PROJECT SUMMARY In low oxygen conditions, such as the hypoxic microenvironment of solid tumors, cells cannot perform mitochondrial respiration and rely solely on glycolysis for ATP generation. Thus, to overcome this deficit in energy production, cells require mechanisms to enhance ATP generation in response to hypoxia. We discovered that in hypoxic yeast cells, the enzymes of the glycolysis pathway, which are diffusely localized in the cytosol under normoxic conditions, organize into non-membrane-bound structures we term Glycolytic (G) bodies. G bodies constitute ribonucleoprotein (RNP) condensates formed by phase separation. G body formation is correlated with increased glucose consumption and cell survival and proliferation under hypoxia. Similar structures have been observed in C. elegans and human cancer cell lines, supporting G body formation as an evolutionarily conserved adaptive response. We hypothesize that G bodies enhance rates of glycolysis by coordinating the multiple steps of the pathway to promote cell survival during hypoxic stress conditions. Using biochemical purification, we have determined the protein and RNA constituents of G bodies and our genome-wide deletion screen identified key signaling pathways that influence G body formation. A major gap in the condensate field is the lack of direct evidence for condensate function. Our preliminary results indicate that G bodies exhibit enhanced glycolytic enzyme activity, thus supporting a functional role for these novel RNP condensates. In this proposal, we will employ a diverse array of experimental strategies to investigate G body activity, physiological impact, biogenesis, biophysical properties, and functional conservation. Mechanisms by which G bodies potentiate glycolytic enzyme activity will be pursued using our purified G body in vitro system and by utilizing novel metabolic biosensors in vivo. We will also determine the global metabolic impact of G bodies through metabolomic approaches and investigate the genetic regulation of G body biogenesis and function mediated by conserved energy-sensing signaling pathways. Molecular and biophysical analysis of G bodies will examine the role of non-canonical RNA binding by glycolysis enzymes in condensate formation via phase separation. Finally, we will take the lessons learned in yeast and apply them to human cancer cell lines and 3D spheroid cultures to study the conservation of G body biophysical properties, biogenesis, and physiological function. Successful outcomes of our research will reveal novel basic principles of RNP condensate regulation and function across species and provide mechanistic insights and potential therapeutic strategies to mitigate hypoxic adaption and cell proliferation in solid tumor microenvironments.