Figure 1.

Reduced endocytosis of Snc2-M42A-HA-6Kp. (A) Snc2-M42A-HA-6Kp in a budding endosomal vesicle. Snc2p has a t-SNARE-binding α-helical domain adjacent to a C-terminal transmembrane anchor. Snc2-HA-6Kp was constructed by appending three HA epitope tags (HA) and six lysines (6K) to the C terminus of Snc2p. The extension is accessible from outside of the cell when Snc2-HA-6Kp is on the plasma membrane, but is protected after internalization of Snc2-HA-6Kp to endosomal vesicles. The M42A mutation is located within the α-helical domain. (B) Endocytosis of wild-type and M42A mutant Snc2-HA-6Kp. Cells expressing wild-type (NY2202) or M42A mutant (NY2203) Snc2-HA-6Kp were surface labeled with NHS-SS-biotin at 4°C. Aliquots of cells were incubated at 30°C for the indicated times (in minutes). Biotin was stripped off proteins remaining at the cell surface by reduction with glutathione. One aliquot of cells (total) was not warmed to 30°C or stripped. The cells were lysed and Snc proteins were collected by immunoprecipitation with anti-Sncp antiserum. A Western blot was probed for biotinylated Sncp with streptavidin-HRP. (C) Endocytosis of wild-type and M43A mutant Snc1-HA-6Kp. Endocytosis was measured in cells expressing Snc1-HA-6K (wt, NY2268) and Snc1-M43A-HA-6K (A, NY2269) as described above.