FIGURE 6.

Vacuoles lacking both Pmc1 and Vcx1 expel Ca²⁺ in an ATP-dependent manner. A, Compares the Ca²⁺ transport profiles of wild type vs the pmc1Δ vcx1Δ double deletion vacuoles. Individual reactions were in the presence of reaction buffer alone, buffer with ATP, or buffer with ATP and 140 μg/mL anti-Sec17 IgG. B, Effect of C8-PI(3,5)P₂ on Ca²⁺ flux by pmc1Δ vcx1Δ double deletion vacuoles. C, Quantitation of relative Ca²⁺ flux into or out of vacuole from wild type and pmc1Δ vcx1Δ double deletion yeast after 5 minutes of incubation. The extraluminal Ca²⁺ in the absence of ATP was set to 1 and the results in the presence of ATP were normalized to the no ATP control. D, Vacuoles from vcx1Δ yeast were incubated with 140 μg/mL anti-Sec17 IgG, 500 μM Verapamil or 125 μM Apilimod added at T = 0 min. E, Quantitation of relative Ca²⁺ uptake wild type and vcx1Δ vacuoles after 10 minutes of incubation. The extraluminal Ca²⁺ in the untreated control was set to 1 and the results with Apilimod and Verapamil was normalized to the untreated control. Error bars are SEM (n = 3). Significant differences were in comparison the untreated control (buffer) **P < .01, ***P < .001 (unpaired t test)