Two different assays reveal a Rab5-dependent Atg17–Snf7 interaction. (A) Vps21-dependent Atg17–Snf7 BiFC interaction occurs on APs. WT and mutant cells expressing mCherry-Atg8 from the chromosome and VN-Atg17 from a plasmid were cotransformed with VC plasmids: VC empty (left panel) or VC-Snf7 (rest of panels). Cells were shifted to SD-N (30 min) before YFP fluorescence was observed using live-cell fluorescence microscopy (bottom, percentage of cells with YFP dots colocalizing with Atg8, and number of red dots used for the analysis). Scale bar, 5 µm. More than 80 cells per strain were examined; data are presented as the mean ± SD of each variable from three independent experiments. The same capital letter at the top right corner of each mean ± SD indicates no statistically significant difference, while different capital letters indicate significant difference (P < 0.05). (B and C) Vps21-dependent GST-pulldown of Atg17 and Snf7 with each other. WT and vps21Δ mutant cells expressing GST as a negative control, GST-Atg17 (from a plasmid), and endogenously tagged Snf7-HA (B) or GST-Snf7(from a plasmid) and endogenously tagged Atg17-HA (C) were grown, shifted to SD-N (30 min), lysed (crude lysate, left), and subjected to a GST-pulldown analysis (right) followed by Western blot analysis (see Materials and methods). Bar graphs (bottom) show percentage of HA-tagged Snf7 (B) or Atg17 (C) that were pulled down by GST-Atg17 or GST-Snf7, respectively, from WT (100%) and vps21Δ mutant cell lysates. Columns represent mean, error bars represent SD. Results represent three independent experiments. ***P < 0.001.