Mitochondrial dysfunction promotes aging, cell death, and ultimately, functional failure and degeneration of the cell. One of the examples is autosomal dominant progressive external ophthalmoplegia (adPEO), an adult-onset neuromuscular degenerative disease caused by mutations in nuclear-encoded proteins, such as the adenine nucleotide translocase isoform 1 (Ant1). Ant1 catalyzes the ADP/ATP exchange across the mitochondrial inner membrane. It is intensively debated that Ant may also play a critical role in the organelle-initiated cell death by participating in the permeabilization of mitochondrial membranes. Our recent investigations have revealed that the pathogenic alleles of Ant1 in adPEO patients cause mitochondrial permeabilization and degenerative cell death in the model yeast system. A parallel study has also indicated that Ant has a novel physiological function distinct from ADP/ATP exchange. We hypothesize that the novel cellular function of Ant is related to a membrane channel, and that a dysregulation of the channel may play a critical role in mitochondrial membrane permeabilization and functional degeneration of adPEO cells. The broad objective of this proposal is to understand the pathogenic mechanism of adPEO and the Ant-induced cell death in general. More specifically, we will (1) determine the mechanism of the Ant-induced cell death, the nature of the Ant-based channel, the origin of the adPEO-associated polydisperse mitochondrial DMA deletions, and the factors (e.g., aging) that affect the onset of the cell death trait in cell populations. (2) We will map the death domains in the Ant molecule and isolate trans-acting elements that modulate the membrane-permeabilizing ability of the mutant Ant. (3) We will test the bifunctionality hypothesis by genetic dissection of Ant molecules from yeast and humans and determine the origin of the pathogenic membrane-permeabilizing channel. The uniqueness of the proposal is the use of the unparalleled yeast system that permits an easy genetic tracking of the cell death mechanism. Identifying the exact pathogenic factor of adPEO could lead to the development of adequate therapeutic strategies for clinical treatment of the disease. Most importantly, success of the proposed studies could contribute to a better understanding of the exact role of Ant in cell death, which in turn, could have a broad applicability to other forms of degenerative disease resulting from excessive cell destruction.