Cholera toxin (CT), covalently attached to
horseradish peroxidase (HRP), is a specific cytochemical marker for
GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of
cyclic AMP in avian erythrocytes. Using a cytochemical
stain for HRP, we found that 9% of control cultured murine
neuroblastoma cells bound
cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of
peroxidase-positive elements probably corresponds to Golgi apparatus-endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of
neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the
ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in
neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1.