The GEM-GECO Calcium Indicator Is Useable in Yeast, but Aggravates Effects of Increased Cytosolic Calcium Levels.

Ca is a ubiquitous second messenger, which allows eukaryotic cells to respond to external stimuli. The use of genetically encoded Ca indicators allows real-time monitoring of cytosolic Ca levels to study such responses. Here we explored the possibility of using the ratiometric Ca indicator GEM-GECO for monitoring cytosolic Ca concentration ([Ca]) in the yeast . High-level production of GEM-GECO led to a severe growth defect in cells lacking the vacuolar Ca ATPase Pmc1, which is involved in [Ca] control, and prompted a phenotype resembling that of Pmc1 deficiency, in a strain with wild-type . This was likely due to the presence of the calmodulin domain in GEM-GECO. In contrast to previous studies of genetically-encoded calcium indicators in neuronal cells, our results suggest that physiological effects of GEM-GECO expression in yeast cells are due not to Ca depletion, but to excessive Ca signaling. Despite these drawbacks, study of fluorescence in individual cells revealed switching of GEM-GECO from the Ca-free to Ca-bound state minutes after external addition of CaCl. This was followed by gradual return of GEM-GECO to a Ca-free-state that was impaired in the mutant. These results demonstrate GEM-GECO usability for [Ca] monitoring in budding yeast.