New Autophagy Regulators: Utilization of a novel autophagy assay to screen components of the mating response pathway

Undergraduates: Lauren Askew, Claire Gordy, PhD

Faculty Advisor: Henrik Dohlman
Department: Biology

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Autophagy is a survival mechanism utilized by all eukaryotic cells during nutrient starvation or stress. During autophagy, the cell eats itself by recycling its cytoplasmic components in the vacuole. Previous research from our lab has shown that some proteins required for autophagy are also required for the G protein-coupled receptor (GPCR)-mediated mating pathway in S. cerevisiae (budding yeast). We are interested in determining whether the GPCR-mediated mating pathway is required for autophagy. To investigate the relationship between the pathways, we have optimized a novel quantitative live-cell fluorimetric assay to dynamically measure autophagic activity as cells are exposed to different stresses or stimuli. This assay utilizes a plasmid that expresses a biosensor composed of a pH-stable red fluorescent protein (dsRed) linked to a pH-sensitive green fluorescent protein (super ecliptic pHluorin, SEP). which allows us to quantify autophagy. S. cerevisiae cells are starved for nitrogen to induce autophagy or stimulated with pheromone to activate GPCR signaling, and the autophagic activity is measured over time in vivo. Using this assay we discovered that pheromone induces autophagy. Currently we are using gene deletions to screen different components of the autophagic pathway and the GPCR-mediated mating response pathway. In this manner, we hope to define the signaling pathway through which GPCR activation induces autophagy.