A platform in yeast to understand oxygen sensing mechanisms in higher eukaryotes

Oxygen perception is essential to most terrestrial organisms. It allows them to switch from aerobic to anaerobic metabolism. Creating an orthogonal oxygen sensing device was achieved by transferring essential modules among species. In this project, we studied the expression of cysteine dioxygenases from plants (PLANT CYSTEINE OXIDASES, PCO) or animals (2-AMINOETHANETHIOL DIOXYGENASE, ADO) in the yeast Saccharomyces cerevisiae. These enzymes grant yeast the ability to confer an oxygen-conditional degradability to a bioluminescent protein module containing a Cys-exposing N-degron from RAP2.12, a well-characterized N-degron from plants involved in oxygen perception. Previously, we had reported the use of the Double Luciferase Oxygen Reporter (DLOR) for the quantitative analysis of oxygen dynamics in yeast. In this work, we present the use of this system to: (1) Characterize the effects of PCO point-mutations in its activity towards the degradation of RAP2.12, (2) Create a functional, high-throughput assay to define chemicals that may affect oxygen perception in plants and (3) Obtain a platform to test candidate proteins to be degraded through the ADO-mediated Cys/Arg N-degron pathway.