Epitope tagging of DAB389IL-2: new insights into C-domain delivery to the cytosol of target cells
van der Spek, J.; Hémard, A.; Dautry-Varsat, A.; Boquet, P.; Murphy, J.R.
Leukemia 8(Suppl): S144-S148
1994
ISSN/ISBN: 0887-6924 PMID: 7512176 Document Number: 429047
The fusion toxin DAB-389 IL-2 is composed of the catalytic (C) and transmembrane (T) domains of native diphtheria toxin to which human interleukin-2 (IL-2) has been genetically fused (1,2). Following binding to the IL-2 receptor, the fusion toxin is internalized by receptor mediated endocytosis, and upon acidification of the endocytic vesicle, the T domain spontaneously inserts into the membrane, and facilitates the delivery of the C domain to the cytosol (3,4). In order to further study the process by which the C domain is delivered to the target cell cytosol, we genetically fused an eleven amino acid epitope derived from the vesicular stomatitis virus (VSV) G protein to the N-terminal end of DAB-389 IL-2. The epitope labelled fusion toxin, VSV-G-DAB-389 IL-2, was found to retain IL-2 receptor specific binding and cytotoxic activity. Target cells were incubated for various times in the presence of VSV-G-DAB-389, fixed and then treated with anti-VSV G and FITC conjugated secondary antibody. Laser scanning confocal microscopy was used to determine the location of the fluorescent signal. The VSV-G epitope tagged fusion toxin was found only to be associated with small vesicles that were situated adjacent to the plasma membrane. These results suggest that the C domain of the fusion toxin is associated with an early intracellular compartment and is rapidly delivered to the cytosol. Since channel formation by the T domain is necessary for the delivery of the C domain, it follows that T domain insertion into the membrane also occurs early in the intoxication pathway.