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Figure S1: Control for Yck3 sorting to vacuoles.

A) Vacuole localization of Yck3 depends on the C-terminal anchor. GFP-tagged Yck3 lacking the cysteine-rich C-terminus (amino acid 517-525) was analysed in the indicated strains by fluorescence microscopy and DIC optics. B) Vac8-GFP localization in the indicated cells was analysed as in (A).

Figure S2: Localization of GFP-Ras2 in the presence of Erf2 cannot be influenced by overexpressed Pfa3.

Cells were analysed by fluorescence microscopy and DIC optics. 5 × Δ = akr1 Δ akr2 Δ pfa3 Δ pfa4 Δ pfa5 Δ

Figure S3: Sorting of Vac8 and Yck3 upon overexpression of Pfa3-C134A.

A) Localization of GFP-Yck3 in cells overexpressing Pfa3-C134A. Analysis was as in Figure S1. B) Palmitoylation of Vac8 G2A-GFP on membrane fractions of cells overexpressing Pfa3. After subcellular fractionation, the pellet (P) was resuspended in the same amount of lysis buffer as supernatant (S) and total lysate (T), and NEM was added in each sample with 0.5% Triton-X-100. The following steps were the same as described above. Labelling and pull down of biotinylated proteins was as described in Methods and Figure 3E

Table S1: Strains used in this study.

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