MORPHOLOGY OF THE YEAST ENDOCYTIC PATHWAY

Positively charged Nanogold (Nanoprobes, Stony Brook, NY) has been dev eloped as a new marker to follow the endocytic pathway in yeast. Posit ively charged Nanogold binds extensively to the surface of yeast spher oplasts and is internalized in an energy-dependent manner. Internaliza tion of gold is blocked in the end3 mutant. During a time course of in cubation of yeast spheroplasts with positively charged Nanogold at 15 degrees C, the gold was detected sequentially in small vesicles, a per ipheral, vesicular/tubular compartment that we designate as an early e ndosome, a multivesicular body corresponding to the late endosome near the vacuole, and in the vacuole. Experiments examining endocytosis in the sec18 mutant showed an accumulation of positively charged Nanogol d in approximately 30-50 nm diameter vesicles. These vesicles most lik ely represent the primary endocytic vesicles as no other intermediates were detected in the mutant cells, and they correspond in size to the first vesicles detected in wild-type spheroplasts at 15 degrees C. Th ese data lend strong support to the idea that the internalization step of endocytosis in yeast involves formation of small vesicles of unifo rm size from the plasma membrane.