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

Ca(2+) is a ubiquitous second messenger, which allows eukaryotic cells to respond to external stimuli. The use of genetically encoded Ca(2+) indicators allows real-time monitoring of cytosolic Ca(2+) levels to study such responses. Here we explored the possibility of using the ratiometric Ca(2+) indicator GEM-GECO for monitoring cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in the yeast Ogataea parapolymorpha. High-level production of GEM-GECO led to a severe growth defect in cells lacking the vacuolar Ca(2+) ATPase Pmc1, which is involved in [Ca(2+)](cyt) control, and prompted a phenotype resembling that of Pmc1 deficiency, in a strain with wild-type PMC1. 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(2+) depletion, but to excessive Ca(2+) signaling. Despite these drawbacks, study of fluorescence in individual cells revealed switching of GEM-GECO from the Ca(2+)-free to Ca(2+)-bound state minutes after external addition of CaCl(2). This was followed by gradual return of GEM-GECO to a Ca(2+)-free-state that was impaired in the pmc1-Δ mutant. These results demonstrate GEM-GECO usability for [Ca(2+)](cyt) monitoring in budding yeast.