Ykt6 is located on autophagosomes. (A) Localization of Atg8 relative to Ykt6 during normal growth and nitrogen starvation. vam3Δ cells expressing GFP-tagged Atg8 and either mCherry-tagged Ykt6 or Vti1 were grown in YPD or SD-N for 2 h and analyzed by fluorescence microscopy. Single planes of stacks are shown. Scale bar, 5 µm. (B) Percentage of Ykt6 or Vti1 puncta colocalizing with Atg8 under both conditions. The data were quantified from A. Error bars represent standard deviation of three independent experiments. **, P < 0.01 (Student’s t test). (C) Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter were transformed with a centromeric plasmid expressing mCherry-Atg8 under the control of the TPI1 promoter. Cells were grown in SGC-LEU or SG-N for 2 h and analyzed by fluorescence microscopy, and are shown as individual slices. Scale bar, 5 µm. (D) Percentage of Ykt6 puncta colocalizing with Atg8 under both conditions. Data were quantified from C. Error bars represent standard deviation of three independent experiments. **, P < 0.01 (Student’s t test). (E) Ykt6 localizes with Atg8 on purified autophagosomes. The vam3Δ mutant expressing 3xFLAG-tagged Atg9, GFP-tagged Atg8, and mCherry-tagged Ykt6 or Vti1 were grown in YPD medium and then starved in SD-N medium for 3 h. Isolated autophagosomes were then incubated with or without an antibody against Ykt6 or Vti1. Scale bar, 2 µm. (F) Quantification of Fig. 4 E. Error bars represent standard deviation of three independent experiments. **, P < 0.01 (Student´s t test).