Monitoring of intracellular calcium in Saccharomyces cerevisiae with an apoaequorin cDNA expression system.

Abstract

A method is described for measuring cytosolic free Ca2+ and its time-dependent changes in the yeast Saccharomyces cerevisiae by using the luminescent protein aequorin as a Ca(2+)-specific indicator. This method with intact yeast cells is labeled "in vivo" to distinguish it from methods with cell extracts, labeled "in vitro." A plasmid in which the apoaequorin cDNA was joined downstream from the glyceraldehyde-3-phosphate dehydrogenase gene promoter was constructed and introduced into yeast cells. The intracellular concentration of apoaequorin expressed by the cDNA was approximately 1 microM, which was high enough to detect the cytosolic Ca2+. Growth of the transformed cells was normal. In the in vitro method, apoaequorin in crude cell extracts was regenerated into aequorin by mixing with coelenterazine, the substrate for the luminescence reaction, whereas in the in vivo method, aequorin was regenerated by incubating intact cells with coelenterazine. Simultaneous addition of 10 mM CaCl2 and 10 microM A23187, a Ca2+ ionophore, to coelenterazine-incorporated cells generated luminescence. Coelenterazine-incorporated cells also responded to native extracellular stimuli. A mating pheromone, alpha-factor, added to cells of mating type a or alpha, generated extracellular Ca(2+)-dependent luminescence specifically in a mating type cells, with maximal intensity occurring 45-50 min after addition of alpha-factor. Glucose added to glucose-starved G0/G1 cells stimulated an increase in extracellular Ca(2+)-dependent luminescence with maximal intensity occurring 2 min after addition. These results show the usefulness of the aequorin system in monitoring [Ca2+]i response to extracellular stimuli in yeast cells.