UC San Diego

The enzymes and reactions of the metabolic network provide cells with a means to utilize the energy stored in substrate chemical bonds and to rearrange those bonds to form biosynthetic building blocks. The chapters of this dissertation are all independent bodies of work exploring how the metabolic network influences and regulates cellular function or dysfunction. Chapter 1, titled "Exploring Metabolic Pathways that Contribute to the Stem Cell Phenotype", is a case study on how the metabolic network exerts control over, or is perturbed by the cellular phenotype, specifically the stem cell phenotype. Substrate and pathway utilization along with molecular signals altering metabolism are key regulators of the stem cell phenotype and influence differentiation status. Chapter 2, titled "Inner Mitochondrial Membrane Transport Regulates Cellular Function" explores an under-appreciated node of metabolic regulation: substrate transport across the inner mitochondrial membrane. Mitochondria are the powerhouses of cells, supplying energy in the form of adenosine triphosphate and crucial building blocks for biosynthesis. Therefore communication with the cytosol via molecular transport exerts exquisite control over the metabolic network. This chapter examines the proteins responsible for molecular exchange across the inner mitochondrial membrane with a focus on their regulatory effects and the methodologies used to investigate them. Many of the highlighted studies examine these transport proteins in isolation, and much work remains on elucidating their full influence over cellular function. Chapter 3, titled "Regulation of Substrate Utilization by the Mitochondrial Pyruvate Carrier" examines how mitochondrial pyruvate transport across the inner mitochondrial membrane influences substrate and pathway utilization. Employing ¹³C tracing allows the regulatory effects of the mitochondrial pyruvate carrier to be examined in whole-cell systems, elucidating their altered reliance on fatty acids and amino acids as fuels and biosynthetic precursors. Finally, Chapter 4, titled "Identification of a Mitochondrial Glutamine Carrier" applies ¹³C tracing to solve the inverse problem. The effects on the metabolic network of inhibiting a mitochondrial carrier are used to deduce its substrate. This previously unannotated carrier is found to transport glutamine across the inner mitochondrial membrane