| Autophagy and apoptosis are two major signaling pathways employed by the cell in response to various stress-induced signals in order to manage such stress and ultimately to determine cellular survival or death. Central to the regulation of both pathways is the BCL-2 protein family. At the endoplasmic reticulum (ER) the pro-survival BCL-2 protein targets multiple protein complexes, which influence Ca2+ homeostasis, as well as the outcome of apoptosis and autophagy pathways. To understand how BCL-2 is partitioned between these complexes, we sought to identify BCL-2-interacting proteins at this site. Here I present the identification and characterization of NAF-1, a novel ER BCL-2-interacting partner. Interestingly, NAF-1 is required for BCL-2 antagonism of autophagy, but is not required by BCL-2 to antagonize apoptosis initiated at the ER by the BH3-only protein BIK. NAF-1 contributes to the physical interaction between BCL-2 and the autophagy effector Beclin 1, which is essential for functional inhibition of autophagy by BCL-2. Thus, NAF-1 targets BCL-2 to the autophagy pathway at the ER.;To study the physiological contribution of the Naf-1 gene, we generated Naf-1 knockout mice. These mice begin to manifest signs of severe muscle degeneration between 2-3 months of age, and exhibit increasingly poor performance status until mortality at 6-9 months. Immortalized MEF cell lines were generated and will be utilized in conjunction with the Naf-1 knockout mice for future elucidation of NAF-1 function.;In summary, this thesis provides a thorough analysis of a novel ER BCL-2-interacting partner, NAF-1, and provides further insight into the mechanism of autophagy regulation by BCL-2.;Furthermore, NAF-1 is found in association with the IP3R ER Ca 2+ efflux channel, and mediates BCL-2 regulation of ER Ca2+ stores. Ectopic expression of ER-targeted BCL-2 reduces ER Ca 2+ content and NAF-1 is required for this process. In response to nutrient deprivation, autophagy induction is accompanied by an early release of ER Ca2+ stores. Cytosolic Ca2+ is required for autophagy in response to starvation, suggesting that the release of Ca 2+ from the ER may be an essential signal for autophagy induction and, in addition to direct sequestration of Beclin 1, a mechanism of inhibition by BCL-2/NAF-1. |
