Project Summary Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite involved in various cellular processes. NAD+ metabolism is also an emerging therapeutic target for several human diseases. The NAD+ pool is maintained by three biosynthesis pathways, which are largely conserved from bacteria to human. The regulation of NAD+ metabolism is incompletely understood due to the dynamic flexibility of NAD+ intermediates, the redundancy of biosynthesis pathways, and the complex interconnections among them. The major goal of this proposal is to uncover novel signaling factors that regulate NAD+ homeostasis and to study the underlying mechanisms. Our studies utilize the genetically tractable budding yeast Saccharomyces cerevisiae that has consistently served as an efficient model system to study cellular mechanisms broadly conserved among eukaryotes. We have recently established an NAD+ intermediate-specific genetic system to identify factors that regulate each branch of the NAD+ biosynthesis pathways. Our studies have uncovered novel NAD+ homeostasis factors including transcription factors, NAD+ intermediates transporters, and nutrient-sensing signaling pathways. The current proposal builds on our recent studies of these factors and the interplay between components in NAD+ metabolism and longevity-related nutrient signaling pathways. Our studies in Project 1 and Project 2 will address specific hypotheses derived from our recent studies to elucidate the mechanisms of regulation. A few major gaps in our knowledge of the mechanisms regulating NAD+ homeostasis will be addressed: 1) Which and how signaling pathways regulate NAD+ homeostasis? 2) Which and how cellular processes contribute to the turnover of NAD+ and its intermediates? 3) What is the molecular basis for the cross-regulation of NAD+ biosynthesis and nutrient-sensing pathways? The long-term goal is to understand how cells maintain NAD+ homeostasis in response to changes in growth conditions. The major hypothesis is that NAD+ homeostasis is co-regulated by nutrient-sensing signaling pathways. Intracellular compartmentalization of NAD+ intermediates and homeostasis factors also contribute to the complex interplay of NAD+ homeostasis factors and nutrient sensing pathways. To achieve theses goals we will employ a combination of molecular genetics and biochemical methods to analyze genes, proteins and pathways involved. These studies will increase our understanding of how eukaryotic cells regulate NAD+ homeostasis in response to changes in growth conditions, and which longevity-related nutrient sensing signaling pathways are involved. Overall Significance: NAD+ preservation helps ameliorate age-associated metabolic disorders. Our findings will contribute to understanding the molecular basis and regulation of NAD+ homeostasis as well as the mechanisms underlying metabolic disorders related to aberrant NAD+ metabolism in human.