Human mitochondrial transcription factor A possesses multiple subcellular targeting signals.

The mitochondrial transcription factor A (TFAM) is a member of a high-mobility group (HMG) family represented mostly by nuclear proteins. Although nuclear localization of TFAM has been demonstrated in some tumors and after treatment of tumor cells with anticancer drugs, the significance of these observations has not been fully elucidated. Here we report that both TFAM overexpression and impairment of its mitochondrial targeting can result in nuclear accumulation of the protein. Both M1 and M7 methionines of human TFAM (hTFAM) can be used for translation initiation with almost equal efficiency resulting in two polypeptides. The shorter polypeptide, however, is not located in the nucleus, despite truncation in the mitochondrial targeting sequence, and both isoforms are targeted to mitochondria with similar efficiency. We further demonstrate that nuclear TFAM confers significant cytoprotection against the chemotherapeutic drugs etoposide, camptothecin, and cisplatin. Three regions of hTFAM [HMG-like domain 1 (HMG1) and HMG-like domain 2 (HMG2), as well as the tail region] can effect nuclear accumulation of enhanced green fluorescent protein (EGFP) fusions. The HMG1 domain contains a bipartite nuclear localization sequence whose identity is supported by site-directed mutagenesis. However, this bipartite nuclear localization sequence is weak, and both N-terminal and C-terminal flanking sequences enhance the nuclear targeting of EGFP. Finally, several mutations in the HMG1 domain increased the mitochondrial targeting of the EGFP fusions, suggesting that the mitochondrial targeting sequence of hTFAM may extend beyond the cleavable presequence.