Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-1.
Cells of Saccharomyces cerevisiae were loaded with indo-1, by incubation in a medium of pH 4.5, which contained penta-potassium indo-1. Cells were then washed and resuspended in a buffer of pH 4.0. The emission fluorescence spectra were recorded between 390 and 500 nm (excitation at 355 nm) and the autofluorescent spectra of the matched controls were subtracted. A 19-fold cellular accumulation of indo-1 was achieved. By permeabilization of plasma membranes, leaving the vacuolar membrane intact, it was proved that indo-1 was accumulated in the cytosol. It was also shown that intracellular indo-1 did not leak out of the cells and was not modified by cellular metabolism. Using the emission fluorescence ratio at 410/480 nm, the concentration of a free cytosolic Ca2+ was found to be 346 nM. Vacuolar Ca2+ concentration, calculated from indo-1 fluorescence after lysis of vacuolar and cellular membranes, was found to be 1.3 mM.
Halachmi
D
D
Department of Bacteriology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Eilam
Y
Y
D002118:Calcium / Q000032:analysis*; D004396:Coloring Agents; D003600:Cytosol / Q000032:analysis*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D007211:Indoles; D012441:Saccharomyces cerevisiae / Q000032:analysis* / Q000648:ultrastructure; D013050:Spectrometry, Fluorescence; D014617:Vacuoles / Q000032:analysis*
D004396:Coloring Agents; D005456:Fluorescent Dyes; D007211:Indoles; C048960:indo-1; D002118:Calcium