Vac17-S222 is required for the ubiquitylation of Vac17. (A) Phosphorylation of Vac17-S222 and Vac17-T240 are independent of each other. (B) Levels of pT240 or pS222 were normalized to GFP, and those ratios were normalized to wild type (WT). AU, arbitrary unit. (C) Dma1 recruitment to the vacuole (FM4-64; arrows) does not require Vac17-S222. Wild-type VAC17 or vac17 point mutants were expressed from plasmids in a DMA-3×GFP vac17Δ strain. DIC, differential interference contrast. (D) Quantification of >21 cells per strain per n. Error bars indicate SEM. n = 3. **, P < 0.01; two-tailed Student’s t test. (E) The vac17-S222A mutant is not ubiquitylated in vivo. vac17Δ and dma1Δ dma2Δ vac17Δ cells were cotransformed with a plasmid encoding myc-ubiquitin under a copper-inducible promoter and plasmids encoding GFP-tagged VAC17 or vac17-S222A. GFP-tagged proteins were immunoprecipitated (IP) using anti-GFP antibodies. Ubiquitylation detected via immunoblotting with anti-myc antibody. Pgk1 was used as a loading control. SA indicates the vac17-S222A-GFP mutant. Molecular mass is shown in kilodaltons.