Summary Fungal infections cause significant morbidity and mortality, particularly in immunocompromised individuals. Most infections are initially treated with Fluconazole or a related azole-class antifungal, which all target sterol biosynthesis enzymes in the endoplasmic reticulum and arrests growth of the pathogen without directly killing it. A serious limitation of these fungistats is the emergence of resistance, in addition to potential for relapse upon withdrawal. Remarkably, azoles can be converted to fungicides by other drugs that specifically inhibit the protein phosphatase calcineurin. The calcineurin inhibitors do not strongly affect resistance mechanisms or change the potency of fungistats. Instead they alter tolerance mechanisms that help the pathogens survive long-term antifungal assaults. Previous studies have focused on how fungistats trigger the activation of calcineurin. This project aims to reveal the downstream effectors of calcineurin that specifically regulate tolerance to the fungistats. Two unbiased screening approaches will be utilized to help define new components of the calcineurin-dependent tolerance mechanism. First, we will utilize a mass spectrometry approach to identify phospho-proteins in a model yeast that change phosphorylation state in response to calcineurin inhibitors during exposure to model fungistats (ER stressors). Second, we will develop a novel genetic approach and conduct the first genome-wide genetic screens in the human opportunistic pathogen Candida glabrata to identify genes that specifically regulate tolerance to Fluconazole. Genes that regulate resistance to Fluconazole also will be identified and categorized. This approach, termed Hermes insertion profiling (HIP), involves in vivo random mutagenesis of the C. glabrata genome using a transposon and Illumina sequencing of the insertion sites. The combination of these unbiased approaches in different yeast species exposed to different fungistat classes provides complementary views of the underlaying tolerance mechanism. Together, a common set of genes/proteins is unveiled whose activities respond to calcineurin in fungistat-stressed cells and regulate tolerance. We propose a series of genetic, biochemical, and cell biological experiments in both yeast species to test several hypotheses about their interactions with one another and their order of action within the calcineurin-dependent tolerance mechanism. These experiments are expected to reveal at least 5 new components in the cascade that act sequentially: the kinases that synthesize inositol pyrophosphates, the protein kinase CK2, the ER enzyme ceramide synthase and its product, and a putative ceramide-activated protein phosphatase. The project therefore provides immediate insights into new therapies that can kill fungal pathogens while establishing a paradigm for tolerance mechanisms that may operate broadly in nature.