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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01081
Research Article |
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
* Author for correspondence (e-mail: ksandvig{at}radium.uio.no)
Accepted 5 January 2004
It has previously been shown that Shiga toxin, despite being bound to a glycolipid receptor, can be efficiently endocytosed from clathrin-coated pits. However, clathrin-independent endocytosis is also responsible for a proportion of the toxin uptake in some cells. After endocytosis the toxin can be transported in retrograde fashion to the Golgi apparatus and the endoplasmic reticulum, and then to the cytosol, where it exerts its toxic effect by inactivating ribosomes. In order to investigate the role of dynamin and clathrin in endosome-to-Golgi transport of Shiga toxin, we have used HeLa dynK44A and BHK antisense clathrin heavy chain (CHC) cells that, in an inducible manner, express mutant dynamin or CHC antisense RNA, respectively. In these cell lines, one can study the role of dynamin and clathrin on endosome-to-Golgi transport because they, as shown here, still internalize Shiga toxin when dynamin- and clathrin-dependent endocytosis is blocked. Butyric acid has been shown to sensitize A431 cells to Shiga toxin by increasing the proportion of cell-associated toxin that is transported to the Golgi apparatus and the endoplasmic reticulum. Here, we find that, in HeLa and BHK cells also, butyric acid also increased toxin transport to the Golgi apparatus and sensitized the cells to Shiga toxin. We have therefore studied the role of dynamin and clathrin in both untreated and butyric-acid-treated cells by measuring the sulfation of a modified Shiga B fragment. Our results indicate that endosome-to-Golgi transport of Shiga toxin is dependent on functional dynamin in both untreated cells and in cells treated with butyric acid. Interestingly, the regulation of Shiga toxin transport in untreated and butyric-acid-treated cells differs when it comes to the role of clathrin, because only cells that are sensitized to Shiga toxin with butyric acid need functional clathrin for endosome-to-Golgi transport.
Key words: Shiga toxin, Clathrin, Dynamin, Golgi apparatus
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