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Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor.
J Cell Sci. 1999 Nov; 112 (Pt 22):3899-909.JC

Abstract

We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.

Authors+Show Affiliations

Institute for Cancer Research at The Norwegian Radium Hospital, Montebello, Norway.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10547351

Citation

Skretting, G, et al. "Endocytic Mechanisms Responsible for Uptake of GPI-linked Diphtheria Toxin Receptor." Journal of Cell Science, vol. 112 (Pt 22), 1999, pp. 3899-909.
Skretting G, Torgersen ML, van Deurs B, et al. Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor. J Cell Sci. 1999;112 (Pt 22):3899-909.
Skretting, G., Torgersen, M. L., van Deurs, B., & Sandvig, K. (1999). Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor. Journal of Cell Science, 112 (Pt 22), 3899-909.
Skretting G, et al. Endocytic Mechanisms Responsible for Uptake of GPI-linked Diphtheria Toxin Receptor. J Cell Sci. 1999;112 (Pt 22):3899-909. PubMed PMID: 10547351.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor. AU - Skretting,G, AU - Torgersen,M L, AU - van Deurs,B, AU - Sandvig,K, PY - 1999/11/5/pubmed PY - 1999/11/5/medline PY - 1999/11/5/entrez SP - 3899 EP - 909 JF - Journal of cell science JO - J. Cell. Sci. VL - 112 (Pt 22) N2 - We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles. SN - 0021-9533 UR - https://www.unboundmedicine.com/medline/citation/10547351/Endocytic_mechanisms_responsible_for_uptake_of_GPI_linked_diphtheria_toxin_receptor_ L2 - http://jcs.biologists.org/cgi/pmidlookup?view=long&pmid=10547351 DB - PRIME DP - Unbound Medicine ER -