Figure 4. LIR motifs in Ccz1 are required for Atg8 binding, but not for the endocytic pathway.

(A–C) Interaction of Ccz1 LIR mutants with Atg8. (A) Analysis of purified Mon1-Ccz1 wild-type and mutant complex by SDS-PAGE and Coomassie staining. All of strains were grown at 30°C for purification. (B–C) Mutations in the LIR motifs impair Mon1-Ccz1 interaction with Atg8. Interaction analysis was done as in Figure 2D, and proteins were analyzed by Western blotting (top) and Coomassie staining (bottom). (D) Comparison of vacuole morphology in LIR mutant cells. Cells were grown at 30 or 37°C in starvation medium, and vacuoles were then stained with CMAC. The number of vacuoles per cell was quantified as indicated. Error bars, SD. (E) Effect of LIR mutants on sorting of vacuolar hydrolases. The indicated cells were grown in starvation medium at the indicated temperature for 2 hr. Total cell lysates were generated and proteins were resolved on SDS-PAGE. Western blots were decorated against CPY and Tom40 (as loading control). (F) Endocytosis analysis in LIR mutants. The indicated cells expressing Mup1-GFP were grown in the absence (-Met) of methionine in minimal medium to an OD₆₀₀ of 1.0 at the 23°C. Where indicated, methionine was added after the temperature shift to 37°C, and cells were analyzed by fluorescence microscopy after 1 hr. Size bar, 5 µm.