FIGURE 8.

The effects of PI(3,5)P₂ levels on Pmc1 interactions. A, Vacuoles from wild type yeast and a strain expressing HA-tagged Pmc1 were compared by immunoprecipitation with anti-HA beads and immunoblotting for isolated complexes. B, Vacuoles expressing Pmc1-HA3 were incubated in large scale reactions (20X) in the presence of 150 μM C8-PI(3,5)P₂, 500 nM ML1-N, 500 μM Apilimod or buffer alone. Reactions were incubated for 30 minutes at 27°C. After incubating, the reactions were place on ice and solubilized and processed for immunoprecipitation with anti-HA agarose. Protein complexes were eluted with sodium thiocyanate and resolved by SDS-PAGE and probed by Western blotting for the presence of Pmc1-HA3 and Nyv1. C, Average quantitation of Nyv1 and Vph1 bound to Pmc1-HA. Western blot band intensities were measured using ImageJ (NIH). Values for the buffer control at 27°C were set to 1 and the other values were normalized relative to this control. Values represent concentrations relative to Pmc1-HA3 at T = 30 minutes. Error bars represent SD (n = 3). Significant differences were in comparison to the buffer control at 27°C *P < .05, **P < .01 (unpaired t test)