ABSTRACT A pivotal point in cellular resource allocation is the point at which glycolytic intermediates are partitioned to lipid biosynthesis versus respiration. For example, hyperlipidemia occurs when cells favor lipid biogenesis and is a major risk factor for cardiovascular disease (CVD) including coronary heart disease, heart attack and stroke, the number one causes of death in the United States. PAS kinase is a serine-threonine protein kinase that is a key regulator of this pivotal point in glucose allocation. PAS kinase-deficient mice (PASK-/-) placed on a high-fat diet or a high-fat high-sugar diet are resistant to liver triglyceride accumulation and display increased whole animal as well as cellular respiration rates when compared to their wild type littermates. Liver triglyceride accumulation and altered metabolic rate are two primary risk factors in the development of CVD as well as related diseases such as type II diabetes. We have recently identified two PAS kinase substrates that may explain its regulation of this pivotal point in metabolism, upstream stimulatory factor 1 (USF1) and Ataxin-2. Our hypothesis is that PAS kinase regulates the pivotal point of partitioning glucose to lipid versus respiratory pathways through phosphorylation of its substrates USF1 and Ataxin-2. USF1 is a transcription factor that directly regulates fatty acid synthase and human mutations in USF1 are associated with familial hypercholesterolemia. PAS kinase phosphorylates and inhibits USF1 in yeast. This phosphorylation leads to decreased respiration and increased lipid biosynthesis. Ataxin-2, on the other hand, associates with and sequesters mRNA and proteins to stress granules, regulating cellular metabolism through their inhibition. PAS kinase-dependent phosphorylation of Ataxin-2 activates the protein by increasing its localization to stress granules in yeast. The focus of this proposal is to further characterize the effects of PAS kinase-dependent phosphorylation on the function of USF1 and Ataxin-2 in yeast and mammalian systems. Our long term goal is to increase our understanding of the regulation of central metabolism while training undergraduates in scientific research, thereby identifying novel targets for the treatment of metabolic disease. Throughout this proposal we will use the genetic and biochemical tools of yeast to investigate diseases for which most undergraduates have a personal connection to, namely hyperlipidemia and diabetes.