Failure to release iron from transferrin in a Chinese hamster ovary cell mutant pleiotropically defective in endocytosis.

Klausner, Richard D.; Renswoude, Jos van; Kempf, Ch.; Rao, K. K.; Bateman, Judith L.; Robbins, April R.

doi:10.1083/jcb.98.3.1098

Cited by 76 publications

(36 citation statements)

References 18 publications

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“…3). We speculate that the higher pH large endosomes are the sorting endosomes; this would be consistent with the inefficiency of the cells in releasing iron from "IT (13,40) and in delivering endocytosed lysosomal enzymes to lysosomes (27,28). Based on this we assign a pH >6.2 to the sorting endosomes and a pH of 5.2-6.0 to the late prelysosomal endosomes in the mutants.…”

Section: Discussionmentioning

confidence: 77%

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

Yamashiro

Maxfield

1987

The Journal of Cell Biology

112

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we have shown that there is rapid acidification of endosomal compartments to pH 6.3 by 3 min in wildtype 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 r 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 >6.5 in the CHO mutants when compared with wildtype 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.I N the preceding paper (40) we examined the kinetics of endosome acidification in wild-type and mutant Chinese hamster ovary (CHO) cells. We found in wild-type cells that there was a rapid (3-5 min) acidification of endosomes to pH 6.2-6.3. With time, ct~-macroglobulin (a2M) ~ and fluorescein-isothiocyanate dextran (F-Dex), molecules that are routed to lysosomes, are found in progressively more acidic endocytic compartments (pH < 6.0), while transferrin Dr. Maxfieid's present address is Departments of Pathology and Physiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.1. Abbreviations used in this paper: a2M, u2-macroglobulin; a2M-gold, a2-macroglobulin adsorbed to colloidal gold; AA/MA, ammonium acetate/methylamine; F-a2M, fluorescein-labeled ct2-macroglobulin; F-Dex, fluorescein isothioeyanate dextran; F-Tf, fluorescein-labeled transferr...

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Section: Discussionmentioning

confidence: 77%

“…Endosomes of the CHO mutants DTG 1-5-4 and DTF 1-5-1 (13,27,28) also show differences in pH, along with a marked decrease in early acidification (40). These and other studies on the kinetics of endosome acidification (12,29,32) show that endosomes within the same cell can vary in their pH.…”

mentioning

confidence: 80%

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

Yamashiro

Maxfield

1987

The Journal of Cell Biology

112

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“…The isolation and characterization of these cells has been published (7,21,22,25). Mutant cell lines were retested for resistance to diphtheria toxin by incubating cells with diphtheria toxin (100 ng/ml) for 24 h. The majority of mutant cells survived toxin treatment, while we estimate that >95 % of WTB cells were killed.…”

Section: Cellsmentioning

confidence: 99%

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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“…Two additional roles for endosome acidification have been proposed based on studies of drug-treated cells or mutant cell lines, but these are not well understood at present. Inhibition of endosome acidification has been associated with slowed endocytic recycling and intracellular retention of recycling receptors (12)(13)(14). In addition, the delivery of endocytosed molecules to late endosomes or lysosomes is affected by treatments that impair acidification (15,16).…”

mentioning

confidence: 99%

Bafilomycin A1 Treatment Retards Transferrin Receptor Recycling More than Bulk Membrane Recycling

Presley

Mayor

McGraw

et al. 1997

Journal of Biological Chemistry

162

120

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After internalization via clathrin-coated pits, endocytosed material rapidly enters sorting endosomes, organelles that sort recycled molecules such as transferrin (Tf) 1 bound to the transferrin receptor (Tf-R) from lysosomally destined molecules such as low density lipoprotein (1-3). We have provided evidence for a sorting mechanism in which components not carrying special retention or targeting signals are removed from the sorting endosome by a default process based on the geometry of the sorting endosome and an iterative fractionation mechanism (1, 3). Most of the fluid brought into the tubulo-vesicular sorting endosome fills a spherical lumen, whereas most of the surface area is on tubular extensions that are involved in the removal of membrane components from the sorting endosome for export along the recycling pathway (1-3). Tf remains attached to its receptor throughout the recycling pathway, but it is converted to apo-Tf by the low pH of endosomes (4, 5). Occupied Tf-R are transported from sorting endosomes to the endocytic recycling compartment, which in CHO cells is a collection of tubules concentrated near the centriole (6). The Tf-R are returned to the cell surface from the endocytic recycling compartment, and apo-Tf dissociates from the receptor upon return to the surface (5). It has been shown that internalized lipid analogs such as 6-[N-[7-nitrobenzo-2-oxa-1,3-diazol-4-yl]-amino]hexoyl-sphingosylphosphorylcholine (C 6 -NBD-SM) follow the same endocytic recycling pathway as Tf, indicating that in the absence of specialized signals membrane molecules will recycle efficiently from endosomes to the cell surface (3). Endocytosed fluid and ligands released from their receptors by low endosomal pH are retained in the lumen of the sorting endosome and accumulate. The vesicular portions of the sorting endosomes become the vesicles that fuse with late endosomes (7-9).Acidification of endosomes by ATP-dependent proton pumps is important for several aspects of endocytic trafficking. Two well established functions of the low pH of endosomes are to dissociate lysosomally destined ligands from membrane-associated receptors and to strip Fe 3ϩ from transferrin (4, 5, 10, 11). Two additional roles for endosome acidification have been proposed based on studies of drug-treated cells or mutant cell lines, but these are not well understood at present. Inhibition of endosome acidification has been associated with slowed endocytic recycling and intracellular retention of recycling receptors (12)(13)(14). In addition, the delivery of endocytosed molecules to late endosomes or lysosomes is affected by treatments that impair acidification (15, 16). Unfortunately, both of these effects appear to vary among cell lines.Several studies have shown that weak bases and ionophores * This work was supported by National Institutes of Health Grant DK27083 (to F. R. M.) and American Cancer Society Grant CB-8 (to T. E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must th...

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Failure to release iron from transferrin in a Chinese hamster ovary cell mutant pleiotropically defective in endocytosis.

Cited by 76 publications

References 18 publications

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

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

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

Bafilomycin A1 Treatment Retards Transferrin Receptor Recycling More than Bulk Membrane Recycling

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