Yck3 palmitoylation is required for LE–Golgi sorting but can be obviated upon substitution with a TMD. (A) Palmitoyltransferase activity is required for LE–Golgi retrieval. Yeast lacking five DHHC palmitoyltransferases (DHHC 5×Δ) and expressing either GFP-YIF1 (top) or KEX2-GFP (bottom) are shown. Note the localization of Yif1 to LE and vacuole and Kex2 to the vacuole membrane. (B) Yck3 is mislocalized to LEs in cells depleted of palmitoyltransferases. DHHC 5×Δ yeast expressing GFP-YCK3 are shown. (C) Depletion of DHHC palmitoyl transferases reduces Sed5 phosphorylation. WT, btn1Δ, and DHHC 5×Δ cells were processed for Western analysis with anti-Sed5 and -Sso antibodies, as described in Fig. 5 A. NP and P indicate the NP and P form of Sed5, respectively. The histogram shows quantification of the Sed5 bands after normalization. The data shown are representative of multiple replicates of the experiment (n = 3). (D) Yif1 is mislocalized to LEs in yck3Δ cells expressing a Yck3-FYVE fusion protein. WT and btn1Δ cells expressing both YCK3-FYVE from the YCK3 locus and GFP-YIF1 from a single-copy plasmid are shown. (E) Expression of Yck3 fused to a TMD bypasses the Btn1 requirement for Yif1 localization to the Golgi. btn1Δ cells and btn1Δ cells expressing YCK3-VAM3 from the YCK3 locus were transformed with a plasmid expressing GFP-Yif1, labeled with FM4-64, and examined. Note the localization of Yif1 to LEs in btn1Δ cells and to smaller puncta (that do not colabel with FM4-64) in cells expressing Yck3-Vam3. (F) Expression of Yck3-Vam3 partially restores Sed phosphorylation. WT, btn1Δ, and btn1Δ cells expressing YCK3-VAM3 were processed for Western analysis and Sed5 quantification, as described in Fig. 5 A. The data shown are representative of multiple replicates of the experiment (n = 2). Bars, 1 µm.