FIGURE 3.

Mutation of Y2 to alanine results in the inability to recruit Mint3 in CD8-APP or APP. A, schematic representation of APP, consisting of the lumenal, transmembrane (TM), and cytosolic (tail) domains showing the primary sequence of only the cytoplasmic tail. The three tyrosines mutated (to alanines) are shown above as Y1, Y2, or Y3 and the three-residue FFE motif, mutated to AAA, is underlined. The lower construct indicates that the lumenal and transmembrane domains of APP were replaced with those of CD8. HeLaM cells were transfected with the cargos indicated and 18 h later were fixed and stained with antibodies against APP and Mint3 (B) or CD8 and Mint3 (C). Confocal images are shown. Wild type and all other mutants recruit Mint3 comparably, but the Y2A mutation uniquely loses Mint3 recruitment. D and E, Mint3 recruitment was quantified in cells treated as described above. Stacks of wide field images were collected and deconvolved, and isosurfaces were generated based on APP staining (D) or CD8 staining (E). The total pixel intensity within each isosurface was calculated and expressed as a ratio of Mint3 intensity/cargo volume. n >7 cells were used per condition. ANOVA was use to compare groups to mock-transfected cells (D) or CD8-APP (E). Asterisks indicate statistical significance compared with mock-transfected (D) or CD8-APP (E) (p < 0.05). F, total cellular Mint3 is unaffected by expression of APP mutants. HeLaM cells were transfected with the CD8-APP (G) or full-length APP (H) constructs indicated and treated as described in F. Immunoblots were probed with an antibody against CD8 (G) or the N terminus of APP (H), as described under “Experimental Procedures.” Overall, none of the mutations cause a change in the level of expression in total cell homogenates. WB, Western blot.