Fig. 2. Analysis of membrane association and palmitoylation of Vac8 mutants. (A) Expression levels of endogenous Vac8 mutants. Proteins were extracted from BJ3505 strains expressing the respective mutants and analyzed by SDS-PAGE and western blotting with antibodies to Vac8 and Vti1. (B,C) Localization of Vac8 mutants to purified vacuoles. Vacuoles were isolated from strains carrying the indicated mutations in Vac8 as described in the Materials and Methods, then washed with 20 mM PIPES (pH 6.8), 150 mM KCl. Isolated vacuoles (30 µg) and total cell lysates were analyzed by SDS-PAGE and western blotting (B). (C) Effect of SH4 mutations in Vac8 on vacuole, binding and inheritance. For vacuole binding, Vac8 bands in B were quantified by laser densitometry (grey bars). Fusion (black bars) was determined by the standard fusion assay (see Materials and Methods). Where indicated, error bars are standard deviations (n=3). To determine vacuole inheritance (white bars), yeast cells of the respective mutants were analyzed by FM4-64 staining and fluorescence microscopy. For each strain at least 80 cells were counted. Inheritance of the wild-type control was on average 72%, which was set to 100% in the Figure. (D) Palmitoylation of Vac8 detected by the biotin-switch method (Hou et al., 2005). Yeast cells were lysed, and free cysteines were quenched by NEM. The extract was then subjected to the biotin-switch procedure using hydroxylamine (HA) and Biotin-BMCC as a crosslinker. A fraction of the lysate (6%) was removed and proteins were TCA precipitated. The remaining modified proteins were captured by Neutravidin-agarose pull-down assay, and analyzed by SDS-PAGE and western blotting with the indicated antibodies. The wild-type control is the same as in Hou et al. (Hou et al., 2005). (E) Yeast cells expressing the respective Vac8 mutant analyzed as in D for palmitoylation.