The complex apoptotic functions of the p53 tumor suppressor are central to its antineoplastic activity in vivo. Besides its well-understood classic action as a transcriptional regulator of multiple apoptotic genes, p53 also exerts a transcription-independent apoptotic activity. In the previous grant cycle we elucidated a mechanism for the latter. We showed that wild type p53 protein has a direct role at the mitochondria by engaging in protein-protein interactions with anti- and pro-apoptotic members of the Bcl2 family of mitochondrial permeability regulators, thereby executing the shortest known circuitry of p53 death signaling: 1) A fraction of stress-induced wild type p53 rapidly translocates to mitochondria during p53-dependent death. This is a universal p53 response and occurs in primary, immortal and transformed cultured cells, and in normal tissues upon the entire gamut of p53-inducing stresses such as DNA damage, hypoxia and oncogene deregulation. 2) The majority of p53 traffics to the outer membrane where wtp53 - but not tumor-associated p53 mutants - interacts with BclXL and Bcl2 via its DNA-binding domain and induces Bak oligomerization and outer membrane permeabilization with release of apoptotic activators like Cytochrome C, Smac etc. 3) Deliberate targeting of p53 to mitochondria is sufficient to induce apoptosis and colony suppression of p53-deficient tumor cells. Tumor-derived transactivation-deficient missense mutants of p53 concomitantly loose the ability to interact with BclXL, suggesting that p53 mutations represent `double-hits' by simultaneously abrogating the transcriptional and mitochondrial apoptotic activity of p53. 4) In irradiated mice, mitochondrial p53 translocation triggers a rapid first wave of cell death in radiosensitive tissues. In thymus - the prototype response tissue - this wave is later fortified by the transcriptional program of p53. 5) As to the mechanism of translocation, monoubiquitylation by Mdm2-type E3 ligases promotes mitochondrial p53 translocation. Rather than the nucleus, the cytoplasm contains a separate and distinct p53 pool that becomes stress-stabilized and serves as the major source for p53 translocation. Upon arrival at mitochondria, p53 undergoes rapid deubiquitylation by mitochondrial HAUSP via a stress-induced p53-HAUSP complex that generates the apoptotically active non-ubiquitylated p53. 6) Retroviral gene transfer of mitochondrial targeted wild-type p53 in a cMyc-driven mouse model of Burkitt's lymphoma shows effective tumor killing of p53-null, ARF-null and p53-mutant tumor cells in vivo. This proposal focuses on the next important phase of this promising research. It will generate relevant animal models and define the participation and extent of the p53 action at mitochondria. Aims 1 and 2 will establish transgenic and switchable mitop53 knock-in mouse models to assess the contribution of the mitochondrial p53 program to p53`s acute genotoxic response and long-term tumor suppression. Aim 3 explores whether the mitochondrial p53 program contributes to the acute pathology of ischemic tissue injury. Aim 4 tests whether mitochondrial p53 - beyond triggering the Bax/Bak- lipid pore - also activates the permeability transition pore (PTP).