Figure 4.

Pfa3p is required for Vac8p palmitoylation and membrane association in vivo. (A) WT, pfa3Δ, pfa3Δerf2Δ, and pfa3Δswf1Δ cells transformed with Vac8p-myc-GFP (pML735) were incubated with [³H]palmitate for 15 min before lysis and immunoprecipitation. Vac8p protein was quantitated from gels stained with SYPRO. Palmitoylation was detected by fluorography and quantitated by densitometry. Protein and [³H]palmitate values were normalized to wild type and expressed as ratios in the bar graph. (B) Whole cell lysates from WT or pfa3Δ cells transformed with Vac8p-myc-GFP underwent differential centrifugation to evaluate Vac8p association with internal membranes (IM, P13), plasma membrane (PM, P100), or cytosol (C, S100). Data shown in both A and B are representative of at least two experiments performed with independent transformants.