Project Summary Copper is an essential micronutrient required for normal cellular functions as evidenced by the finding that copper deficiency and overload result in devastating human disorders. Despite the fundamental importance of copper in cellular and organismal health, the intracellular copper trafficking and distribution is poorly understood. For example, copper is required for the assembly and activity of the terminal enzyme of the mitochondrial respiratory chain, however, how copper is delivered to the mitochondria is not known. To address this gap in our understanding of intracellular copper trafficking to the mitochondria, a genome-wide “copper-sensitized” screen was performed, which identified multiple proteins involved in vacuolar function as putative regulators of mitochondrial copper homeostasis. The yeast vacuole has long been implicated in maintaining cellular metal homeostasis, but exactly how vacuolar function impacts mitochondrial metal homeostasis is not understood. The hits from the screen are likely to provide the molecular mechanism underlying this connection. Top “hits” from our screen included multiple components of the adaptor protein 3 complex (AP-3), as regulators of respiratory growth and copper homeostasis. This is a novel finding because AP-3 has not been linked to either metal homeostasis or mitochondrial respiratory function. Additionally, we identified components of the pH-sensing pathway- Rim20 and Rim21, which like AP-3 impact vacuolar function, as novel regulators of copper-dependent mitochondrial function. The loss of either Rim20 or Rim21 causes a decrease in the levels of a copper requiring subunit of the mitochondrial respiratory chain. This result suggests that a perturbation in vacuolar function disrupts mitochondrial copper homeostasis. To investigate the role of the vacuole in copper trafficking to mitochondria, I propose the following aims: (1) Determine the role of AP-3 in intracellular metal homeostasis and mitochondrial function; and (2) Determine the mechanism by which Rim20 and Rim21 promotes expression of copper-containing subunits of the mitochondrial respiratory chain. Since mutations in genes encoding the subunits of AP-3 cause Hermansky-Pudlak syndrome (HPS) in humans, I will experimentally test the hypothesis that like yeast mutants, copper supplementation can alleviate biochemical defects observed in HPS patient cell lines. Thus, the proposed project will not only address a fundamental question in cell biology of intracellular metal trafficking but may also provide new insights on the pathology associated with a rare human disease.