Figure 4.

Atg41 localizes to peripheral Atg9-containing sites. (A) Representative images for colocalization of Atg41 and MitoTracker Red. ATG41-GFP (ZYY107) cells were grown to mid-log phase and stained with MitoTracker Red in YPD medium. Cells were then washed and shifted to starvation conditions (SD-N) before fluorescence microscopy analysis; images are representative pictures from single Z-sections. Scale bar: 5 µm. (B) Representative images and the quantification for Atg41-GFP and Atg9-mCherry (ZYY125) colocalization. Cells were starved in SD-N medium prior to microscopy. Scale bar: 5 µm. (C) Representative images for a BiFC assay of strains expressing Atg41-VC and either Atg2-VN (ZYY114), Atg9-VN (ZYY113) or Atg27-VN (ZYY115). Cells were starved in SD-N medium prior to microscopy. Scale bar: 5 µm. (D) Quantitative analysis of cells showing BiFC signals described in (C). The result was examined by 2-way analysis of variance (ANOVA). p values derived from the Sidak post test are reported for the comparisons between ZYY113 and 2 other strains. **, p<0 .01. (E) Images for the yeast 2-hybrid result. The ATG41-AD and AD empty prey plasmids were cotransformed with the ATG9-BD bait plasmid. Cells were grown on SMD –Leu -Ura plates selecting for the presence of both plasmids. Cells were also grown on SMD –Leu –Ura -His plates with 10-fold serial dilution for the selection of strains having protein interactions. (F) Quantitative analysis of Atg9 and prApe1 (PAS) colocalization. WT (SKB171) and mutant (ZYY126) cells were transformed with a plasmid expressing temperature-sensitive Atg1 and cultured at the permissive temperature (24°C) with SMD medium (+N) until the mid-log phase. Samples were then shifted to SD-N and cultured at the nonpermissive temperature (37°C) for 1 h. The colocalization of Atg9–3GFP and RFP-Ape1 were counted and the frequency was determined. The result was examined by ANOVA. p values derived from the Sidak post test are reported for the comparison between wild type and mutant. *, p < 0.05.