Morphology of the yeast endocytic pathway

Abstract

Positively charged Nanogold (Nanoprobes, Stony Brook, NY) has been developed as a new marker to follow the endocytic pathway in yeast. Positively charged Nanogold binds extensively to the surface of yeast spheroplasts and is internalized in an energy-dependent manner. Internalization of gold is blocked in the end3 mutant. During a time course of incubation of yeast spheroplasts with positively charged Nanogold at 15 degrees C, the gold was detected sequentially in small vesicles, a peripheral, vesicular/tubular compartment that we designate as an early endosome, 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 Nanogold in approximately 30-50 nm diameter vesicles. These vesicles most likely 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. These data lend strong support to the idea that the internalization step of endocytosis in yeast involves formation of small vesicles of uniform size from the plasma membrane.

Figure 1

Positively charged Nanogold can be used as an endocytic marker. Wild-type and end3 mutant strains were grown overnight, converted to spheroplasts, and incubated under various conditions on ice for 15 min followed by a 30-min incubation at RT. The samples were fixed and embedded and thin sections were cut. (A) Sections from wild-type cells incubated with positively charged Nanogold were enhanced with HQ Silver. (B) Sections from end3 mutant cells incubated with positively charged Nanogold and enhanced. (C) Sections from wild-type cells incubated in the absence of positively charged Nanogold, but enhanced. (D) Sections from wild-type cells incubated with positively charged Nanogold in the presence of NaN₃ and NaF and enhanced. The cells were visualized in the electron microscope as described in MATERIALS AND METHODS. Bar, 200 nm.

Figure 1

Positively charged Nanogold can be used as an endocytic marker. Wild-type and end3 mutant strains were grown overnight, converted to spheroplasts, and incubated under various conditions on ice for 15 min followed by a 30-min incubation at RT. The samples were fixed and embedded and thin sections were cut. (A) Sections from wild-type cells incubated with positively charged Nanogold were enhanced with HQ Silver. (B) Sections from end3 mutant cells incubated with positively charged Nanogold and enhanced. (C) Sections from wild-type cells incubated in the absence of positively charged Nanogold, but enhanced. (D) Sections from wild-type cells incubated with positively charged Nanogold in the presence of NaN₃ and NaF and enhanced. The cells were visualized in the electron microscope as described in MATERIALS AND METHODS. Bar, 200 nm.

Figure 2

Quantitation of internalization by end3 spheroplasts. Positively charged Nanogold was incubated with wild-type and end3 spheroplasts as described in Figure 1, A and B. Gold particles were counted for five sections each on the plasma membrane and over the cell interior. The percentage of internal gold particles was calculated and plotted. The total number of cell surface particles counted was 3680 for wild-type spheroplasts and 4692 for end3 spheroplasts.

Figure 3

Endocytosis at early time points. Wild-type cells were grown overnight, converted to spheroplasts, and incubated with positively charged Nanogold on ice for 15 min (A) and subsequently warmed up to 15°C for 8 min (B and C). The spheroplasts were prepared for electron microscopy and visualized as in Figure 1A. Bar, 200 nm.

Figure 4

Quantitation of labeled profiles. Wild-type cells were grown overnight, converted to spheroplasts, and incubated with positively charged Nanogold on ice for 15 min and subsequently warmed up to 15°C for 0, 8, 12, or 20 min. Labeled vesicles, vesicular/tubular structures (early), oval or spherical structures (late), or vacuoles were counted. The numbers represent an average of two independent countings of the total number of labeled profiles over 20 spheroplast profiles.

Figure 5

Early and late endocytic structures. Wild-type cells were grown overnight, converted to spheroplasts, and incubated with positively charged Nanogold on ice for 15 min and subsequently warmed up to 15°C for 12 min (A and B) or 20 min (C and D). The spheroplasts were prepared for electron microscopy and visualized as in Figure 1A. Bar, 200 nm.

Figure 6

Early endosome profiles. Early endosomal profiles were taken from wild-type cells incubated and treated as in Figure 5. Bar, 200 nm.

Figure 7

Late endocytic structures. Wild-type cells were grown overnight, converted to spheroplasts, and incubated with positively charged Nanogold on ice for 15 min and subsequently warmed up to 15°C for 90 min. The spheroplasts were prepared for electron microscopy and visualized as for Figure 1A. Bar, 200 nm.

Figure 8

Late endosome profiles. Late endosome profiles were taken from wild-type cells treated as in Figure 5, C and D, or Figure 7. Bar, 200 nm.

Figure 9

Small endocytic vesicles are seen in sec18 spheroplasts. Wild-type (A) or sec18 (B and C) cells were grown overnight, converted to spheroplasts, preincubated at 32°C for 10 min, and then incubated with positively charged Nanogold at 32°C for 30 min. The spheroplasts were prepared for electron microscopy and visualized as for Figure 1A. Bar, 200 nm.

Figure 10

Small endocytic vesicles accumulate in sec18 spheroplasts. Mutant (sec18) cells were grown overnight at 24°C, converted to spheroplasts, and incubated with positively charged Nanogold on ice for 15 min and subsequently warmed up to RT or nonpermissive temperature (32°C) for 0, 5, 10, or 40 min. Labeled vesicles, vesicular/tubular structures (early), and oval or spherical structures (late) were counted from 20 spheroplast sections at each time point and temperature.

Figure 11

Putative primary endocytic vesicle profiles. Profiles of putative primary endocytic vesicles were taken from sec18 spheroplasts incubated for 30 min at 32°C with positively charged Nanogold.