||Mitochondria are complex organelles that have important roles in energy production, intermediary metabolism, signal transduction, and apoptosis. Given these critical cellular functions, major efforts have been made to determine the identity of the mitochondrial proteome. Remarkably, about one fifth of mitochondrial proteins have unknown functions, and many of these are conserved through evolution, suggesting that they play a critical role in mitochondrial biology. We have focused on characterizing a subset of these evolutionarily conserved genes through a collaborative effort in Drosophila and yeast model systems. Through this work, we identified novel components of metabolic pathways involved in energy production, represented by the mitochondrial pyruvate carrier (MPC) and two succinate dehydrogenase complex assembly factors (SDHAFs). The MPC is a multimeric complex comprising the MPC1 and MPC2 proteins. Loss of MPC1 in yeast, Drosophila, and humans results in impaired pyruvate metabolism stemming from a defect in pyruvate import through the inner mitochondrial membrane. A point mutation generated in yeast MPC1 confers resistance to a known inhibitor of the MPC, thus linking our discovery to classic biochemical analysis of mitochondrial pyruvate transport. Moreover, Drosophila MPC1 mutants display hallmarks of diabetes, including hyperglycemia, reduced glucose tolerance, and defects in insulin signaling. Taken together, our results demonstrate that the MPC proteins are iv required for mitochondrial pyruvate transport and, further, that this function is critical for maintaining carbohydrate homeostasis in vivo. SDHAF3 and SDHAF4 are mitochondrial matrix proteins that are required for maximal SDH activity in yeast, Drosophila, and mammalian cells. Both of these proteins interact directly with SDH complex subunits to promote normal assembly of the SDH complex. Moreover, Drosophila Sdhaf3 and Sdhaf4 mutants display hallmarks of mitochondrial dysfunction, including muscular and neuronal dysfunction. These studies provide novel candidate causative loci for human diseases characterized by SDH deficiency. Overall, the work described in this thesis yields key insights into mitochondrial pyruvate transport and succinate dehydrogenase complex assembly, using Drosophila to highlight their importance for energy metabolism and animal physiology.