinese hamster ovary (CHO) cell mutants defective in acidification, which cannot efficiently extract iron from transferrin (10)(11)(12). In addition, DautryVarsat et al (13) have demonstrated that exposure of cells to pH 5.5 results in release of 70% of transferrin-bound iron from receptor-bound transferrin.…”

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High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling.

Sipe

Murphy

1987

Proc. Natl. Acad. Sci. U.S.A.

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The kinetics of acidification of diferric human transferrin in BALB/c mouse 3T3 cells were determined by flow cytometry using a modification of the fluorescein-rhodamine fluorescence ratio technique. For cells labeled at 0°C and warmed to 37C, the minimum pH observed was 6.1 ± 0.2, occurring 5 min after warmup. This step was followed by a slower alkalinization to the pH of the external medium, occurring with a half-time of 5 min. Warmup to 24°C or 17°C resulted in slowing of the time of onset of acidification such that the minimum pH was 6.3 ± 0.2, attained 15 and 25 min after warmup, respectively; the alkalinization step was completely blocked. The limited acidification observed for transferrin corresponds to the initial phase of acidification normally observed for other (nonrecycled) ligands. Since transferrin is not further acidified, the results confirm the existence of two phases of acidification during endocytosis. Measurements of transferrin dissociation at neutral pH after exposure to mildly acidic pH support the conclusion that the transferrin cycle may be completed without exposure of transferrin to a pH below 6. The mildly acidic pH of the endocytic compartments involved in recycling may play a role in regulating enzymatic processing of endocytosed material.Iron delivery to vertebrate cells is primarily mediated by serum transferrin. Uptake of diferric transferrin has been shown to occur through receptor-mediated endocytosis in many different cell types (1-3), and both transferrin and receptor undergo subsequent acidification in the endocytic compartment (4, 5). Unlike most ligands, transferrin does not dissociate from its receptor upon acidification and therefore avoids lysosomal degradation by recycling with the receptor.Several lines of evidence point to the importance of a low endosomal pH in the effective delivery of iron: the increase in the rate of iron release from transfemn upon acidification in vitro (6); the inhibition of iron release caused by lysosomotropic amines known to raise intravesicular pH in vivo (1, 7-9); and the existence of Chinese hamster ovary (CHO) cell mutants defective in acidification, which cannot efficiently extract iron from transferrin (10-12 MATERIALS AND METHODS Preparation of Transferrin Conjugates. Human diferric transfemn was obtained from Miles Scientific (>98% pure). FITC and lissamine rhodamine sulfonyl chloride (LRSC) were obtained from Molecular Probes (Junction City, OR). Transferrin was conjugated with either FITC (FITC-Tf) or LRSC (LRSC-Tf) in 0.1 M sodium carbonate (pH 9.6). Free dye was removed using G-25 columns. The fluorochrome/ protein molar ratio was 4.3 for the FITC conjugate and 5.9 for the LRSC conjugate. These ratios were found to yield optimal specificity (90% or greater), and a signal/noise ratio (ratio of fluorescence from bound labeled transferrin to autofluorescence of unlabeled cells) of -3.0 for each conjugate. The mean number of transferrin binding sites per cell was determined to be 13,000 by using FITC-conjugated calibration b...

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

Abstract: We describe a mutant derived from Chinese hamster ovary cells that is heatsensitive for viability and for resistance to certain protein toxins. This mutant, termed G.7

Cited by 59 publications

References 38 publications

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

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

A chloroquine-resistant Swiss 3T3 cell line with a defect in late endocytic acidification

High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling.

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