Palmitoylation of Snc1 requires Swf1. (A) Immunoblot of myc-tagged Snc1 expressed in the indicated strains. (B) Extracts of cells expressing myc-tagged Snc1 or the Cys–Ala mutant (Snc1ΔC) were treated with hydoxylamine (H) or, as a control, Tris (T) before gel electrophoresis and immunoblotting. Equal amounts of total protein were loaded in each lane in both (A) and (B), as confirmed by staining of the blot.

Swf1-dependent modification of Syn8 and Tlg1. (A) Extracts were treated with hydroxylamine (H) or Tris (T) as in Figure 1B, and immunoblotted using anti-Syn8 antiserum. (B) Syn8 mobility was compared in the indicated deletion mutants by immunoblotting. (C) Tlg1 was analysed as in panel A, using anti-Tlg1 antiserum. (D) The indicated strains were transformed with an empty vector (−), or a plasmid expressing either C-terminally GFP-tagged WT Swf1 or the equivalent DHHC mutant (Swf1^*). In the left panel, extracts were treated with the cysteine-specific biotinylation reagents, and biotinylated proteins recovered with streptavidin beads before immunoblotting. In the right panel, samples were treated sequentially with N-ethylmaleimide to block free cysteines, then with hydroxylamine to remove palmitate, then with the biotinylation reagent. The loading control represents an example of a cysteine-containing protein detected by probing the same blot with fluorescent streptavidin, to demonstrate the reproducible recovery of biotinylated protein. Controls in which biotinylation was omitted resulted in no detectable Tlg1 being recovered from the streptavidin beads.

Unpalmitoylated Tlg1 is recognised by Tul1 and Bsd2. (A) GFP-tagged Tlg1 mutants with the cysteines replaced by serines (Tlg1M2) or leucines (Tlg1M7) were visualised in the indicated strains. (B) Immunoblotting with anti-GFP antibodies of the strains shown in (A) expressing GFP-Tlg1M2. (C) GFP-Tlg1M7 analysed as in (B) in the indicated strains. (D) GFP-Tlg1 and GFP-Tlg1M2 analysed in an swf1 tul1 double mutant. Sequences of the C termini of the various forms of Tlg1 are shown below.

TMD sequences of yeast and human SNAREs. Cysteine residues are highlighted, and the hydrophobic TMD sequences underlined. The major locations of the proteins are indicated: PM, plasma membrane; endo, endosomes.

Vacuolar targeting of Tlg1 in swf1Δ cells. (A) GFP-Tlg1 visualised in WT and swf1Δ cells, and GFP-Tlg1M2, in which the two cysteine residues are replaced with serines, together with the single cysteine mutants M3 (CC-CS) and M4 (CC-SC), in WT cells. With the mutant proteins, or in swf1 mutant cells, GFP is visible inside vacuoles. (B) Immunoblotting with anti-GFP shows the generation of free (vacuolar) GFP from GFP-Tlg1 in swf1Δ cells, and from GFP-Tlg1M2 in WT cells. The lower panel shows the same samples after immunoprecipitation with anti-Tlg1 antibodies. Note that full-length GFP-Tlg1M2 is not abundant enough to be clearly visible unless concentrated by immunoprecipitation. Numbers indicate apparent sizes in kilodaltons. (C) Immunoblotting shows that degradation of GFP-Tlg1 occurs preferentially in swf1Δ cells. (D) Immunoblotting with anti-Tlg1 shows reduced levels of endogenous protein in swf1Δ cells, and the appearance of bands with reduced mobility corresponding approximately in size to the addition of one and two ubiquitins. Equivalent loading was demonstrated by probing the same blot with antibodies to Pgk1. Tlg1 was also immunoprecipitated from the cells, and the precipitates probed with anti-Tlg1 (centre panel) and anti-ubiquitin (right panel). (E) WT and swf1Δ cells were treated with cycloheximide and samples taken at intervals for the assay of Tlg1 by immunoblotting. Note the logarithmic scale of the vertical axis.

Localisation of Swf1. (A) Tagged Swf1 is functional. Immunoblotting of GFP-Tlg1 in swf1Δ cells transformed also with an empty plasmid (PRS316), or plasmids expressing Swf1 tagged with HA at the N-terminus (HA-Swf1) or C-terminus (Swf1-HA), or GFP at the N-terminus (GFP-Swf1). (B) Fluorescence and immunofluorescence of the N-terminally GFP-tagged Swf1 in swf1Δ cells, and HA-tagged Swf1 in WT cells. Two different fields are shown for Swf1-HA. Identical results were obtained with GFP and HA tags at either the N- or C-terminus, in either swf1Δ or WT cells.