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The p53 tumor suppressor induces apoptosis in response to genotoxic and environmental stresses.Separately from its functions as a transcription factor, it is also capable to be translocated to the mitochondria and plays a critical role in transcription-independent mitochondrial apoptosis;however, the mechanism that regulates its translocation to the mitochondria has been unknown.We show that the tumor suppressor Tid1 forms a complex with p53 under hypoxic conditions that directs p53 translocation to the mitochondria and the subsequent initiation of the mitochondrial apoptosis pathway.Loss of Tid1 expression abrogated p53 translocation to the mitochondria and inhibited apoptosis, whereas the overexpression of Tid1 promoted p53 mitochondrial localization and apoptosis.Tid1s mitochondrial signal sequence and DnaJ domain were both required for the movement of the p53-Tid1 complex from the cytosol to the mitochondria.When Tid is overexpressed in cancer cell lines expressing mutant p53 isoforms defective in transcriptional activity,mitochondrial localization and pro-apoptotic activities of the mutant p53 proteins was restored.Also with far western analyses, we demonstrated that Tid1 directly interacted with p53.Using domain deletion mutant constructs, we determined that DnaJ domain of Tid1 was necessary for the interaction, while either N-or C-terminal domains of p53 were sufficient for the interaction.In breast cancer cells, depletion of Tid1 by short hairpin RNA (shRNA) lead to absence of p53 accumulation at mitochondria and resistance to apoptosis under hypoxic or genotoxic stresses.Our studies imply that Tid1 could be important in the potential combination chemotherapies ofp53-related cancers.