Characterization of the Human Adenine Nucleotide Translocase (ANT) Isoform-1 (ANT-1) and Isoform-3 (ANT-3) Effects on Mitochondrial Bioenergetics and Transmission of Apoptotic Signaling in Vitro

This dissertation addresses the role of the adenine nucleotide translocase (ANT) protein in mitochondria, and how different isoforms of the protein affect cellular homeostasis. All isoforms of the human ANT protein: ANT-1, ANT-2 and ANT-3 transport ADP fiom the cytosol into the mitochondria1 matrix, and transport synthesized ATP fiom the matrix to the cytosol. In so doing, the ANT helps maintain rates of oxidative phosphorylation proportional to energy demands of the cell. It has also been postulated that the ANT acts as a key component of apoptotic signaling by allowing the inner mitochondrial membrane to become permeable to solutes greater than 1,500 MW, causing rupture of mitochondria and release of pro-apoptotic factors such as cytochrome c into the cytosol. Functional characterization of the human isoforms has been limited by difficulty in expressing recombinant ANT proteins. To this end, the current work describes the cloning and successful expression in eukaryotic cell lines of two of the three human isoforms (ANT-1 and ANT-3). Through the use of a novel radioligand, binding affinities of ligands to the ANT were measured in mitochondria isolated from an insect cell line expressing human ANT-3. Homologous and heterologous binding assays revealed that binding was comparable to that measured in mitochondria from bovine heart, a tissue previously shown to express the bovine ANT-1 isoform. More in-depth studies of ANT function were performed on ANT-1 and ANT-3 isoforms over-expressed in the human SH-SYSY cell line. ANT-1 and ANT-3 expressing cells were compared for differences in ANT ligand binding, susceptibility to apoptosis, ADP transport, mitochondria1 respiration and calcium uptake. While ligand binding and ADP transport activity did not differ between isoforms, ANT-3 expression led to an increased susceptibility to BAX-mediated apoptosis. At the same time, ANT-3 expression rendered mitochondria more resistant to calcium-induced collapse of the membrane potential - an event preceding the release of cytochrome c and downstream apoptotic events. Collectively, data on the cell lines provides a model wherein the ANT- 3 isoform more readily interacts either directly or indirectly with BAX, which in turn may be related to known tissue-specific expression levels of the isoforms in vivo