Lipidation of Atg8 is reduced in atg21Δ cells. (A) Atg8 lipidation in rich medium versus nitrogen starvation medium. Cells were grown in YPD to early mid-log phase and then starved for 3 h in SD-N. Atg8-PE was separated from Atg8 by 12% SDS page gels containing 6% urea (Kirisako et al., 2000). All strains are of BY4742 background. (B) Deletion of ATG21 antagonizes the accumulation of Atg8-PE in autophagy mutants. WT (SEY6210), atg21Δ (PSY7), atg18Δ (JGY3), atg18Δ atg21Δ (PSY167), atg9Δ (JKY007), and atg9Δ atg21Δ (PSY191) cells were starved for 4 h in SD-N, and Atg8-PE was analyzed by Western blot. (C) Deletion of ATG21 antagonizes the accumulation of GFP-Atg8 at the PAS in autophagy mutants. Strains used in B containing a plasmid expressing GFP-Atg8 from the CUP1 promoter were grown to mid-log phase in SMD and then starved for 4 h in SD-N. (D) atg21Δ cells have a reduced rate of Atg8 lipidation. Cells of BY4742 background were labeled and Atg8 immunoprecipitated as described in MATERIALS AND METHODS and analyzed by 12% SDS-urea gels.