FIGURE 5.

PI(3,5)P₂ regulates Ca²⁺ transport through Pmc1 during fusion. A, Vacuoles isolated from wild type as well as pmc1Δ, yvc1Δ and vcx1Δ strains were used in Ca²⁺ transport assays as described. Reactions were incubated with 140 μg/mL anti-Sec17, buffer or 116 μM C8-PI(3,5)P₂ added after 5 minutes of incubation. Reactions were incubated for a total of 40 minutes and fluorescence was measured every 60 seconds. B, Average Ca²⁺ transport levels for multiple experiments with wild type, pmc1Δ, yvc1Δ or vcx1Δ vacuoles. After 10 minutes of incubation reactions were incubated with buffer, DMSO, 116 μM C8-PI(3,5)P₂, 500 μM Verapamil or C8-PI(3,5)P₂ and Verapamil together and incubated for a total of 40 minutes. The extraluminal Ca²⁺ at the end of 40 minutes was normalized to 1 for the buffer and DMSO controls. The amount of extraluminal Ca²⁺ for each treatment was then normalized to the controls. Error bars are SEM (n = 3). Significant differences were in comparison the untreated control (buffer) *P < .05, ***P < .001 (unpaired t test)