Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 in budding yeast and its application for a positive screening for drugs inhibiting calcineurin

Abstract

Calcineurin, which is a Ca2+/calmodulindependent protein phosphatase, is a key mediator in calcium signaling in diverse biological processes and of clinical importance as the target of the immunosuppressant FK506. To identify a mutant(s) in which calcineurin is activated, inhibiting cellular growth as a result, we screened for a mutant(s) whose temperature sensitivity would be suppressed by FK506 from the budding yeast non-essential gene deletion library. We found that the temperature sensitivity of cells in which the conserved Verprolin VRP1 gene had been deleted, which gene is required for actin organization and endocytosis, was suppressed by either FK506 or by cnb1 deletion. Indeed, the calcineurin activity increased significantly in the Δvrp1 cells. Finally, we demonstrated that the Δvrp1 strain to be useful as an indicator in a positive screening for bioactive compounds inhibiting calcineurin.