A single mutation in Chinese hamster ovary cells impairs both Golgi and endosomal functions.

Robbins, April R.; Oliver, Constance; Bateman, Judith L.; Krag, Sharon S.; Galloway, Cynthia J.; Mellman, Ira

doi:10.1083/jcb.99.4.1296

Cited by 121 publications

(99 citation statements)

References 57 publications

(68 reference statements)

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“…This is consistent with penetration occurring from large endosomes. The resistance of the mutant cell lines to enveloped viruses (27,28) would appear to be in conflict with our finding that many of the large endosomes in the mutants have a pH <6.2. However, studies of the kinetics of Semliki Forest virus uncoating and penetration have shown that these processes are very similar in the mutants and wild-type cells (Marsh, M., A. Helenius, and I. Mellman, personal communication).…”

Section: Discussioncontrasting

confidence: 55%

“…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: 76%

“…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: 79%

“…Acid phosphatase in contrast to other acid hydrolases is not deficient in I-cell fibroblasts, suggesting that this enzyme is not routed to lysosomes via the mannose-6-phosphate (man-6-P) receptor (14). Use of a lysosomal enzyme that is not carried by the man-6-P receptor was necessary since those enzymes are decreased in the CHO mutants (27,28). We confirmed that acid phosphatase is not depleted in the mutant cells by measuring the total cell enzyme levels.…”

Section: Lntraceuular Localization Of A2mmentioning

confidence: 99%

“…The mean pH of endosomes identified by our image processing method at this time was pH 5.8 in WTB, pH 6.0 in DTG 1-5-4, and pH 6.0 in DTF 1-5-1. This population of vesicles is endosomal, since after 10 min of endocytosis, the vast majority of F-Dex was in a light density endosomal fraction after Percoll density centrifugation (28). The pH of lysosomes was also determined by incubating cells with F-Dex for 10 min, and then reincubated for 30--45 min.…”

Section: Ph Of Endosomes and Lysosomes Containing F-dexmentioning

confidence: 99%

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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: Discussioncontrasting

confidence: 55%

Section: Discussionmentioning

confidence: 76%

mentioning

confidence: 79%

Section: Lntraceuular Localization Of A2mmentioning

confidence: 99%

Section: Ph Of Endosomes and Lysosomes Containing F-dexmentioning

confidence: 99%

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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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The influenza hemagglutinin precursor as an acid-sensitive probe of the biosynthetic pathway.

et al. 1987

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The hemagglutinin of influenza virus (HA), an acid‐activated membrane fusion protein, is synthesized in the endoplasmic reticulum and transported through the Golgi complex to the cell surface of infected cells as an uncleaved, fusion‐incompetent precursor, HA0. The mature, proteolytically activated HA is known to undergo a rapid, irreversible, acid‐induced conformational change which mediates membrane fusion and virus penetration. On the basis of antigenic modifications and the acquisition of trypsin susceptibility, we demonstrate here that HA0, while unable to cause fusion, is acid sensitive. It undergoes irreversible conformational changes quite similar to those of HA at mildly acidic pH (pH less than 6.0). The ectodomain of HA0 does not, however, acquire hydrophobic properties and the changes occur in a less concerted manner (the pH dependence is much broader and the rate of conversion slower). These differences are likely to account for the inability of acid‐treated HA0 to trigger membrane fusion. It was shown, moreover, that HA0 acquired its acid‐sensitive properties immediately following trimerization in the endoplasmic reticulum. Since HA0 did not convert to the acid form at any point during its intracellular transport, we concluded that the trans‐Golgi compartment, known to be more acidic than the cytosol and involved in constitutive membrane transport, is not likely to have a pH less than 6.0.

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Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: Evidence for a maturation model for the formation of lysosomes

Bowser¹,

Murphy²

1987

Journal Cellular Physiology

127

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The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11 degrees C; acidification was limited to pH 6-6.5 at temperatures between 13 degrees C and 17 degrees C. In contrast, a rapid drop to pH 6-6.5 followed by acidification to pH 5-5.5 was observed at temperatures above 19 degrees C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98: 1757-1762). The timing of exposure of material internalized by fluid-phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37 degrees C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13 degrees (and 21 degrees C, the rate of hydrolysis was reduced to 31-44% of that at 37 degrees C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20 degrees C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low-density organelle containing lysosomal enzymes.

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A single mutation in Chinese hamster ovary cells impairs both Golgi and endosomal functions.

Cited by 121 publications

References 57 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.

The influenza hemagglutinin precursor as an acid-sensitive probe of the biosynthetic pathway.

Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: Evidence for a maturation model for the formation of lysosomes

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