Accumulation of cholera toxin and GM1 ganglioside in the early endosome ofNiemann-Pick C1-deficient cells

We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pic k C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(-) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTS) a ccumulated in GM1-enriched intracellular vesicles of NPC1(-) cells. CTB-lab eled vesicles contained the early endosome marker RAS but not lysosome-asso ciated membrane protein 2 and were not labeled with either Texas red-transf errin or Lysotracker, indicating that they represent early endosomes. Simil arly, CT accumulated in intracellular vesicles of human NPC fibroblasts tha t contained both Rab5 and early endosomal antigen 1. CTB accumulation in NP C1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing do main. A part of these mutant NPC1 proteins expressed in NPC1(-) cells was l ocalized on CTB-labeled vesicles. U18666A treatment of "knock in" cells [NP C1(-) cells that stably expressed wild-type NPC1] caused CTB accumulation s imilar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experimen ts in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subuni ts. In accordance with the latter result, CT was more effective in stimulat ing cAMP formation in NPC1(-) than in wild-type cells. These results sugges t that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi ap paratus/endoplasmic reticulum does not.